| 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." [Abstract] 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]
CHRISTOPHE CROCHEMORE,
THEOLOGOS M. MICHAELIDIS, DIETER FISCHER, JEAN-PHILIPPE LOEFFLER, and OSBORNE
F. X. ALMEIDA
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
Mediation of Nerve Growth Factor-driven
Cell Cycle Arrest in PC12 Cells by p53. SIMULTANEOUS DIFFERENTIATION
AND PROLIFERATION SUBSEQUENT TO p53 FUNCTIONAL INACTIVATION
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
REGULATION
OF TRANSCRIPTION FACTORS BY NEURONAL ACTIVITY
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, Yos |
