neuronal differentiation


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(Updated 4th quarter 2002)

Laifenfeld, Daphna, Klein, Ehud, Ben-Shachar, Dorit
Norepinephrine alters the expression of genes involved in neuronal sprouting and differentiation: relevance for major depression and antidepressant mechanisms
J Neurochem 2002 83: 1054-1064
"Recent research into depression has focused on the involvement of long-term intracellular processes, leading to abnormal neuronal plasticity in brains of depressed patients, and reversed by antidepressant treatment. Given a suggested decrease in noradrenergic transmission in depression, and an antidepressant induced increase in norepinephrine (NE) level, a possible role for NE in mediating alterations in neuronal morphology and plasticity was examined. Human neuroblastoma SH-SY5Y cells treated with 10-5 m NE presented an elongated granule-rich cell-body and increased number of neurites, when compared with non-treated cells. Moreover, cell survival was enhanced in the presence of NE, while proliferation was inhibited. The above effects suggest a role for NE in cell differentiation. Indeed similar effects on cell survival and neurite outgrowth were induced in SH-SY5Y cells by retinoic acid (RA), an established differentiating agent. Finally, NE treatment resulted in a progressive decrease in the pluripotent marker Oct4 and an increase in the neuronal growth cone marker, growth-associated-protein 43 (GAP-43). Alongside these effects, NE-treated cells presented alterations in the expression of 44 genes as observed in a neurobiology cDNA microarray. Among the altered genes, an increase in the expression level of two neurite-outgrowth promoting genes, neural cell adhesion molecule L1 and laminin, was confirmed by RT-PCR. Taken together, the results support a role for NE in processes of synaptic connectivity, and may point to a role for this neurotransmitter in mediating the suggested neuronal plasticity in depression and in antidepressant treatment." [Abstract]

Lunyak VV, Burgess R, Prefontaine GG, Nelson C, Sze SH, Chenoweth J, Schwartz P, Pevzner PA, Glass C, Mandel G, Rosenfeld MG.
Corepressor-dependent silencing of chromosomal regions encoding neuronal genes.
Science 2002 Nov 29;298(5599):1747-52
"The molecular mechanisms by which central nervous system-specific genes are expressed only in the nervous system and repressed in other tissues remain a central issue in developmental and regulatory biology. Here, we report that the zinc-finger gene-specific repressor element RE-1 silencing transcription factor/neuronal restricted silencing factor (REST/NRSF) can mediate extraneuronal restriction by imposing either active repression via histone deacetylase recruitment or long-term gene silencing using a distinct functional complex. Silencing of neuronal-specific genes requires the recruitment of an associated corepressor, CoREST, that serves as a functional molecular beacon for the recruitment of molecular machinery that imposes silencing across a chromosomal interval, including transcriptional units that do not themselves contain REST/NRSF response elements." [Abstract]

Adams, J. Paige, Sweatt, J. David
MOLECULAR PSYCHOLOGY: Roles for the ERK MAP Kinase Cascade in Memory
Annu. Rev. Pharmacol. Toxicol. 2002 42: 135-163
"In this review we describe an emerging understanding of the roles of the Extracellular-signal regulated kinase/mitogen-activated protein kinase (ERK/MAPK) cascade in learning and memory. We begin by describing several behavioral memory paradigms and review data implicating ERK as an essential component of the signal transduction mechanisms subserving these processes. We then describe evidence implicating ERK as a critical player in synaptic and neuronal plasticity—a cellular role likely to underlie ERK's behavioral role in the animal. We then proceed to parsing the complexities of biochemical regulation of ERK in neurons and to a description of a few likely cellular targets of ERK. This is in order to begin discussing the possible molecular basis of ERK-mediated behavioral change. We close our review with speculations concerning how the complexities and idiosyncrasies of ERK regulation may allow for sophisticated information processing at the biochemical level in neurons—attributes that may make the ERK cascade well-suited for triggering complex and long-lasting behavioral change. Our goal in this review is not so much to portray ERK as unique regarding its role as a signal transducter in memory, but rather to use ERK as one specific example of recent studies beginning to address the molecules and signal transduction pathways subserving cognition." [Abstract]

Ying, Shui-Wang, Futter, Marie, Rosenblum, Kobi, Webber, Mark J., Hunt, Stephen P., Bliss, Timothy V. P., Bramham, Clive R.
Brain-Derived Neurotrophic Factor Induces Long-Term Potentiation in Intact Adult Hippocampus: Requirement for ERK Activation Coupled to CREB and Upregulation of Arc Synthesis
J. Neurosci. 2002 22: 1532-1540
"Brain-derived neurotrophic factor (BDNF) is implicated in long-term synaptic plasticity in the adult hippocampus, but the cellular mechanisms are little understood. Here we used intrahippocampal microinfusion of BDNF to trigger long-term potentiation (BDNF-LTP) at medial perforant path-granule cell synapses in vivo. BDNF infusion led to rapid phosphorylation of the mitogen-activated protein (MAP) kinases ERK (extracellular signal-regulated protein kinase) and p38 but not JNK (c-Jun N-terminal protein kinase). These effects were restricted to the infused dentate gyrus; no changes were observed in microdissected CA3 and CA1 regions. Local infusion of MEK (MAP kinase kinase) inhibitors (PD98059 and U0126) during BDNF delivery abolished BDNF-LTP and the associated ERK activation. Application of MEK inhibitor during established BDNF-LTP had no effect. Activation of MEK-ERK is therefore required for the induction, but not the maintenance, of BDNF-LTP. BDNF-LTP was further coupled to ERK-dependent phosphorylation of the transcription factor cAMP response element-binding protein. Finally, we investigated the expression of two immediate early genes, activity-regulated cytoskeleton-associated protein (Arc) and Zif268, both of which are required for generation of late, mRNA synthesis-dependent LTP. BDNF infusion resulted in selective upregulation of mRNA and protein for Arc. In situ hybridization showed that Arc transcripts are rapidly and extensively delivered to granule cell dendrites. U0126 blocked Arc upregulation in parallel with BDNF-LTP. The results support a model in which BDNF triggers long-lasting synaptic strengthening through MEK-ERK and selective induction of the dendritic mRNA species Arc."
[Full Text]

Rosenblum, Kobi, Futter, Marie, Voss, Karen, Erent, Muriel, Skehel, Paul A., French, Pim, Obosi, Louis, Jones, Matt W., Bliss, Tim V. P.
The Role of Extracellular Regulated Kinases I/II in Late-Phase Long-Term Potentiation
J. Neurosci. 2002 22: 5432-5441
"Extracellular regulated kinases (ERKI/II), members of the mitogen-activated protein kinase family, play a role in long-term memory and long-term potentiation (LTP). ERKI/II is required for the induction of the early phase of LTP, and we show that it is also required for the late phase of LTP in area CA1 in vitro, induced by a protocol of brief, repeated 100 Hz trains. We also show that ERKI/II is necessary for the upregulation of the proteins encoded by the immediate early genes Zif268 and Homer after the induction of LTP in the dentate gyrus by tetanic stimulation of the perforant path in vivo or by BDNF stimulation of primary cortical cultures. To test whether the induction of persistent synaptic plasticity by stimuli such as BDNF is associated with nuclear translocation of ERKI/II, we expressed enhanced green fluorescent protein (EGFP)-ERKII in PC12 cell lines and primary cortical cultures. In both preparations, we observed translocation of EGFP-ERKII from the cytoplasm to the nucleus in cells exposed to neurotrophic factors. Our results suggest that the induction of late LTP involves translocation of ERKI/II to the nucleus in which it activates the transcription of immediate early genes. The ability to visualize the cellular redistribution of ERKII after induction of long-term synaptic plasticity may provide a method for visualizing neuronal circuits underlying information storage in the brain in vivo."

Joan Arehart-Treichel
Size of Hippocampus Has Implications for Depression
Psychiatric News August 16, 2002 Volume 37 Number 16 p. 24 [Article]

Dwivedi, Y., Rizavi, H. S., Roberts, R. C., Conley, R. C., Tamminga, C. A., Pandey, G. N.
Reduced activation and expression of ERK1/2 MAP kinase in the post-mortem brain of depressed suicide subjects
J Neurochem 2001 77: 916-928 [Abstract]

Jonathan D. Cooper, Ahmad Salehi, Jean-Dominique Delcroix, Charles L. Howe, Pavel V. Belichenko, Jane Chua-Couzens, Joshua F. Kilbridge, Elaine J. Carlson, Charles J. Epstein, and William C. Mobley
Failed retrograde transport of NGF in a mouse model of Down's syndrome: Reversal of cholinergic neurodegenerative phenotypes following NGF infusion
PNAS 98: 10439-10444; published online before print as 10.1073/pnas.181219298
"Age-related degeneration of basal forebrain cholinergic neurons (BFCNs) contributes to cognitive decline in Alzheimer's disease and Down's syndrome. With aging, the partial trisomy 16 (Ts65Dn) mouse model of Down's syndrome exhibited reductions in BFCN size and number and regressive changes in the hippocampal terminal fields of these neurons with respect to diploid controls. The changes were associated with significantly impaired retrograde transport of nerve growth factor (NGF) from the hippocampus to the basal forebrain. Intracerebroventricular NGF infusion reversed well established abnormalities in BFCN size and number and restored the deficit in cholinergic innervation. The findings are evidence that even BFCNs chronically deprived of endogenous NGF respond to an intervention that compensates for defective retrograde transport. We suggest that age-related cholinergic neurodegeneration may be a treatable disorder of failed retrograde NGF signaling."
[Full Text]

Markus Klinger, Oliver Kudlacek, Markus G. Seidel, Michael Freissmuth, and Veronika Sexl
MAP Kinase Stimulation by cAMP Does Not Require RAP1 but SRC Family Kinases
J. Biol. Chem. 277: 32490-32497, September 2002.
"The small G protein RAP1 and the kinase B-RAF have been proposed to link elevations of cAMP to activation of ERK/mitogen-activated protein (MAP) kinase. In order to delineate signaling pathways that link receptor-generated cAMP to the activation of MAP kinase, the human A2A-adenosine receptor, a prototypical Gs-coupled receptor, was heterologously expressed in Chinese hamster ovary cells (referred as CHO-A2A cells). In CHO-A2A cells, the stimulation of the A2A-receptor resulted in an activation of RAP1 and formation of RAP1-B-RAF complexes. However, overexpression of a RAP1 GTPase-activating protein (RAP1GAP), which efficiently clamped cellular RAP1 in the inactive GDP-bound form, did not affect A2A-agonist-mediated MAP kinase stimulation. In contrast, the inhibitor of protein kinase A H89 efficiently suppressed A2A-agonist-mediated MAP kinase stimulation. Neither dynamin-dependent receptor internalization nor receptor-promoted shedding of matrix-bound growth factors accounted for A2A-receptor-dependent MAP kinase activation. PP1, an inhibitor of SRC family kinases, blunted both the A2A-receptor- and the forskolin-induced MAP kinase stimulation (IC50 = 50 nM); this was also seen in PC12 cells, which express the A2A-receptor endogenously, and in NIH3T3 fibroblasts, in which cAMP causes MAP kinase stimulation. In the corresponding murine fibroblast cell line SYF, which lacks the ubiquitously expressed SRC family kinases SRC, YES, and FYN, forskolin barely stimulated MAP kinase; this reduction was reversed in cells in which c-SRC had been reintroduced. These findings show that activation of MAP kinase by cAMP requires a SRC family kinase that lies downstream of protein kinase A. A role for RAP1, as documented for the {beta}2-adrenergic receptor, is apparently contingent on receptor endocytosis." [Abstract]

Hansen, Thomas v. O., Rehfeld, Jens F., Nielsen, Finn C.
Cyclic AMP-Induced Neuronal Differentiation via Activation of p38 Mitogen-Activated Protein Kinase
J Neurochem 2000 75: 1870-1877
"The p38 mitogen-activated protein kinase (MAPK) pathway mediates cellular responses to inflammatory cytokines and environmental stress, but recent studies have indicated that p38 MAPK may be involved in a more widespread set of cellular functions. Here we show that activation of the cyclic AMP (cAMP) pathway induces a rapid, dose-dependent phosphorylation and activation of p38 MAPK and that combined stimulation with forskolin and growth factors results in additive stimulation of p38 MAPK. Forskolin-stimulated neurite out-growth in rat pheochromocytoma PC12 cells was inhibited by the p38 MAPK inhibitor SB203580. With the combination of forskolin and nerve growth factor, neurite outgrowth was additively increased, and this effect was also inhibited by SB203580. Finally, transfection of p38AGF, which exhibits a mutated activation loop, inhibited cAMP-mediated neuronal differentiation. The results indicate that p38 MAPK is a downstream target of the cAMP signaling pathway and that p38 MAPK plays a key role in neuronal differentiation induced by cAMP and growth factors by integration of signals from both pathways." [Abstract]

Curtis J, Finkbeiner S.
Sending signals from the synapse to the nucleus: possible roles for CaMK, Ras/ERK, and SAPK pathways in the regulation of synaptic plasticity and neuronal growth.
J Neurosci Res 1999 Oct 1;58(1):88-95
"The ability to learn and form memories depends on specific patterns of synaptic activity and is in part transcription dependent. However, the signal transduction pathways that connect signals generated at synapses with transcriptional responses in the nucleus are not well understood. In the present report, we discuss three signal transduction pathways: the Ca(2+)/calmodulin-dependent kinase (CaMK) pathway, the Ras/ERK pathway, and the SAPK pathways that might function to couple synaptic activity to long-term adaptive responses, in part through the regulation of new gene expression. Evidence suggests that these pathways become activated in response to stimuli that regulate synaptic function such as the influx of extracellular Ca(2+) and certain neurotrophin growth factors such as brain-derived neurotrophic factor. Once activated, the CaMK, Ras/ERK, and SAPK pathways lead to the phosphorylation and activation of transcription factors in the nucleus such as the cyclic AMP response element binding protein (CREB). Genes regulated by CREB or other transcription factor targets of the CaMK, Ras/ERK, and SAPK pathways could mediate important adaptive responses to changes in synaptic activity such as changes in synaptic strength and the regulation of neuronal survival and death. Copyright 1999 Wiley-Liss, Inc" [Abstract]

Wansong Qiu, Shunhui Zhuang, Friederike C. von Lintig, Gerry R. Boss, and Renate B. Pilz
Cell Type-specific Regulation of B-Raf Kinase by cAMP and 14-3-3 Proteins
J. Biol. Chem. 275: 31921-31929, October 2000
"Cyclic AMP can either activate or inhibit the mitogen-activated protein kinase (MAPK) pathway in different cell types; MAPK activation has been observed in B-Raf-expressing cells and has been attributed to Rap1 activation with subsequent B-Raf activation, whereas MAPK inhibition has been observed in cells lacking B-Raf and has been attributed to cAMP-dependent protein kinase (protein kinase A)-mediated phosphorylation and inhibition of Raf-1 kinase. We found that cAMP stimulated MAPK activity in CHO-K1 and PC12 cells but inhibited MAPK activity in C6 and NB2A cells. In all four cell types, cAMP activated Rap1, and the 95- and 68-kDa isoforms of B-Raf were expressed. cAMP activation or inhibition of MAPK correlated with activation or inhibition of endogenous and transfected B-Raf kinase. Although all cell types expressed similar amounts of 14-3-3 proteins, approximately 5-fold less 14-3-3 was associated with B-Raf in cells in which cAMP was inhibitory than in cells in which cAMP was stimulatory. We found that the cell type-specific inhibition of B-Raf could be completely prevented by overexpression of 14-3-3 isoforms, whereas expression of a dominant negative 14-3-3 mutant resulted in partial loss of B-Raf activity. Our data suggest that 14-3-3 bound to B-Raf protects the enzyme from protein kinase A-mediated inhibition; the amount of 14-3-3 associated with B-Raf may explain the tissue-specific effects of cAMP on B-Raf kinase activity." [Full Text]

Melanie C. MacNicol, Anthony J. Muslin, and Angus M. MacNicol
Disruption of the 14-3-3 Binding Site within the B-Raf Kinase Domain Uncouples Catalytic Activity from PC12 Cell Differentiation
J. Biol. Chem. 275: 3803-3809, February 2000.
"A number of Raf-associated proteins have recently been identified, including members of the 14-3-3 family of phosphoserine-binding proteins. Although both positive and negative regulatory functions have been ascribed for 14-3-3 interactions with Raf-1, the mechanisms by which 14-3-3 binding modulates Raf activity have not been fully established. We report that mutational disruption of 14-3-3 binding to the B-Raf catalytic domain inhibits B-Raf biological activity. Expression of the isolated B-Raf catalytic domain (B-Rafcat) induces PC12 cell differentiation in the absence of nerve growth factor. By contrast, the B-Rafcat 14-3-3 binding mutant, B-Rafcat S728A, was severely compromised for the induction of PC12 cell differentiation. Interestingly, the B-Rafcat 14-3-3 binding mutant retained significant in vitro catalytic activity. In Xenopus oocytes, the analogous full-length B-Raf 14-3-3 binding mutant blocked progesterone-stimulated maturation and the activation of endogenous mitogen-activated protein kinase kinase and mitogen-activated protein kinase. Similarly, the full-length B-Raf 14-3-3 binding mutant inhibited nerve growth factor-stimulated PC12 cell differentiation. We conclude that 14-3-3 interaction with the catalytic domain is not required for kinase activity per se but is essential to couple B-Raf catalytic activity to downstream effector activation." [Full Text]

Priam Villalonga, Cristina López-Alcalá, Antonio Chiloeches, Joan Gil, Richard Marais, Oriol Bachs, and Neus Agell
Calmodulin Prevents Activation of Ras by PKC in 3T3 Fibroblasts
J. Biol. Chem. 277: 37929-37935, October 2002.
"We have shown previously (Villalonga, P., López- Alcalá, C., Bosch, M., Chiloeches, A., Rocamora, N., Gil, J., Marais, R., Marshall, C. J., Bachs, O., and Agell, N. (2001) Mol. Cell. Biol. 21, 7345-7354) that calmodulin negatively regulates Ras activation in fibroblasts. Hence, anti-calmodulin drugs (such as W13, trifluoroperazine, or W7) are able to induce Ras/ERK pathway activation under low levels of growth factors. We show here that cell treatment with protein kinase C (PKC) inhibitors abolishes W13-induced activation of Ras, Raf-1, and ERK. Consequently, PKC activity is essential for achieving the synergism between calmodulin inhibition and growth factors to activate Ras. Furthermore, whereas the activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) does not induce Ras activation in 3T3 cells, activation is observed if calmodulin is simultaneously inhibited. This indicates that calmodulin is preventing Ras activation by PKC. Treatment of cells with epidermal growth factor receptor or platelet-derived growth factor receptor tyrosine kinase inhibitors does not abrogate the activation of Ras by calmodulin inhibition. This implies that epidermal growth factor receptor and platelet-derived growth factor receptor tyrosine kinase activities are dispensable for the activation of Ras by TPA plus W13, and, therefore, Ras activation is not a consequence of the transactivation of those receptors by the combination of the anti-calmodulin drug plus TPA. Furthermore, K-Ras, the isoform previously shown to bind to calmodulin, is the only one activated by TPA when calmodulin is inhibited. These data suggest that direct interaction between K-Ras and calmodulin may account for the inability of PKC to activate Ras in 3T3 fibroblasts. In vitro experiments showed that the phosphorylation of K-Ras by PKC was inhibited by calmodulin, suggesting that calmodulin-dependent modulation of K-Ras phosphorylation by PKC could be the mechanism underlying K-Ras activation in fibroblasts treated with TPA plus W13."
[Abstract/Full Text]

Villalonga, Priam, Lopez-Alcala, Cristina, Bosch, Marta, Chiloeches, Antonio, Rocamora, Nativitat, Gil, Joan, Marais, Richard, Marshall, Christopher J., Bachs, Oriol, Agell, Neus
Calmodulin Binds to K-Ras, but Not to H- or N-Ras, and Modulates Its Downstream Signaling
Mol. Cell. Biol. 2001 21: 7345-7354
"Activation of Ras induces a variety of cellular responses depending on the specific effector activated and the intensity and amplitude of this activation. We have previously shown that calmodulin is an essential molecule in the down-regulation of the Ras/Raf/MEK/extracellularly regulated kinase (ERK) pathway in cultured fibroblasts and that this is due at least in part to an inhibitory effect of calmodulin on Ras activation. Here we show that inhibition of calmodulin synergizes with diverse stimuli (epidermal growth factor, platelet-derived growth factor, bombesin, or fetal bovine serum) to induce ERK activation. Moreover, even in the absence of any added stimuli, activation of Ras by calmodulin inhibition was observed. To identify the calmodulin-binding protein involved in this process, calmodulin affinity chromatography was performed. We show that Ras and Raf from cellular lysates were able to bind to calmodulin. Furthermore, Ras binding to calmodulin was favored in lysates with large amounts of GTP-bound Ras, and it was Raf independent. Interestingly, only one of the Ras isoforms, K-RasB, was able to bind to calmodulin. Furthermore, calmodulin inhibition preferentially activated K-Ras. Interaction between calmodulin and K-RasB is direct and is inhibited by the calmodulin kinase II calmodulin-binding domain. Thus, GTP-bound K-RasB is a calmodulin-binding protein, and we suggest that this binding may be a key element in the modulation of Ras signaling." [Full Text]

Egea, Joaquim, Espinet, Carme, Soler, Rosa M., Peiro, Sandra, Rocamora, Nativitat, Comella, Joan X.
Nerve Growth Factor Activation of the Extracellular Signal-Regulated Kinase Pathway Is Modulated by Ca2+ and Calmodulin
Mol. Cell. Biol. 2000 20: 1931-1946
"Nerve growth factor is a member of the neurotrophin family of trophic factors that have been reported to be essential for the survival and development of sympathetic neurons and a subset of sensory neurons. Nerve growth factor exerts its effects mainly by interaction with the specific receptor TrkA, which leads to the activation of several intracellular signaling pathways. Once activated, TrkA also allows for a rapid and moderate increase in intracellular calcium levels, which would contribute to the effects triggered by nerve growth factor in neurons. In this report, we analyzed the relationship of calcium to the activation of the Ras/extracellular signal-regulated kinase pathway in PC12 cells. We observed that calcium and calmodulin are both necessary for the acute activation of extracellular signal-regulated kinases after TrkA stimulation. We analyzed the elements of the pathway that lead to this activation, and we observed that calmodulin antagonists completely block the initial Raf-1 activation without affecting the function of upstream elements, such as Ras, Grb2, Shc, and Trk. We have broadened our study to other stimuli that activate extracellular signal-regulated kinases through tyrosine kinase receptors, and we have observed that calmodulin also modulates the activation of such kinases after epidermal growth factor receptor stimulation in PC12 cells and after TrkB stimulation in cultured chicken embryo motoneurons. Calmodulin seems to regulate the full activation of Raf-1 after Ras activation, since functional Ras is necessary for Raf-1 activation after nerve growth factor stimulation and calmodulin-Sepharose is able to precipitate Raf-1 in a calcium-dependent manner." [Full Text]

Egea, Joaquim, Espinet, Carme, Soler, Rosa M., Peiro, Sandra, Rocamora, Nativitat, Comella, Joan X.
Nerve Growth Factor Activation of the Extracellular Signal-Regulated Kinase Pathway Is Modulated by Ca2+ and Calmodulin
Mol. Cell. Biol. 2000 20: 1931-1946
"Nerve growth factor is a member of the neurotrophin family of trophic factors that have been reported to be essential for the survival and development of sympathetic neurons and a subset of sensory neurons. Nerve growth factor exerts its effects mainly by interaction with the specific receptor TrkA, which leads to the activation of several intracellular signaling pathways. Once activated, TrkA also allows for a rapid and moderate increase in intracellular calcium levels, which would contribute to the effects triggered by nerve growth factor in neurons. In this report, we analyzed the relationship of calcium to the activation of the Ras/extracellular signal-regulated kinase pathway in PC12 cells. We observed that calcium and calmodulin are both necessary for the acute activation of extracellular signal-regulated kinases after TrkA stimulation. We analyzed the elements of the pathway that lead to this activation, and we observed that calmodulin antagonists completely block the initial Raf-1 activation without affecting the function of upstream elements, such as Ras, Grb2, Shc, and Trk. We have broadened our study to other stimuli that activate extracellular signal-regulated kinases through tyrosine kinase receptors, and we have observed that calmodulin also modulates the activation of such kinases after epidermal growth factor receptor stimulation in PC12 cells and after TrkB stimulation in cultured chicken embryo motoneurons. Calmodulin seems to regulate the full activation of Raf-1 after Ras activation, since functional Ras is necessary for Raf-1 activation after nerve growth factor stimulation and calmodulin-Sepharose is able to precipitate Raf-1 in a calcium-dependent manner."
[Full Text]

Egea, Joaquim, Espinet, Carme, Soler, Rosa M., Dolcet, Xavier, Yuste, Victor J., Encinas, Mario, Iglesias, Montserrat, Rocamora, Nativitat, Comella, Joan X.
Neuronal survival induced by neurotrophins requires calmodulin
J. Cell Biol. 2001 154: 585-598
"It has been reported that phosphoinositide 3-kinase (PI 3-kinase) and its downstream target, protein kinase B (PKB), play a central role in the signaling of cell survival triggered by neurotrophins (NTs). In this report, we have analyzed the involvement of Ca2+ and calmodulin (CaM) in the activation of the PKB induced by NTs. We have found that reduction of intracellular Ca2+ concentration or functional blockade of CaM abolished NGF-induced activation of PKB in PC12 cells. Similar results were obtained in cultures of chicken spinal cord motoneurons treated with brain-derived neurotrophic factor (BDNF). Moreover, CaM inhibition prevented the cell survival triggered by NGF or BDNF. This effect was counteracted by the transient expression of constitutive active forms of the PKB, indicating that CaM regulates NT-induced cell survival through the activation of the PKB. We have investigated the mechanisms whereby CaM regulates the activation of the PKB, and we have found that CaM was necessary for the proper generation and/or accumulation of the products of the PI 3-kinase in intact cells." [Full Text]

Yu Cheng, Igor Zhizhin, Robert L. Perlman, and Dimitra Mangoura
Prolactin-induced Cell Proliferation in PC12 Cells Depends on JNK but Not ERK Activation
J. Biol. Chem. 275: 23326-23332, July, 2000.
"The effects of pituitary and extrapituitary prolactin include cellular proliferation and differentiation. PC12 cells was used as a model to delineate respective signaling of prolactin. Prolactin acted as a mitogen for undifferentiated PC12 cells, as measured by significant increases in bromodeoxyuridine incorporation and in cell numbers, with an efficacy equal to epidermal growth factor. Both the long and short form of the prolactin receptor was expressed, yet only the long isoform was tyrosine-phosphorylated upon agonist binding. Functional prolactin receptor signaling was further demonstrated in the activation of JAK2 and phosphorylation activation of the transcription factors Stat1, -3, and -5a. Surprisingly, prolactin stimulated a sustained activation of Raf-B, without activation of the MAP kinases ERK1 or -2. Instead, in solid phase kinase assays using a glutathione S-transferase-c-Jun fusion protein (amino acids 1-79) as the substrate, a significant activation of the mitogen-activated protein Janus kinase (c-Jun N-terminal kinase; JNK) was observed. The prolactin-induced activation of JNK was prolonged and accompanied by a significant increase in c-Jun mRNA abundance and c-Jun protein synthesis. Moreover, analysis of bromodeoxyuridine incorporation at the single cell level revealed that epidermal growth factor-dependent incorporation was inhibited by PD98059 and independent of SB203580, whereas prolactin-induced incorporation was ERK and mitogen-activated protein kinase p38 independent but was abolished with JNK inhibition by 30 µM SB203580. Our studies suggest that prolactin may have a role in the growth of PC12 cells, where it stimulates concurrent mitogenic and differentiation-promoting signaling pathways." [Full Text]

Enhancement of p53 activity and inhibition of neural cell proliferation by glucocorticoid receptor activation
FASEB J. 16: 761-770, 2002.
"In analyzing the molecular mechanisms underlying glucocorticoid-induced apoptosis in neural cells, we observed that dexamethasone, by activating glucocorticoid receptors, causes arrest of HT-22 cells in the G1 phase of the cell cycle; upon withdrawal of the agonist, cells resume proliferation. Our investigations revealed that glucocorticoid treatment, although having no effects on endogenous p53 protein stability, induces rapid translocation of p53 to the nucleus and enhances its transcriptional activity. Consistently, transfection studies with p53-responsive promoters revealed a substantial stimulation of the trans-activation potential of exogenous p53 by dexamethasone. Cells arrested in G1 failed to show signs of apoptosis even after overexpression of p53. Although dexamethasone induced transcription of the proapoptotic gene bax, there was no increase of Bax protein levels. We conclude that glucocorticoid receptor-induced neural cell cycle arrest is associated with an increase in nuclear translocation and transcriptional activity of p53, and suggest that potentiation of p53 may serve as a brake on cell proliferation and may prime cells for differentiation or death induced by other signals." [Abstract]

Bao-Hong Zhang, Eric D. Tang, Tianqing Zhu, Michael E. Greenberg, Anne B. Vojtek, and Kun-Liang Guan
Serum- and Glucocorticoid-inducible Kinase SGK Phosphorylates and Negatively Regulates B-Raf
J. Biol. Chem. 276: 31620-31626, August 2001.
"Phosphorylation can both positively and negatively regulate activity of the Raf kinases. Akt has been shown to phosphorylate and inhibit C-Raf activity. We have recently reported that Akt negatively regulates B-Raf kinase activation by phosphorylating multiple residues within its amino-terminal regulatory domain. Here we investigated the regulation of B-Raf by serum and glucocorticoid-inducible kinase, SGK, which shares close sequence identity with the catalytic domain of Akt but lacks the pleckstrin homology domain. We observed that SGK inhibits B-Raf activity. A comparison of substrate specificity between SGK and Akt indicates that SGK is a potent negative regulator of B-Raf. In contrast to Akt, SGK negatively regulates B-Raf kinase activity by phosphorylating only a single Akt consensus site, Ser364. Under similar experimental conditions, SGK displays a measurably stronger inhibitory effect on B-Raf kinase activity than Akt, whereas Akt exhibits a more inhibitory effect on the forkhead transcription factor, FKHR. The selective substrate specificity is correlated with an enhanced association between Akt or SGK and their preferred substrates, FKHR and B-Raf, respectively. These results indicate that B-Raf kinase activity is negatively regulated by Akt and SGK, suggesting that the cross-talk between the B-Raf and other signaling pathways can be mediated by both Akt and SGK." [Full Text]

Wojciech Poluha, Christopher M. Schonhoff, Kimberly S. Harrington, Mahesh B. Lachyankar, Nancy E. Crosbie, Dylan A. Bulseco, and Alonzo H. Ross
A Novel, Nerve Growth Factor-activated Pathway Involving Nitric Oxide, p53, and p21WAF1 Regulates Neuronal Differentiation of PC12 Cells
J. Biol. Chem. 272: 24002-24007, September 1997.
"During development, neuronal differentiation is closely coupled with cessation of proliferation. We use nerve growth factor (NGF)-induced differentiation of PC12 pheochromocytoma cells as a model and find a novel signal transduction pathway that blocks cell proliferation. Treatment of PC12 cells with NGF leads to induction of nitric oxide synthase (NOS) (Peunova, N., and Enikolopov, G. (1995) Nature 375, 68-73). The resulting nitric oxide (NO) acts as a second messenger, activating the p21WAF1 promoter and inducing expression of p21WAF1 cyclin-dependent kinase inhibitor. NO activates the p21WAF1 promoter by p53-dependent and p53-independent mechanisms. Blocking production of NO with an inhibitor of NOS reduces accumulation of p53, activation of the p21WAF1 promoter, expression of neuronal markers, and neurite extension. To determine whether p21WAF1 is required for neurite extension, we prepared a PC12 line with an inducible p21WAF1 expression vector. Blocking NOS with an inhibitor decreases neurite extension, but induction of p21WAF1 with isopropyl-1-thio--D-galactopyranoside restored this response. Levels of p21WAF1 induced by isopropyl-1-thio--D-galactopyranoside were similar to those induced by NGF. Therefore, we have identified a signal transduction pathway that is activated by NGF; proceeds through NOS, p53, and p21WAF1 to block cell proliferation; and is required for neuronal differentiation by PC12 cells."
[Full Text]

Scotto, Christian, Delphin, Christian, Deloulme, Jean Christophe, Baudier, Jacques
Concerted Regulation of Wild-Type p53 Nuclear Accumulation and Activation by S100B and Calcium-Dependent Protein Kinase C
Mol. Cell. Biol. 1999 19: 7168-7180 [Full Text]

Eizenberg, O, Faber-Elman, A, Gottlieb, E, Oren, M, Rotter, V, Schwartz, M
p53 plays a regulatory role in differentiation and apoptosis of central nervous system-associated cells
Mol. Cell. Biol. 1996 16: 5178-5185
"This study demonstrated the involvement of the tumor suppressor protein p53 in differentiation and programmed cell death of neurons and oligodendrocytes, two cell types that leave the mitotic cycle early in development and undergo massive-scale cell death as the nervous system matures. We found that primary cultures of rat oligodendrocytes and neurons, as well as of the neuronal PC12 pheochromocytoma cell line, constitutively express the p53 protein. At critical points in the maturation of these cells in vitro, the subcellular localization of p53 changes: during differentiation it appears mainly in the nucleus, whereas in mature differentiated cells it is present mainly in the cytoplasm. These subcellular changes were correlated with changes in levels of immunoprecipitated p53. Infection of cells with a recombinant retrovirus encoding a C-terminal p53 miniprotein (p53 DD), previously shown to act as a dominant negative inhibitor of endogenous wild-type p53 activity, inhibited the differentiation of oligodendrocytes and of PC12 cells and protected neurons from spontaneous apoptotic death. These findings suggest that p53, upon receiving appropriate signals, is recruited into the nucleus, where it plays a regulatory role in directing primary neurons', oligodendrocytes, and PC12 cells toward either differentiation or apoptosis in vitro." [Abstract/Full Text]

Allison L. Hughes, Lakshmi Gollapudi, Todd L. Sladek, and Kenneth E. Neet
J. Biol. Chem. 275: 37829-37837, December 2000.
"Upon stimulation with nerve growth factor (NGF), PC12 cells extend neurites and cease to proliferate by influencing cell cycle proteins. Previous studies have shown that neuritogenesis and a block at the G1/S checkpoint correlate with the nuclear translocation of and an increase in the p53 tumor suppressor protein. This study was designed to determine if p53 plays a direct role in mediating NGF-driven G1 arrest. A retroviral vector that overexpresses a temperature-sensitive p53 mutant protein (p53ts) was used to extinguish the function of endogenous p53 in PC12 cells in a dominant-negative manner at the nonpermissive temperature. NGF treatment led to transactivation of a p53 response element in a luciferase reporter construct in PC12 cells, whereas this response to NGF was absent in PC12(p53ts) cells at the nonpermissive temperature. With p53 functionally inactivated, NGF failed to activate growth arrest, as measured by bromodeoxyuridine incorporation, and also failed to induce p21/WAF1 expression, as measured by Western blotting. Since neurite outgrowth proceeded unharmed, 50% of the cells simultaneously demonstrated neurite morphology and were in S phase. Both PC12 cells expressing SV40 T antigen and PC12 cells treated with p53 antisense oligonucleotides continued through the cell cycle, confirming the dependence of the NGF growth arrest signal on a p53 pathway. Activation of Ras in a dexamethasone-inducible PC12 cell line (GSRas1) also caused p53 nuclear translocation and growth arrest. Therefore, wild-type p53 is indispensable in mediating the NGF antiproliferative signal through the Ras/MAPK pathway that regulates the cell cycle of PC12 cells." [Full Text]

Hsin-yi Tang, Kathy Zhao, Joseph F. Pizzolato, Maxim Fonarev, Jessica C. Langer, and James J. Manfredi
Constitutive Expression of the Cyclin-dependent Kinase Inhibitor p21 Is Transcriptionally Regulated by the Tumor Suppressor Protein p53
J. Biol. Chem. 273: 29156-29163, October 1998. [Full Text]

Michieli, P, Chedid, M, Lin, D, Pierce, JH, Mercer, WE, Givol, D
Induction of WAF1/CIP1 by a p53-independent pathway
Cancer Res 1994 54: 3391-3395
"The p53-inducible gene WAF1/CIP1 encodes a M(r) 21,000 protein (p21) that has been shown to arrest cell growth by inhibition of cyclin-dependent kinases. Induction of WAF1/CIP1 in cells undergoing p53-dependent G1 arrest or apoptosis supports the idea that WAF1/CIP1 is a critical downstream effector of p53. In the present study, we used embryonic fibroblasts from p53 "knock-out" mice to demonstrate p53-independent induction of WAF1/CIP1. We show that serum or individual growth factors such as platelet-derived growth factor, fibroblast growth factor, and epidermal growth factor but not insulin are able to induce WAF1/CIP1 in quiescent p53-deficient cells as well as in normal cells. The kinetics of this transient induction, which is enhanced by cycloheximide, demonstrates that WAF1/CIP1 is an immediate-early gene the transcript of which reaches a peak at approximately 2 h following serum or growth factor stimulation. On the other hand, DNA damage elicited by gamma-irradiation induces WAF1/CIP1 in normal human and mouse fibroblasts but does not affect WAF1/CIP1 expression in p53-deficient cells. These results suggest the existence of two separate pathways for the induction of WAF1/CIP1, a p53-dependent one activated by DNA damage and a p53-independent one activated by mitogens at the entry into the cell cycle." [Abstract]

Piyajit Watcharasit, Gautam N. Bijur, Jaroslaw W. Zmijewski, Ling Song, Anna Zmijewska, Xinbin Chen, Gail V. W. Johnson, and Richard S. Jope
Direct, activating interaction between glycogen synthase kinase-3 and p53 after DNA damage
PNAS 99: 7951-7955, June 11, 2002 [Abstract]

N Billon, LA van Grunsven, and BB Rudkin
The CDK inhibitor p21WAF1/Cip1 is induced through a p300-dependent mechanism during NGF-mediated neuronal differentiation of PC12 cells.
Oncogene, Nov 1996; 13(10): 2047-54.
"The block of cell proliferation elicited by the addition of nerve growth factor (NGF) to exponentially-growing PC12 cells results, in part, from the inhibition of cyclin D1-associated kinase activity by p21WAF1/CIP1. NGF treatment of PC12 cells provokes the accumulation of p21 mRNA, due to transcriptional activation of the p21 promoter in a p53-independent manner. Transient expression of a mutated form of the adenovirus E1A protein (E1A dCR2), which retains its capacity to bind the transcriptional co-activator p300, completely abolishes the NGF-mediated stimulation of p21 promoter activity. This phenomenon can be reversed by ectopic expression of p300, suggesting that p300 is necessary for the induction of p21 by NGF. In addition, stable expression of E1A dCR2 in PC12 cells results in the inhibition of the NGF response, i.e. it prevents activation of the p21 promoter, cell cycle arrest, and neuronal differentiation. The signalling pathway from the TrkA receptor via the MAP kinase pathway is not altered in these cells. Together, these data indicate that p300 could play a pivotal role in the triggering of the anti-mitogenic effect of NGF and of neuronal differentiation." [Abstract]

Goodman, Richard H., Smolik, Sarah
CBP/p300 in cell growth, transformation, and development
Genes Dev. 2000 14: 1553-1577
"Because adenovirus E1A blocks CBP/p300 function (see below), it has been suggested that at least some of its effects on cell transformation might occur by inhibiting the actions of p53. Conversely, the growth suppression activities of CBP and p300 have been attributed to their ability to augment p53-mediated transcription. In addition to its transcriptional activation functions, p53 negatively regulates genes whose promoters do not contain a suitable binding site." [Full Text]

Perkins ND, Felzien LK, Betts JC, Leung K, Beach DH, Nabel GJ.
Regulation of NF-kappaB by cyclin-dependent kinases associated with the p300 coactivator.
Science 1997 Jan 24;275(5299):523-7
"The nuclear factor kappaB (NF-kappaB) transcription factor is responsive to specific cytokines and stress and is often activated in association with cell damage and growth arrest in eukaryotes. NF-kappaB is a heterodimeric protein, typically composed of 50- and 65-kilodalton subunits of the Rel family, of which RelA(p65) stimulates transcription of diverse genes. Specific cyclin-dependent kinases (CDKs) were found to regulate transcriptional activation by NF-kappaB through interactions with the coactivator p300. The transcriptional activation domain of RelA(p65) interacted with an amino-terminal region of p300 distinct from a carboxyl-terminal region of p300 required for binding to the cyclin E-Cdk2 complex. The CDK inhibitor p21 or a dominant negative Cdk2, which inhibited p300-associated cyclin E-Cdk2 activity, stimulated kappaB-dependent gene expression, which was also enhanced by expression of p300 in the presence of p21. The interaction of NF-kappaB and CDKs through the p300 and CBP coactivators provides a mechanism for the coordination of transcriptional activation with cell cycle progression." [Full Text]

Maggirwar, Sanjay B., Ramirez, Servio, Tong, Ning, Gelbard, Harris A., Dewhurst, Stephen
Functional Interplay Between Nuclear Factor-{kappa}B and c-Jun Integrated by Coactivator p300 Determines the Survival of Nerve Growth Factor-Dependent PC12 Cells
J Neurochem 2000 74: 527-539
"Nerve growth factor (NGF) activates the transcription factors nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1) in sympathetic neurons. Whereas NGF-inducible NF-kappaB is required for the survival of neurons, c-Jun has the ability to promote neuronal death. In this report, we have examined the effect of NGF withdrawal on c-Jun and NF-kappaB transcription factors in PC12 cells differentiated to a neuronal phenotype. We show that the withdrawal of NGF from these cultures results in de novo synthesis of c-Jun, increase in AP-1 activity, and down-regulation of NF-kappaB activity. To investigate how the signal transduction pathways activating c-Jun and NF-kappaB are differentially regulated by NGF, we performed transcriptional analyses. Expression of ReIA (NF-kappaB) suppressed the c-Jun-dependent transcription of c-jun, and this effect was reversed by overexpression of the coactivator p300. RelA's effects on c-Jun transcription were mediated by competitive binding of the carboxy-terminal region of RelA to the CH1 domain of p300, which also binds to c-Jun; deletion of this region abrogated the ability of RelA to inhibit c-Jun activity. Furthermore, the inhibition of endogenous NF-kappaB in NGF-maintained neuronal PC12 cells led to the induction of c-Jun synthesis and a marked increase in cell death. Together, these studies demonstrate a functional interaction between NF-kappaB and c-Jun and suggest a novel mechanism of NF-kappaB-mediated neuroprotection." [Abstract]

Ying Li, Donald Dowbenko, and Laurence A. Lasky
AKT/PKB Phosphorylation of p21Cip/WAF1 Enhances Protein Stability of p21Cip/WAF1 and Promotes Cell Survival
J. Biol. Chem. 277: 11352-11361, December 2001.
"p21Cip1/WAF1 (p21), a p53-inducible protein, is a critical regulator of cell cycle and cell survival. p21 binds to and inhibits both the DNA synthesis regulator proliferating cell nuclear antigen and cyclin A/E-CDK2 complexes. Recently, p21 has also been shown to be a positive regulator of cell cycle progression as p21 is necessary for the assembly and activation of cyclin D1-CDK4/6 complexes. Furthermore, elevated p21 protein levels have been observed in various aggressive tumors as well as linked to chemoresistance. Here we demonstrate that p21 is directly phosphorylated by AKT/PKB, a survival kinase that is hyperactivated in many late stage tumors. Two sites (Thr145 and Ser146) in the carboxyl terminus of p21 are phosphorylated by AKT/PKB in vitro and in vivo. Phosphorylation of Thr145 inhibits PCNA binding, whereas phosphorylation of Ser146 significantly increases p21 protein stability. Glioblastoma cell lines with activated AKT/PKB show enhanced p21 stability, and they are more resistant to taxol-mediated toxicity. Finally, AKT/PKB controls the assembly of cyclin D1-CDK4 complexes through modulation of p21 and cyclin D1 levels. These data imply that enhanced levels of p21 in tumors are due, in part, to phosphorylation by activated AKT/PKB. Furthermore, they suggest that one mechanism of AKT/PKB regulation of tumor cell survival and/or proliferation is to stabilize p21 protein." [Abstract]

Hashimoto, Keiko, Guroff, Gordon, Katagiri, Yasuhiro
Delayed and Sustained Activation of p42/p44 Mitogen-Activated Protein Kinase Induced by Proteasome Inhibitors Through p21ras in PC12 Cells
J Neurochem 2000 74: 92-98
"Proteolysis by the ubiquitin/proteasome pathway regulates the intracellular level of several proteins, some of which control cell proliferation and cell cycle progression. To determine what kinds of signaling cascades are activated or inhibited by proteasome inhibition, we treated PC12 cells with specific proteasome inhibitors and subsequently performed in-gel kinase assays. N-Acetyl-Leu-Leu-norleucinal and lactacystin, which inhibit the activity of the proteasome, induced the activation of p42/p44 mitogen-activated protein (MAP) kinases [extracellular signal-regulated kinases (ERKs) 1 and 2]. In contrast, N-acetyl-Leu-Leu-methional, which inhibits the activity of calpains, but not of the proteasome, failed to induce ERK activation. Uniquely, the kinetics of MAP kinase activation induced by proteasome inhibitors are very slow compared with those resulting from activation by nerve growth factor; ERK activation is detectable only after a 5-h treatment with the inhibitors, and its activity remained unchanged for at least until 27 h. Proteasome inhibitor-initiated ERK activation is inhibited by pretreatment with the ERK kinase inhibitor PD 98059, as well as by overexpression of a dominant-negative form of Ras. Thus, proteasome inhibitors induce sustained ERK activation in a Ras-dependent manner. Proteasome inhibitor-induced neurite outgrowth, however, is not inhibited by PD 98059, indicating that sustained activation of ERKs is not the factor responsible for proteasome inhibitor-induced morphological differentiation. Our data suggest the presence of a novel mechanism for activation of the MAP kinase cascade that involves proteasome activity." [Abstract]

Personett, David, Fass, Uwe, Panickar, Kiran, McKinney, Michael
Retinoic Acid-Mediated Enhancement of the Cholinergic/Neuronal Nitric Oxide Synthase Phenotype of the Medial Septal SN56 Clone: Establishment of a Nitric Oxide-Sensitive Proapoptotic State
J Neurochem 2000 74: 2412-2424
"It is unclear what mechanisms lead to the degeneration of basal forebrain cholinergic neurons in Alzheimer's or other human brain diseases. Some brain cholinergic neurons express neuronal nitric oxide (NO) synthase (nNOS), which produces a free radical that has been implicated in some forms of neurodegeneration. We investigated nNOS expression and NO toxicity in SN56 cells, a clonal cholinergic model derived from the medial septum of the mouse basal forebrain. We show here that, in addition to expressing choline acetyltransferase (ChAT), SN56 cells express nNOS. Treatment of SN56 cells with retinoic acid (RA; 1 µM) for 48 h increased ChAT mRNA (+126%), protein (+88%), and activity (+215%) and increased nNOS mRNA (+98%), protein (+400%), and activity (+15%). After RA treatment, SN56 cells became vulnerable to NO excess generated with S-nitro-N-acetyl-DL-penicillamine (SNAP) and exhibited increased nuclear DNA fragmentation that was blocked with a caspase-3 inhibitor. Treatment with dexamethasone, which largely blocked the RA-mediated increase in nNOS expression, or inhibition of nNOS activity with methylthiocitrulline strongly potentiated the apoptotic response to SNAP in RA-treated SN56 cells. Caspase-3 activity was reduced when SNAP was incubated with cells or cell lysates, suggesting that NO can directly inhibit the protease. Thus, whereas RA treatment converts SN56 cells to a proapoptotic state sensitive to NO excess, endogenously produced NO appears to be anti-apoptotic, possibly by tonically inhibiting caspase-3." [Abstract]

Jiwei Wang, Lora W. Barsky, Chung H. Shum, Ambrose Jong, Kenneth I. Weinberg, Steven J. Collins, Timothy J. Triche, and Lingtao Wu
Retinoid-induced G1 arrest and differentiation activation are associated with a switch to CAK hypophosphorylation of RARa
JBC Papers in Press published on September 3, 2002 as 10.1074/jbc.M206792200 [Abstract/Full Text]

Anne E. West, Eric C. Griffith & Michael E. Greenberg
Nature Reviews Neuroscience 3, 921-931 (2002); doi:10.1038/nrn987 [Full Text]

Takai, Yoshimi, Sasaki, Takuya, Matozaki, Takashi
Small GTP-Binding Proteins
Physiol. Rev. 2001 81: 153-208 [Full Text]

Gray Pearson, Fred Robinson, Tara Beers Gibson, Bing-e Xu, Mahesh Karandikar, Kevin Berman, and Melanie H. Cobb
Mitogen-Activated Protein (MAP) Kinase Pathways: Regulation and Physiological Functions
Endocr. Rev. 22: 153-183, 2001. [Full Text]

Kyriakis, John M., Avruch, Joseph
Mammalian Mitogen-Activated Protein Kinase Signal Transduction Pathways Activated by Stress and Inflammation
Physiol. Rev. 2001 81: 807-869 [Full Text]

Stefan Strack
Overexpression of the protein phosphatase 2A regulatory subunit B[gamma] promotes neuronal differentiation by activating the MAP kinase cascade
JBC Papers in Press published on August 20, 2002 as 10.1074/jbc.M203767200
"Protein serine/threonine phosphatase 2A (PP2A) is a multifunctional regulator of cellular signaling. Variable regulatory subunits associate with a core dimer of scaffolding and catalytic subunits and are postulated to dictate substrate specificity and subcellular location of the heterotrimeric PP2A holoenzyme. The role of brain-specific regulatory subunits in neuronal differentiation and signaling was investigated in the PC6-3 subline of PC12 cells. Endogenous B[beta], B[gamma], and B’[beta] protein expression was induced during nerve growth factor (NGF)-mediated neuronal differentiation. Transient expression of B[gamma], but not other PP2A regulatory subunits, facilitated neurite outgrowth in the absence and presence of NGF. Tetracycline-inducible expression of B[gamma] caused growth arrest and neurofilament expression, further evidence that PP2A/B[gamma] can promote differentiation. In PC6-3 cells, but not non-neuronal cell lines, B[gamma] specifically promoted long lasting activation of the MAP (mitogen-activated protein) kinase cascade, a key mediator of neuronal differentiation. Pharmacological and dominant-negative inhibition and kinase assays indicate that B[gamma] promotes neuritogenesis by stimulating the MAP kinase cascade downstream of the trkA NGF receptor, but upstream or at the level of the B-Raf kinase. Mutational analyses demonstrate that the divergent N-terminus is critical for B[gamma] activity. These studies implicate PP2A/B[gamma] as a positive regulator of MAP kinase signaling in neurons."
[Abstract/Full Text]

Susan O. Meakin, James I. S. MacDonald, Ela A. Gryz, Christopher J. Kubu, and Joseph M. Verdi
The Signaling Adapter FRS-2 Competes with Shc for Binding to the Nerve Growth Factor Receptor TrkA. A MODEL FOR DISCRIMINATING PROLIFERATION AND DIFFERENTIATION
J. Biol. Chem. 274: 9861-9870, April 1999. [Full Text]

Zeng, Guoqian, Meakin, Susan O.
Overexpression of the signaling adapter FRS2 reconstitutes the cell cycle deficit of a nerve growth factor non-responsive TrkA receptor mutant
J Neurochem 2002 81: 820-831
"We have characterized the cell cycle deficit of a novel TrkA receptor mutant (TrkAS3) that fails to support nerve growth factor (NGF)-dependent cell cycle arrest and neurite outgrowth. TrkAS3 receptors fail to support an NGF-dependent increase in the expression of cyclin D1 and the cell cycle inhibitor, p21Waf1/Cip1 , two important regulators of G1 /S transition, and do not down-regulate expression of the G2 /M phase marker, cdc2/cdk1, or the S phase marker, proliferating cell nuclear antigen. Moreover, NGF-activated TrkAS3 receptors do not down-regulate cyclin-dependent kinase 4 phosphorylation of the retinoblastoma protein, essential for G1 arrest, in comparison to NGF-activated wild-type TrkA. Collectively these data indicate that TrkAS3 receptors fail to support NGF-dependent G1 arrest. Interestingly, ectopic expression of regulators of G1 /S arrest, such as cyclin D1 or inhibitors of cell cycle (p21Waf1/Cip1 , p16INK4A ), or the fibroblast growth factor (FGF) receptor substrate-2 (FRS2) in cells expressing TrkAS3 reconstitutes NGF-dependent neurite outgrowth. Collectively, these data suggest a model in which NGF-stimulated TrkA-dependent activation of FRS2 supports neurite outgrowth through a mechanism that likely involves the induction of p21Waf1/Cip1 expression and the arrest of cells at G1 /S." [Abstract]

Hadari, Y. R., Kouhara, H., Lax, I., Schlessinger, J.
Binding of Shp2 Tyrosine Phosphatase to FRS2 Is Essential for Fibroblast Growth Factor-Induced PC12 Cell Differentiation
Mol. Cell. Biol. 1998 18: 3966-3973
"FRS2 is a lipid-anchored docking protein that plays an important role in linking fibroblast growth factor (FGF) and nerve growth factor receptors with the Ras/mitogen-activated protein (MAP) kinase signaling pathway. In this report, we demonstrate that FRS2 forms a complex with the N-terminal SH2 domain of the protein tyrosine phosphatase Shp2 in response to FGF stimulation. FGF stimulation induces tyrosine phosphorylation of Shp2, leading to the formation of a complex containing Grb2 and Sos1 molecules. In addition, a mutant FRS2 deficient in both Grb2 and Shp2 binding induces a weak and transient MAP kinase response and fails to induce PC12 cell differentiation in response to FGF stimulation. Furthermore, FGF is unable to induce differentiation of PC12 cells expressing an FRS2 point mutant deficient in Shp2 binding. Finally, we demonstrate that the catalytic activity of Shp2 is essential for sustained activation of MAP kinase and for potentiation of FGF-induced PC12 cell differentiation. These experiments demonstrate that FRS2 recruits Grb2 molecules both directly and indirectly via complex formation with Shp2 and that Shp2 plays an important role in FGF-induced PC12 cell differentiation." [Abstract]

Zhang, Bao-Hong, Guan, Kun-Liang
Activation of B-Raf kinase requires phosphorylation of the conserved residues Thr598 and Ser601
EMBO J. 2000 19: 5429-5439
"The Raf kinase family serves as a central intermediate to relay signals from Ras to ERK. The precise molecular mechanism for Raf activation is still not fully understood. Here we report that phosphorylation of Thr598 and Ser601, which lie between kinase subdomains VII and VIII, is essential for B-Raf activation by Ras. Substitution of these residues by alanine (B-RafAA) abolished Ras-induced B-Raf activation without altering the association of B-Raf with other signaling proteins. Phosphopeptide mapping and immunoblotting with phospho-specific antibodies confirmed that Thr598 and Ser601 are in vivo phosphorylation sites induced by Ras." [Abstract]

Toshihisa Ohtsuka, Kazuya Shimizu, Bunpei Yamamori, Shinya Kuroda, and Yoshimi Takai
Activation of Brain B-Raf Protein Kinase by Rap1B Small GTP-binding Protein
J. Biol. Chem. 271: 1258-1261, January 1996.
"Rap1 small GTP-binding protein has the same amino acid sequence at its effector domain as that of Ras. Rap1 has been shown to antagonize the Ras functions, such as the Ras-induced transformation of NIH 3T3 cells and the Ras-induced activation of the c-Raf-1 protein kinase-dependent mitogen-activated protein (MAP) kinase cascade in Rat-1 cells, whereas we have shown that Rap1 as well as Ras stimulates DNA synthesis in Swiss 3T3 cells. We have established a cell-free assay system in which Ras activates bovine brain B-Raf protein kinase. Here we have used this assay system and examined the effect of Rap1 on the B-Raf activity to phosphorylate recombinant MAP kinase kinase (MEK). Recombinant Rap1B stimulated the activity of B-Raf, which was partially purified from bovine brain and immunoprecipitated by an anti-B-Raf antibody. The GTP-bound form was active, but the GDP-bound form was inactive. The fully post-translationally lipid-modified form was active, but the unmodified form was nearly inactive. The maximum B-Raf activity stimulated by Rap1B was nearly the same as that stimulated by Ki-Ras. Rap1B enhanced the Ki-Ras-stimulated B-Raf activity in an additive manner. These results indicate that not only Ras but also Rap1 is involved in the activation of the B-Raf-dependent MAP kinase cascade." [Full Text]

Michael L. Spencer, Haipeng Shao, H. Michael Tucker, and Douglas A. Andres
Nerve Growth Factor-dependent Activation of the Small GTPase Rin
J. Biol. Chem. 277: 17605-17615, May 2002.
"The Rit and Rin proteins comprise a distinct and evolutionarily conserved subfamily of Ras-related small GTPases. Although we have defined a role for Rit-mediated signal transduction in the regulation of cell proliferation and transformation, the function of Rin remains largely unknown. Because we demonstrate that Rin is developmentally regulated and expressed in adult neurons, we examined its role in neuronal signaling. In this study, we show that stimulation of PC6 cells with either epidermal growth factor or nerve growth factor (NGF) results in rapid activation of Rin. This activation correlates with the onset of Ras activation, and dominant-negative Ras completely inhibits Rin activation induced by NGF. Further examination of Ras-mediated Rin activation suggests that this process is dependent upon neuronally expressed regulatory factors. Expression of mutationally activated H-Ras fails to activate Rin in non-neuronal cells, but results in potent stimulation of Rin-GTP levels in a variety of neuronal cell lines. Furthermore, although constitutively activated Rin does not induce neurite outgrowth on its own, both NGF-induced and oncogenic Ras-induced neurite outgrowth were inhibited by the expression of dominant-negative Rin. Together, these studies indicate that Rin activation is a direct downstream effect of growth factor-dependent signaling in neuronal cells and suggest that Rin may function to transduce signals within the mature nervous system."
[Full Text]

Michael L. Spencer, Haipeng Shao, and Douglas A. Andres
Induction of Neurite Extension and Survival in Pheochromocytoma Cells by the Rit GTPase
J. Biol. Chem. 277: 20160-20168, June 2002.
"The Rit, Rin, and Ric proteins comprise a distinct and evolutionarily conserved subfamily of the Ras-like small G-proteins. Although these proteins share the majority of core effector domain residues with Ras, recent studies suggest that Rit uses novel effector pathways to regulate NIH3T3 cell proliferation and transformation, while the functions of Rin and Ric remain largely unknown. Since we demonstrate that Rit is expressed in neurons, we investigated the role of Rit signaling in promoting the differentiation and survival of pheochromocytoma cells. In this study, we show that expression of constitutively active Rit (RitL79) in PC6 cells results in neuronal differentiation, characterized by the elaboration of an extensive network of neurite-like processes that are morphologically distinct from those mediated by the expression of oncogenic Ras. Although activated Rit fails to stimulate mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) signaling pathways in COS cells, RitL79 induced the phosphorylation of ERK1/2 in PC6 cells. We also find that Rit-mediated effects on neurite outgrowth can be blocked by co-expression of dominant-negative mutants of C-Raf1 or mitogen-activated protein kinase kinase 1 (MEK1). Moreover, expression of dominant-negative Rit is sufficient to inhibit NGF-induced neurite outgrowth. Expression of active Rit inhibits growth factor-withdrawal mediated apoptosis of PC6 cells, but does not induce phosphorylation of Akt/protein kinase B, suggesting that survival does not utilize the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Instead, pharmacological inhibitors of MEK block Rit-stimulated cell survival. Taken together, these studies suggest that Rit represents a distinct regulatory protein, capable of mediating differentiation and cell survival in PC6 cells using a MEK-dependent signaling pathway to achieve its effects." [Abstract/Full Text]

Kimmelman, Alec C., Rodriguez, Nelson Nunez, Chan, Andrew M.-L.
R-Ras3/M-Ras Induces Neuronal Differentiation of PC12 Cells through Cell-Type-Specific Activation of the Mitogen-Activated Protein Kinase Cascade
Mol. Cell. Biol. 2002 22: 5946-5961
"R-Ras3/M-Ras is a novel member of the Ras subfamily of GTP-binding proteins which has a unique expression pattern highly restricted to the mammalian central nervous system. In situ hybridization using an R-Ras3 cRNA probe revealed high levels of R-Ras3 transcripts in the hippocampal region of the mouse brain as well as a pattern of expression in the cerebellum that was distinct from that of H-Ras. We found that R-Ras3 was activated by nerve growth factor (NGF) and basic fibroblast growth factor as well as by the guanine nucleotide exchange factor GRP but not by epidermal growth factor. Ectopic expression of either R-Ras3 or GRP in PC12 cells induced efficient neuronal differentiation. The ability of NGF as well as GRP to promote differentiation of PC12 cells was attenuated by an R-Ras3 dominant-negative mutant. Furthermore, the biological action of R-Ras3 in PC12 cells was dependent on the mitogen-activated protein kinase (MAPK). Interestingly, whereas R-Ras3 was unable to mediate efficient activation of MAPK activity in NIH 3T3 cells, it was able to do so in PC12 cells. This cell-type specificity is in stark contrast to that of H-Ras, which can stimulate the MAPK pathway in both cell types. Indeed, this pattern of MAPK activation could be explained by the fact that R-Ras3 was unable to activate c-Raf, while it bound and stimulated the neuronal Raf isoform, B-Raf, in PC12 cells. Thus, R-Ras3 is implicated in a novel pathway of neuronal differentiation by coupling specific trophic factors to the MAPK cascade through the activation of B-Raf." [Full Text]

Hidekazu Yasui, Hironori Katoh, Yoshiaki Yamaguchi, Junko Aoki, Hirotada Fujita, Kazutoshi Mori, and Manabu Negishi
Differential Responses to Nerve Growth Factor and Epidermal Growth Factor in Neurite Outgrowth of PC12 Cells Are Determined by Rac1 Activation Systems
J. Biol. Chem. 276: 15298-15305, May 2001.
"Neurite outgrowth of PC12 cells is induced by nerve growth factor (NGF) but not by epidermal growth factor (EGF). This differential response has been explained by the duration of mitogen-activated protein kinase (MAPK) activation; NGF induces sustained MAPK activation but EGF leads short-lived activation. However, precise mechanisms have not yet been understood. Here we demonstrate the difference between NGF and EGF in regulation of Rac1, a small GTPase involved in neurite outgrowth, in PC12 cells. NGF phosphoinositide 3-kinase dependently induces transient activation of Rac1 and accumulation of active Rac1 at protrusion sites on the cell surface, inducing filamentous actin-rich protrusions and subsequent neurite formation in a Rac1-dependent manner. On the other hand, EGF phosphoinositide 3-kinase independently induces more transient Rac1 activation but neither accumulates active Rac1 nor forms Rac1- and filamentous actin-rich protrusions. Difference in the Rac1 localization between NGF and EGF was also observed with the localization of exogenously expressed green fluorescent protein-tagged Rac1. The Rac1-mediated protrusion by NGF is independent of MAPK cascade, but the subsequent neurite extension requires the cascade. Thus, the differential activation of Rac1 and localization of active Rac1 contribute to the difference in the ability of NGF and EGF to induce neurite outgrowth, and we propose that the MAPK cascade-independent prompt activation of Rac1 and recruitment of active Rac1 at the protrusion sites trigger the initiation of neurite formation." [Abstract]

Lee, Jaewon, Duan, Wenzhen, Mattson, Mark P.
Evidence that brain-derived neurotrophic factor is required for basal neurogenesis and mediates, in part, the enhancement of neurogenesis by dietary restriction in the hippocampus of adult mice
J Neurochem 2002 82: 1367-1375
"To determine the role of brain-derived neurotrophic factor (BDNF) in the enhancement of hippocampal neurogenesis resulting from dietary restriction (DR), heterozygous BDNF knockout (BDNF +/–) mice and wild-type mice were maintained for 3 months on DR or ad libitum (AL) diets. Mice were then injected with bromodeoxyuridine (BrdU) and killed either 1 day or 4 weeks later. Levels of BDNF protein in neurons throughout the hippocampus were decreased in BDNF +/– mice, but were increased by DR in wild-type mice and to a lesser amount in BDNF +/– mice. One day after BrdU injection the number of BrdU-labeled cells in the dentate gyrus of the hippocampus was significantly decreased in BDNF +/– mice maintained on the AL diet, suggesting that BDNF signaling is important for proliferation of neural stem cells. DR had no effect on the proliferation of neural stem cells in wild-type or BDNF +/– mice. Four weeks after BrdU injection, numbers of surviving labeled cells were decreased in BDNF +/– mice maintained on either AL or DR diets. DR significantly improved survival of newly generated cells in wild-type mice, and also improved their survival in BDNF +/– mice, albeit to a lesser extent. The majority of BrdU-labeled cells in the dentate gyrus exhibited a neuronal phenotype at the 4-week time point. The reduced neurogenesis in BDNF +/– mice was associated with a significant reduction in the volume of the dentate gyrus. These findings suggest that BDNF plays an important role in the regulation of the basal level of neurogenesis in dentate gyrus of adult mice, and that by promoting the survival of newly generated neurons BDNF contributes to the enhancement of neurogenesis induced by DR." [Abstract]

Iida, Naoyuki, Namikawa, Kazuhiko, Kiyama, Hiroshi, Ueno, Hikaru, Nakamura, Shun, Hattori, Seisuke
Requirement of Ras for the Activation of Mitogen-Activated Protein Kinase by Calcium Influx, cAMP, and Neurotrophin in Hippocampal Neurons
J. Neurosci. 2001 21: 6459-6466
"Mitogen-activated protein (MAP) kinase plays important roles in the establishment of long-term potentiation both in vitro and in living animals. MAP kinase is activated in response to a broad range of stimuli, including calcium influx through NMDA receptor and L-type calcium channel, cAMP, and neurotrophins. To investigate the role of Ras in the activation of MAP kinase and cAMP response element-binding protein (CREB) in hippocampal neurons, we inhibited Ras function by overexpressing a Ras GTPase-activating protein, Gap1m, or dominant negative Ras by means of adenovirus vectors. Gap1m expression almost completely suppressed MAP kinase activation in response to NMDA, calcium ionophore, membrane depolarization, forskolin, and brain-derived neurotrophic factor (BDNF). Dominant negative Ras also showed similar effects. On the other hand, Rap1GAP did not significantly inhibit the forskolin-induced activation of MAP kinase. In contrast to MAP kinase activation, the inactivation of Ras activity did not inhibit significantly NMDA-induced CREB phosphorylation, whereas BDNF-induced CREB phosphorylation was inhibited almost completely. These results demonstrate that Ras transduces signals elicited by a broad range of stimuli to MAP kinase in hippocampal neurons and further suggest that CREB phosphorylation depends on multiple pathways." [Full Text]

Schonhoff, Christopher M., Bulseco, Dylan A., Brancho, Deborah M., Parada, Luis F., Ross, Alonzo H.
The Ras-ERK pathway is required for the induction of neuronal nitric oxide synthase in differentiating PC12 cells
J Neurochem 2001 78: 631-639
"We have studied the role of MAP kinase pathways in neuronal nitric oxide synthase (nNOS) induction during the differentiation of PC12 cells. In nerve growth factor (NGF)-treated PC12 cells, we find nNOS induced at RNA and protein levels, resulting in increased NOS activity. We note that neither nNOS mRNA, nNOS protein nor NOS activity is induced by NGF treatment in cells that have been infected with a dominant negative Ras adenovirus. We have also used drugs that block MAP kinase pathways and assessed their ability to inhibit nNOS induction. Even though U0126 and PD98059 are both MEK inhibitors, we find that U0126, but not PD98059, blocks induction of nNOS protein and NOS activity in NGF-treated PC12 cells. Also, the p38 kinase inhibitor, SB203580, does not block nNOS induction in our clone of PC12 cells. Since the JNK pathway is not activated in NGF-treated PC12 cells, we conclude that the Ras–ERK pathway and not the p38 or JNK pathway is required for nNOS induction in NGF-treated PC12 cells. We find that U0126 is much more effective than PD98059 in blocking the Ras–ERK pathway, thereby explaining the discrepancy in nNOS inhibition. We conclude that the Ras–ERK pathway is required for nNOS induction." [Abstract]

Bang, OS, Park, EK, Yang, SI, Lee, SR, Franke, TF, Kang, SS
Overexpression of Akt inhibits NGF-induced growth arrest and neuronal differentiation of PC12 cells
J Cell Sci 2001 114: 81-88
"To investigate the role of Akt in nerve growth factor (NGF)-induced neuronal differentiation, PC12 cells ectopically expressing wild-type or dominant-inhibitory forms of Akt were analyzed. NGF-induced neurite outgrowth was greatly accelerated in cells expressing dominant-inhibitory Akt, compared to parental PC12 cells, but was almost completely blocked in cells expressing wild-type Akt. Since neuronal differentiation requires an arrest of cell growth, several aspects of cell growth of the different cell lines were compared. Cells expressing wild-type Akt were not susceptible to the growth-arresting effect of NGF, whereas parental PC12 cells and notably cells expressing mutant Akt were so affected. Accompanying this, the expressions of CDKs and p21(WAF1) were down- and up-regulated, respectively, in both parental PC12 cells and cells expressing mutant Akt. When treated with some growth arrest-inducing agents such as sodium nitroprusside, forskolin and butyrolactone I, cells expressing wild-type Akt regained their responsiveness to the effects of NGF on differentiation. In summary, our results indicate that Akt overrides the growth-arresting effect of NGF and thereby, negatively regulates neuronal differentiation." [Abstract/Full Text]

York, Randall D., Molliver, Derek C., Grewal, Savraj S., Stenberg, Paula E., McCleskey, Edwin W., Stork, Philip J. S.
Role of Phosphoinositide 3-Kinase and Endocytosis in Nerve Growth Factor-Induced Extracellular Signal-Regulated Kinase Activation via Ras and Rap1
Mol. Cell. Biol. 2000 20: 8069-8083
"Neurotrophins promote multiple actions on neuronal cells including cell survival and differentiation. The best-studied neurotrophin, nerve growth factor (NGF), is a major survival factor in sympathetic and sensory neurons and promotes differentiation in a well-studied model system, PC12 cells. To mediate these actions, NGF binds to the TrkA receptor to trigger intracellular signaling cascades. Two kinases whose activities mediate these processes include the mitogen-activated protein (MAP) kinase (or extracellular signal-regulated kinase [ERK]) and phosphoinositide 3-kinase (PI3-K). To examine potential interactions between the ERK and PI3-K pathways, we studied the requirement of PI3-K for NGF activation of the ERK signaling cascade in dorsal root ganglion cells and PC12 cells. We show that PI3-K is required for TrkA internalization and participates in NGF signaling to ERKs via distinct actions on the small G proteins Ras and Rap1. In PC12 cells, NGF activates Ras and Rap1 to elicit the rapid and sustained activation of ERKs respectively. We show here that Rap1 activation requires both TrkA internalization and PI3-K, whereas Ras activation requires neither TrkA internalization nor PI3-K. Both inhibitors of PI3-K and inhibitors of endocytosis prevent GTP loading of Rap1 and block sustained ERK activation by NGF. PI3-K and endocytosis may also regulate ERK signaling at a second site downstream of Ras, since both rapid ERK activation and the Ras-dependent activation of the MAP kinase kinase kinase B-Raf are blocked by inhibition of either PI3-K or endocytosis. The results of this study suggest that PI3-K may be required for the signals initiated by TrkA internalization and demonstrate that specific endocytic events may distinguish ERK signaling via Rap1 and Ras." [Full Text]

Schaeffer, Hans J., Weber, Michael J.
Mitogen-Activated Protein Kinases: Specific Messages from Ubiquitous Messengers
Mol. Cell. Biol. 1999 19: 2435-2444
"Signal transduction networks allow cells to perceive changes in the extracellular environment and to mount an appropriate response. Mitogen-activated protein kinase (MAPK) cascades are among the most thoroughly studied of signal transduction systems and have been shown to participate in a diverse array of cellular programs, including cell differentiation, cell movement, cell division, and cell death. A key question in studies of this cascade is, how does a ubiquitously activated regulatory enzume generate a specific and biologically appropriate cellular response? In this review we describe recent findings that provide insight into ways that the regulation, structure, and localization of MAPKs and the participation of adapters and scaffolds can help determine biological outcomes." [Full Text]

Shih-chu Kao, Rama K. Jaiswal, Walter Kolch, and Gary E. Landreth
Identification of the Mechanisms Regulating the Differential Activation of the MAPK Cascade by Epidermal Growth Factor and Nerve Growth Factor in PC12 Cells
J. Biol. Chem. 276: 18169-18177, May 2001.
"In PC12 cells, epidermal growth factor (EGF) transiently stimulates the mitogen-activated protein (MAP) kinases, ERK1 and ERK2, and provokes cellular proliferation. In contrast, nerve growth factor (NGF) stimulation leads to the sustained activation of the MAPKs and subsequently to neuronal differentiation. It has been shown that both the magnitude and longevity of MAPK activation governs the nature of the cellular response. The activations of MAPKs are dependent upon two distinct small G-proteins, Ras and Rap1, that link the growth factor receptors to the MAPK cascade by activating c-Raf and B-Raf, respectively. We found that Ras was transiently stimulated upon both EGF and NGF treatment of PC12 cells. However, EGF transiently activated Rap1, whereas NGF stimulated prolonged Rap1 activation. The activation of the ERKs was due almost exclusively (>90%) to the action of B-Raf. The transient activation of the MAPKs by EGF was a consequence of the formation of a short lived complex assembling on the EGF receptor itself, composed of Crk, C3G, Rap1, and B-Raf. In contrast, NGF stimulation of the cells resulted in the phosphorylation of FRS2. FRS2 scaffolded the assembly of a stable complex of Crk, C3G, Rap1, and B-Raf resulting in the prolonged activation of the MAPKs. Together, these data provide a signaling link between growth factor receptors and MAPK activation and a mechanistic explanation of the differential MAPK kinetics exhibited by these growth factors." [Full Text]

Goi, Takanori, Rusanescu, Gabriel, Urano, Takeshi, Feig, Larry A.
Ral-Specific Guanine Nucleotide Exchange Factor Activity Opposes Other Ras Effectors in PC12 Cells by Inhibiting Neurite Outgrowth
Mol. Cell. Biol. 1999 19: 1731-1741
"Ras proteins can activate at least three classes of downstream target proteins: Raf kinases, phosphatidylinositol-3 phosphate (PI3) kinase, and Ral-specific guanine nucleotide exchange factors (Ral-GEFs). In NIH 3T3 cells, activated Ral-GEFs contribute to Ras-induced cell proliferation and oncogenic transformation by complementing the activities of Raf and PI3 kinases. In PC12 cells, activated Raf and PI3 kinases mediate Ras-induced cell cycle arrest and differentiation into a neuronal phenotype. Here, we show that in PC12 cells, Ral-GEF activity acts opposite to other Ras effectors. Elevation of Ral-GEF activity induced by transfection of a mutant Ras protein that preferentially activates Ral-GEFs, or by transfection of the catalytic domain of the Ral-GEF Rgr, suppressed cell cycle arrest and neurite outgrowth induced by nerve growth factor (NGF) treatment. In addition, Rgr reduced neurite outgrowth induced by a mutant Ras protein that preferentially activates Raf kinases. Furthermore, inhibition of Ral-GEF activity by expression of a dominant negative Ral mutant accelerated cell cycle arrest and enhanced neurite outgrowth in response to NGF treatment. Ral-GEF activity may function, at least in part, through inhibition of the Rho family GTPases, CDC42 and Rac. In contrast to Ras, which was activated for hours by NGF treatment, Ral was activated for only ~20 min. These findings suggest that one function of Ral-GEF signaling induced by NGF is to delay the onset of cell cycle arrest and neurite outgrowth induced by other Ras effectors. They also demonstrate that Ras has the potential to promote both antidifferentiation and prodifferentiation signaling pathways through activation of distinct effector proteins. Thus, in some cell types the ratio of activities among Ras effectors and their temporal regulation may be important determinants for cell fate decisions between proliferation and differentiation." [Full Text]

Amanda M. Vanhoose, Megan Emery, Lismary Jimenez, and Danny G. Winder
ERK Activation by G-protein-coupled Receptors in Mouse Brain Is Receptor Identity-specific
J. Biol. Chem. 277: 9049-9053, March 2002. [Abstract]

Ulrik Gether
Uncovering Molecular Mechanisms Involved in Activation of G Protein-Coupled Receptors
Endocr. Rev. 21: 90-113, 2000.
"G protein-coupled, seven-transmembrane segment receptors (GPCRs or 7TM receptors), with more than 1000 different members, comprise the largest superfamily of proteins in the body. Since the cloning of the first receptors more than a decade ago, extensive experimental work has uncovered multiple aspects of their function and challenged many traditional paradigms. However, it is only recently that we are beginning to gain insight into some of the most fundamental questions in the molecular function of this class of receptors. How can, for example, so many chemically diverse hormones, neurotransmitters, and other signaling molecules activate receptors believed to share a similar overall tertiary structure? What is the nature of the physical changes linking agonist binding to receptor activation and subsequent transduction of the signal to the associated G protein on the cytoplasmic side of the membrane and to other putative signaling pathways? The goal of the present review is to specifically address these questions as well as to depict the current awareness about GPCR structure-function relationships in general." [Full Text]

Yehia Daaka, Louis M. Luttrell, Seungkirl Ahn, Gregory J. Della Rocca, Stephen S. G. Ferguson, Marc G. Caron, and Robert J. Lefkowitz
Essential Role for G Protein-coupled Receptor Endocytosis in the Activation of Mitogen-activated Protein Kinase
J. Biol. Chem. 273: 685-688. [Full Text]

Rakhit, Soma, Pyne, Susan, Pyne, Nigel J.
Nerve Growth Factor Stimulation of p42/p44 Mitogen-Activated Protein Kinase in PC12 Cells: Role of Gi/o, G Protein-Coupled Receptor Kinase 2, beta -Arrestin I, and Endocytic Processing
Mol Pharmacol 2001 60: 63-70
"In this study, we have shown that nerve growth factor (NGF)-dependent activation of the p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) pathway in PC12 cells can be partially blocked by pertussis toxin (which inactivates the G proteins Gi/o). This suggests that the Trk A receptor may use a G protein-coupled receptor pathway to signal to p42/p44 MAPK. This was supported by data showing that the NGF-dependent activation of p42/p44 MAPK is potentiated in cells transfected with G protein-coupled receptor kinase 2 (GRK2) or beta-arrestin I. Moreover, GRK2 is constitutively bound with the Trk A receptor, whereas NGF stimulates the pertussis toxin-sensitive binding of beta-arrestin I to the TrkA receptor-GRK2 complex. Both GRK2 and beta-arrestin I are involved in clathrin-mediated endocytic signaling to p42/p44 MAPK. Indeed, inhibitors of clathrin-mediated endocytosis (e.g., monodansylcadaverine, concanavalin A, and hyperosmolar sucrose) reduced the NGF-dependent activation of p42/p44 MAPK. Finally, we have found that the G protein-coupled receptor-dependent component regulating p42/p44 MAPK is required for NGF-induced differentiation of PC12 cells. Thus, NGF-dependent inhibition of DNA synthesis was partially blocked by PD098059 (inhibitor of MAPK kinase-1 activation) and pertussis toxin. Our findings are the first to show that the Trk A receptor uses a classic G protein-coupled receptor-signaling pathway to promote differentiation of PC12 cells." [Full Text]

Ng, Tony, Shima, David, Squire, Anthony, Bastiaens, Philippe I.H., Gschmeissner, Steve, Humphries, Martin J., Parker, Peter J.
PKCalpha regulates beta 1 integrin-dependent cell motility through association and control of integrin traffic
EMBO J. 1999 18: 3909-3923 [Abstract]

Valeria Antonelli, Francesca Bernasconi, Yung H. Wong, and Lucia Vallar
Activation of B-Raf and Regulation of the Mitogen-activated Protein Kinase Pathway by the G(o)alpha chain
Mol. Biol. Cell 2000 11: 1129-1142.
"Many receptors coupled to the pertussis toxin-sensitive G(i/o) proteins stimulate the mitogen-activated protein kinase (MAPK) pathway. The role of the alpha chains of these G proteins in MAPK activation is poorly understood. We investigated the ability of Galpha(o) to regulate MAPK activity by transient expression of the activated mutant Galpha(o)-Q205L in Chinese hamster ovary cells. Galpha(o)-Q205L was not sufficient to activate MAPK but greatly enhanced the response to the epidermal growth factor (EGF) receptor. This effect was not associated with changes in the state of tyrosine phosphorylation of the EGF receptor. Galpha(o)-Q205L also potentiated MAPK stimulation by activated Ras. In Chinese hamster ovary cells, EGF receptors activate B-Raf but not Raf-1 or A-Raf. We found that expression of activated Galpha(o) stimulated B-Raf activity independently of the activation of the EGF receptor or Ras. Inactivation of protein kinase C and inhibition of phosphatidylinositol-3 kinase abolished both B-Raf activation and EGF receptor-dependent MAPK stimulation by Galpha(o). Moreover, Galpha(o)-Q205L failed to affect MAPK activation by fibroblast growth factor receptors, which stimulate Raf-1 and A-Raf but not B-Raf activity. These results suggest that Galpha(o) can regulate the MAPK pathway by activating B-Raf through a mechanism that requires a concomitant signal from tyrosine kinase receptors or Ras to efficiently stimulate MAPK activity. Further experiments showed that receptor-mediated activation of Galpha(o) caused a B-Raf response similar to that observed after expression of the mutant subunit. The finding that Galpha(o) induces Ras-independent and protein kinase C- and phosphatidylinositol-3 kinase-dependent activation of B-Raf and conditionally stimulates MAPK activity provides direct evidence for intracellular signals connecting this G protein subunit to the MAPK pathway." [Full Text]

Gregory J. Della Rocca, Tim van Biesen, Yehia Daaka, Deirdre K. Luttrell, Louis M. Luttrell, and Robert J. Lefkowitz
Ras-dependent Mitogen-activated Protein Kinase Activation by G Protein-coupled Receptors. CONVERGENCE OF Gi- AND Gq-MEDIATED PATHWAYS ON CALCIUM/CALMODULIN, Pyk2, AND Src KINASE
J. Biol. Chem. 272: 19125-19132, August 1997.
"Many receptors that couple to heterotrimeric guanine-nucleotide binding proteins (G proteins) have been shown to mediate rapid activation of the mitogen-activated protein kinases Erk1 and Erk2. In different cell types, the signaling pathways employed appear to be a function of the available repertoire of receptors, G proteins, and effectors. In HEK-293 cells, stimulation of either alpha1B- or alpha2A-adrenergic receptors (ARs) leads to rapid 5-10-fold increases in Erk1/2 phosphorylation. Phosphorylation of Erk1/2 in response to stimulation of the alpha2A-AR is effectively attenuated by pretreatment with pertussis toxin or by coexpression of a Gbetagamma subunit complex sequestrant peptide (betaARK1ct) and dominant-negative mutants of Ras (N17-Ras), mSOS1 (SOS-Pro), and Raf (DeltaN-Raf). Erk1/2 phosphorylation in response to alpha1B-AR stimulation is also attenuated by coexpression of N17-Ras, SOS-Pro, or DeltaN-Raf, but not by coexpression of betaARK1ct or by pretreatment with pertussis toxin. The alpha1B- and alpha2A-AR signals are both blocked by phospholipase C inhibition, intracellular Ca2+ chelation, and inhibitors of protein-tyrosine kinases. Overexpression of a dominant-negative mutant of c-Src or of the negative regulator of c-Src function, Csk, results in attenuation of the alpha1B-AR- and alpha2A-AR-mediated Erk1/2 signals. Chemical inhibitors of calmodulin, but not of PKC, and overexpression of a dominant-negative mutant of the protein-tyrosine kinase Pyk2 also attenuate mitogen-activated protein kinase phosphorylation after both alpha1B- and alpha2A-AR stimulation. Erk1/2 activation, then, proceeds via a common Ras-, calcium-, and tyrosine kinase-dependent pathway for both Gi- and Gq/11-coupled receptors. These results indicate that in HEK-293 cells, the Gbetagamma subunit-mediated alpha2A-AR- and the Galphaq/11-mediated alpha1B-AR-coupled Erk1/2 activation pathways converge at the level of phospholipase C. These data suggest that calcium-calmodulin plays a central role in the calcium-dependent regulation of tyrosine phosphorylation by G protein-coupled receptors in some systems." [Full Text]

David C. Budd, Gary B. Willars, John E. McDonald, and Andrew B. Tobin
Phosphorylation of the Gq/11-coupled M3-Muscarinic Receptor Is Involved in Receptor Activation of the ERK-1/2 Mitogen-activated Protein Kinase Pathway
J. Biol. Chem. 276: 4581-4587, February 2001.
"We investigated the role played by agonist-mediated phosphorylation of the G(q/11)-coupled M(3)-muscarinic receptor in the mechanism of activation of the mitogen-activated protein kinase pathway, ERK-1/2, in transfected Chinese hamster ovary cells. A mutant of the M(3)-muscarinic receptor, where residues Lys(370)-Ser(425) of the third intracellular loop had been deleted, showed a reduced ability to activate the ERK-1/2 pathway. This reduction was evident despite the fact that the receptor was able to couple efficiently to the phospholipase C second messenger pathway. Importantly, the ERK-1/2 responses to both the wild-type M(3)-muscarinic receptor and DeltaLys(370)-Ser(425) receptor mutant were dependent on the activity of protein kinase C. Our results, therefore, indicate the existence of two mechanistic components to the ERK-1/2 response, which appear to act in concert. First, the activation of protein kinase C through the diacylglycerol arm of the phospholipase C signaling pathway and a second component, absent in the DeltaLys(370)-Ser(425) receptor mutant, that is independent of the phospholipase C signaling pathway. The reduced ability of the DeltaLys(370)-Ser(425) receptor mutant to activate the ERK-1/2 pathway correlated with an approximately 80% decrease in the ability of the receptor to undergo agonist-mediated phosphorylation. Furthermore, we have previously shown that M(3)-muscarinic receptor phosphorylation can be inhibited by a dominant negative mutant of casein kinase 1alpha and by expression of a peptide corresponding to the third intracellular loop of the M(3)-muscarinic receptor. Expression of these inhibitors of receptor phosphorylation reduced the wild-type M(3)-muscarinic receptor ERK-1/2 response. We conclude that phosphorylation of the M(3)-muscarinic receptor on sites in the third intracellular loop by casein kinase 1alpha contributes to the mechanism of receptor activation of ERK-1/2 by working in concert with the diacylglycerol/PKC arm of the phospholipase C signaling pathway." [Full Text]

Shigetomo Fukuhara, Maria Julia Marinissen, Mario Chiariello, and J. Silvio Gutkind
Signaling from G Protein-coupled Receptors to ERK5/Big MAPK 1 Involves G{alpha}q and G{alpha}12/13 Families of Heterotrimeric G Proteins. EVIDENCE FOR THE EXISTENCE OF A NOVEL Ras AND Rho-INDEPENDENT PATHWAY
J. Biol. Chem. 275: 21730-21736, July 2000.
"In conclusion, the present study demonstrates that the ERK5 pathway can be functionally activated by the stimulation of GPCRs depending on their coupling specificity and that the Gq and G12/13 families of heterotrimeric G proteins can mediate this effect. As ERK5 regulates the activity of a growing number of nuclear transcription factors, these findings may help explain the distinct ability of Gq- and G12/13-coupled receptors to promote the expression of growth related genes. Furthermore, our observations raise the possibility of the existence of a novel signaling pathway whereby GPCRs enhance the activity of ERK5. Although the precise nature of this biochemical route is still unknown, we provide evidence that the pathway linking GPCRs to the MEK5-ERK5 kinase cascade is distinct from those utilized by these cell surface receptors to stimulate MAPK, JNK, and Rho GTPases." [Full Text]

Hiroki Chikumi, José Vázquez-Prado, Joan-Marc Servitja, Hiroshi Miyazaki, and J. Silvio Gutkind
Potent Activation of RhoA by Gq and Gq-coupled Receptors
J. Biol. Chem. 277: 27130-27134, July 2002.
"Heterotrimeric G proteins of the G(i), G(s), and G(q) family control a wide array of physiological functions primarily by regulating the activity of key intracellular second messenger-generating systems. alpha subunits of the G(12) family, Galpha(12) and Galpha(13), however, can promote cellular responses that are independent of conventional second messengers but that result from the activation of small GTP-binding proteins of the Rho family and their downstream targets. These findings led to the identification of a novel family of guanine-nucleotide exchange factors (GEFs) that provides a direct link between Galpha(12/13) and Rho stimulation. Recent observations suggest that many cellular responses elicited by Galpha(q) and its coupled receptors also require the functional activity of Rho. However, available evidence suggests that Galpha(q) may act on pathways downstream from Rho rather than by promoting Rho activation. These seemingly conflicting observations and the recent development of sensitive assays to assess the in vivo levels of active Rho prompted us to ask whether Galpha(q) and its coupled receptors can stimulate endogenous Rho. Here we show that the expression of activated forms of Galpha(q) and the stimulation of G(q)-coupled receptors or chimeric Galpha(q) molecules that respond to G(i)-linked receptors can promote a robust activation of endogenous Rho in HEK-293T cells. Interestingly, this response was not prevented by molecules interfering with the ability of Galpha(13) to stimulate its linked RhoGEFs, together suggesting the existence of a novel molecular mechanism by which Galpha(q) and the large family of G(q)-coupled receptors can regulate the activity of Rho and its downstream signaling pathways." [Abstract/Full Text]

Jingzhen Yuan, Lee W. Slice, and Enrique Rozengurt
Activation of Protein Kinase D by Signaling through Rho and the alpha Subunit of the Heterotrimeric G Protein G13
J. Biol. Chem. 276: 38619-38627, October 19, 2001. [Full Text]

Riad, Mustapha, Watkins, Kenneth C., Doucet, Edith, Hamon, Michel, Descarries, Laurent
Agonist-Induced Internalization of Serotonin-1A Receptors in the Dorsal Raphe Nucleus (Autoreceptors) But Not Hippocampus (Heteroreceptors)
J. Neurosci. 2001 21: 8378-8386
"Serotonin-1A (5-HT1A) receptors in the CNS are a major target for psychotropic drugs. In nucleus raphe dorsalis (NRD) and hippocampus (CA3), the selective 5-HT1A agonist (+)-8-hydroxy-2-(di-N-propylamino) tetralin (8-OH-DPAT) reduces the firing activity of serotoninergic (5-HT) and pyramidal neurons, respectively. When located on 5-HT (autoreceptors), but not on non-5-HT (heteroreceptors) neurons, 5-HT1A receptors are known to be subject to desensitization. Using quantitative electron microscopy after pre-embedding immunogold labeling with specific antibodies, we examined the subcellular distribution of these receptors after acute administration of 8-OH-DPAT (0.5 mg/kg, i.v.). Silver-intensified immunogold particles associated with the plasma membrane or the cytoplasm were counted in somata and dendrites within the NRD, 15 min, 1 hr and 24 hr after 8-OH-DPAT injection, and in hippocampal dendrites 1 hr after the same treatment. Significant decrease in the density of membrane labeling and concomitant increase of cytoplasmic labeling were demonstrated in the NRD, 15 min and 1 hr after 8-OH-DPAT administration, with a return to baseline level at 24 hr. Internalization was blocked by previous administration of the 5-HT1A antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane-carboxamide (WAY 100635), which, by itself, was without apparent effect. In hippocampus (CA3), there were no apparent changes in the distribution of the receptor after 8-OH-DPAT administration. These findings are in line with earlier results showing a desensitization of 5-HT1A autoreceptors but not heteroreceptors after treatment with 5-HT1A receptor agonist. They suggest that this desensitization is the result of autoreceptor internalization."

John M. Schmitt, and Philip J. S. Stork
2-Adrenergic Receptor Activates Extracellular Signal-regulated Kinases (ERKs) via the Small G Protein Rap1 and the Serine/Threonine Kinase B-Raf
J. Biol. Chem. 275: 25342-25350, August 2000. [Full Text]

Wenbo Yang, Yu Holly Hong, Xi-Qiang Shen, Christy Frankowski, Heidi S. Camp, and Todd Leff
Regulation of Transcription by AMP-activated Protein Kinase. PHOSPHORYLATION OF p300 BLOCKS ITS INTERACTION WITH NUCLEAR RECEPTORS
J. Biol. Chem. 276: 38341-38344, August 2001.
"AMP-activated protein kinase (AMP-kinase) modulates many metabolic processes in response to fluctuations in cellular energy status. Although most of its known targets are metabolic enzymes, it has been proposed that AMP-kinase might also regulate gene expression. Here we demonstrate that the transcriptional coactivator p300 is a substrate of AMP-kinase. Phosphorylation of p300 at serine 89 by AMP-kinase dramatically reduced its interaction, in vitro and in vivo, with the nuclear receptors peroxisome proliferator-activated receptor , thyroid receptor, retinoic acid receptor, and retinoid X receptor, but did not affect its interaction with the non-nuclear receptor transcription factors E1a, p53, or GATA4. These findings indicate that the AMP-kinase signaling pathway selectively modulates a subset of p300 activities and represent the first example of a transcriptional component regulated by AMP-kinase. Our results suggest a direct link between cellular energy metabolism and gene expression." [Full Text]

Scharfman, Helen E., Goodman, Jeffrey H., Sollas, Anne L.
Actions of Brain-Derived Neurotrophic Factor in Slices from Rats with Spontaneous Seizures and Mossy Fiber Sprouting in the Dentate Gyrus
J. Neurosci. 1999 19: 5619-5631 [Full Text]

Bolton, M. McLean, Pittman, Andrew J., Lo, Donald C.
Brain-Derived Neurotrophic Factor Differentially Regulates Excitatory and Inhibitory Synaptic Transmission in Hippocampal Cultures
J. Neurosci. 2000 20: 3221-3232 [Full Text]

Tyler, William J., Pozzo-Miller, Lucas D.
BDNF Enhances Quantal Neurotransmitter Release and Increases the Number of Docked Vesicles at the Active Zones of Hippocampal Excitatory Synapses
J. Neurosci. 2001 21: 4249-4258 [Full Text]

Carter, Alexandre R., Chen, Chinfei, Schwartz, Phillip M., Segal, Rosalind A.
Brain-Derived Neurotrophic Factor Modulates Cerebellar Plasticity and Synaptic Ultrastructure
J. Neurosci. 2002 22: 1316-1327 [Abstract]

Chen, Chu, Magee, Jeffery C., Bazan, Nicolas G.
Cyclooxygenase-2 Regulates Prostaglandin E2 Signaling in Hippocampal Long-Term Synaptic Plasticity
J Neurophysiol 2002 87: 2851-2857 [Abstract]

Smith, MA, Makino, S, Kvetnansky, R, Post, RM
Stress and glucocorticoids affect the expression of brain-derived neurotrophic factor and neurotrophin-3 mRNAs in the hippocampus
J. Neurosci. 1995 15: 1768-1777 [Abstract]

ES Levine, CF Dreyfus, IB Black, and MR Plummer
Brain-Derived Neurotrophic Factor Rapidly Enhances Synaptic Transmission in Hippocampal Neurons via Postsynaptic Tyrosine Kinase Receptors
PNAS 92: 8074-8077, 1995. [Abstract/Full Text]

Lodovichi, Claudia, Berardi, Nicoletta, Pizzorusso, Tommaso, Maffei, Lamberto
Effects of Neurotrophins on Cortical Plasticity: Same or Different?
J. Neurosci. 2000 20: 2155-2165 [Full Text]

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