| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 274, Issue 19, 13193-13197, May 7, 1999
From the Department of Medicine and the Committee on Cancer Biology, The University of Chicago, Chicago, Illinois 60637
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
The cAMP-dependent protein kinase
(PKA) exhibits both inhibitory and stimulatory effects upon growth
factor signaling mediated by the mitogen-activated protein kinase
signaling pathway. PKA has been demonstrated to inhibit Raf-1-mediated
cellular proliferation. PKA can both prevent Ras-dependent
Raf-1 activation and directly inhibit Raf-1 catalytic activity. In
contrast to the inhibitory effect of PKA on Raf-1-dependent
processes, PKA potentiates nerve growth factor-stimulated PC12 cell
differentiation, a B-Raf mediated process. This potentiation, rather
than inhibition, of PC12 cell differentiation is curious in light of
the ability of PKA to inhibit Raf-1 catalytic activity. The kinase
domains of Raf-1 and B-Raf are highly conserved, and it has been
predicted that B-Raf catalytic activity would also be inhibited by PKA.
In this study we examined the ability of PKA to regulate the kinase
activity of the B-raf proto-oncogene. We report that nerve
growth factor-stimulated B-Raf activity is not inhibited by PKA. By
contrast, an N-terminally truncated, constitutively active form of
B-Raf is inhibited by PKA both in vitro and in transfected
PC12 cells. These results suggest that the N-terminal regulatory domain
interferes with the ability of PKA to modulate B-Raf catalytic activity
and provide an explanation for the observed resistance of
B-Raf-dependent processes to PKA inhibition.
Members of the Raf family of serine/threonine protein kinases
(Raf-1, B-Raf, and A-Raf) have been shown to be key regulators of
growth factor signaling in diverse biological systems. Raf can directly
phosphorylate and activate
MEK1 (1-3), which in turn
leads to the activation of mitogen-activated or extracellular
signal-regulated protein kinase (MAPK) (4). In response to a variety of
extracellular stimuli, the Raf/MEK/MAPK cascade mediates a signal relay
from the plasma membrane to the nucleus, resulting in cell
type-specific responses that include proliferation and differentiation
(5-7). An early step in Raf-1 activation involves the binding of Ras
and the recruitment of Raf-1 to the plasma membrane. However,
subsequent events are then required to generate full Raf-1 activity
(reviewed in Ref. 8).
Diverse extracellular signals are integrated within the cell to elicit
a tailored cellular response to different environmental cues. Signal
integration is achieved through cross-talk between intracellular
signaling pathways. It has been demonstrated that inhibitory signal
integration occurs between the cAMP-dependent protein
kinase (PKA) pathway and the MAPK signaling pathway in a variety of
cell types (9-18) through inhibition of Raf-1 activity. PKA has been
shown to inhibit Raf-1 by two mechanisms: (i) inhibition of Ras binding
(9) and (ii) direct inhibition of Raf-1 catalytic activity (17, 19,
20). Raf-1 signal transduction is also subject to negative regulation
by the Ras-related small GTPase, Rap1. Elevation of intracellular cAMP
levels and consequent PKA activation results in Rap1 GTP loading (21).
The GTP-bound form of Rap1 can bind to the N-terminal regulatory domain
of Raf-1 (22-24) and block Ras-dependent Raf-1 activation
(25, 26). Thus, cAMP-dependent signaling can inhibit Raf-1
activity by several mechanisms including the direct phosphorylation of
Raf-1 and the stimulation of the inhibitory Rap1 protein.
By contrast to Raf-1, less is known regarding the activation and
regulation of B-Raf. B-Raf has been demonstrated to be required for
early mouse development and is essential for vasculogenesis (27). B-Raf
has been shown to be expressed at high levels in neural tissue (28) and
has been most extensively characterized in the PC12 cell line. Raf-1
and B-Raf are closely related and share extensive homology between
their catalytic domains (29). Raf-1 and B-Raf both phosphorylate and
activate MEK1 and MEK2 the isoforms, albeit with different specific
activities (30). In a manner similar to Raf-1 regulation, PKA can
phosphorylate B-Raf and block Ras-dependent B-Raf
activation (31). However, in contrast to the inhibitory effect of PKA
on Raf-1-dependent proliferation, it has been demonstrated
that PKA can potentiate B-Raf-dependent PC12 cell
differentiation. This stimulatory effect of PKA appears to be indirect
and mediated through Rap1 (26, 32, 33). Thus, Rap1 is inhibitory to
Raf-1 but stimulatory to B-Raf.
The observation that NGF-stimulated PC12 cell neuronal differentiation
is not inhibited by PKA (34, 35) is curious in light of the ability of
PKA to inhibit Raf-1 catalytic activity. The high degree of homology
between the kinase domains of Raf-1 and B-Raf has led to the prediction
that B-Raf catalytic activity would also be inhibited by PKA (19). In
this study we have directly tested the ability of PKA to regulate B-Raf
catalytic activity. We report that even though the activity of the
isolated C-terminal catalytic domain is negatively regulated by PKA,
PKA did not inhibit the catalytic activity of NGF-stimulated,
full-length B-Raf. Our data suggest that the N-terminal regulatory
domain interferes with the ability of PKA to modulate B-Raf catalytic
activity. This finding resolves the apparent discrepancy between the
putative inhibitory effect of PKA upon B-Raf catalytic activity and the observed stimulatory effect of PKA on B-Raf-mediated PC12 cell differentiation.
Plasmid Constructs, Polymerase Chain Reaction Mutagenesis, and
RNA Preparation--
A plasmid encoding the full-length human
B-raf (pSL B-raf) was digested with
StyI and NdeI to isolate the C-terminal catalytic domain (B-rafcat). The B-rafcat was subcloned
into SmaI-digested pXen2, a Xenopus expression
vector containing a glutathione S-transferase (GST) motif
(20) to generate pXen B-rafcat-GST. For studies in PC12
cells, the same StyI/NdeI fragment was subcloned
into EcoRV-digested SR Protein Expression in Xenopus Oocytes--
Defolliculated
Xenopus oocytes were microinjected with in vitro
transcribed RNA (10 ng of RNA per embryo) encoding GST or GST-Raf
fusion proteins as indicated. Pools of 20 injected oocytes were lysed
in Nonidet P-40 lysis buffer (10 mM Tris, pH7.5, 137 mM NaCl, 1 mM EDTA, 50 mM NaF, 10 mM NaPPi, 1% Nonidet P-40, 2 mM
phenylmethylsulfonyl fluoride, 0.2 units of aprotinin (Sigma) /ml, and
25 µM leupeptin) 12 h after microinjection (37).
PC12 Cell Culture and Transfections--
PC12 cells were grown
in Dulbecco's modified Eagle's medium with 10% equine serum and 5%
fetal calf serum. For transfections PC12 cells were plated on 35-mm
polylysine-coated dishes at 50% confluency and transfected by the
LipofectAMINE method (Life Technologies, Inc.). Transfected cells were
washed with cold phosphate-buffered saline and lysed in Nonidet P-40
lysis buffer 20 h after transfection. Cell debris was removed by
centrifugation (12,000 × g, 5 min, 4 °C), and the
resulting supernatant was assayed for total protein by the Bradford
assay (Pierce). Prior to stimulation, PC12 cells were incubated in low
serum medium (Dulbecco's modified Eagle's medium with 2% equine
serum and 1% fetal calf serum) for 20 h. NGF (Roche Molecular
Biochemicals, 100 ng/ml final concentration) was then added to the
medium for 5 min. Cell lysate was prepared as described above.
Glutathione-Sepharose Affinity Purification, Immunoprecipitation,
and Kinase Assays--
Raf-GST fusion proteins were partially purified
from equivalent amounts (100 µg) of total cell lysate protein by
incubation with 20 µl of a 1:1 slurry of glutathione-Sepharose
(Amersham Pharmacia Biotech) in a total volume of 250 µl (20). After
rocking at 4 °C for 1 h, the samples were spun at 6,000 × g for 5 s. The pellets were washed twice in lysis
buffer and once in Tris-buffered saline (20 mM Tris, pH7.5,
150 mM NaCl). PC12 B-Raf was immunopurified from
NGF-stimulated PC12 cells by incubating 100 µg of total cell lysate
protein with 2 µg of anti-B-Raf IgG (Santa Cruz Biotechnology) and 30 µl of a 1:1 slurry of protein G Plus-Agarose (Santa Cruz Biotechnology) at 4 °C for 1 h. The immune complex was washed as described above. Raf-1 was immunopurified from Xenopus
oocytes by incubating 500 µg of total cell lysate protein with 2 µg
of anti-Raf-1 IgG (Santa Cruz Biotechnology) and 30 µl of a 1:1
slurry of protein G Plus-agarose (Santa Cruz Biotechnology) at 4 °C
for 1 h. PKC activation of Raf-1 was performed using recombinant
PKC (Upstate Biotechnology) following the supplier's protocol. For PKA
pre-phosphorylation, agarose- or Sepharose-bound Raf proteins were
incubated in the presence of 5 units of PKA (Sigma) in 50 mM PIPES, pH7.0, 10 mM MgCl2, 1 mM dithiothreitol, and 100 µM unlabeled ATP
for 15 min at 30 °C. The reaction was terminated by adding 1 ml of
lysis buffer. The Sepharose beads were then washed once in lysis buffer
and once in Tris-buffered saline at 4 °C. The GST fusion proteins
were then assayed for Raf activity in 20 mM Tris, pH7.5, 10 mM MnCl2, 10 mM MgCl2,
25 mM The B-Raf and Raf-1 proteins are highly homologous within their
catalytic domains (81% amino acid identity). Because PKA can directly
phosphorylate the C-terminal domain and inhibit the catalytic activity
of Raf-1 (17, 19, 20), we wished to determine whether the catalytic
activity of B-Raf was subject to similar regulation by PKA.
We first tested whether the catalytic domain of B-Raf was a substrate
for phosphorylation by PKA. The catalytic domain of B-Raf (amino acids
385-765) was fused to an N-terminal glutathione S-transferase moiety (designated B-Rafcat-GST). A point
mutation was then introduced within the ATP binding site of B-Raf to
render the enzyme catalytically inactive and prevent
autophosphorylation (designated B-Rafcat K482M-GST). The B-Rafcat
K482M-GST protein was expressed in Xenopus oocytes and
partially purified by glutathione-Sepharose affinity chromatography.
Fig. 1 demonstrates that the B-Raf
catalytic domain (B-Rafcat K482M-GST), like the Raf-1 catalytic domain
(Rafcat K375M), is a substrate for PKA. No phosphorylation of B-Rafcat K482M-GST was observed in the absence of PKA (data not shown). The
B-Rafcat K482M-GST protein migrates as a doublet under these assay
conditions. The GST moiety when expressed alone was not phosphorylated
by PKA (GST, Fig. 1), demonstrating that PKA phosphorylates site(s)
within the Raf-1 and B-Raf catalytic domains.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
to generate
SR-B-rafcat. pSL B-raf was digested with
HindIII and NdeI to isolate the full-length
B-raf and Klenow-treated and subcloned into
EcoRV-digested SR to generate SR-B-raf.
Standard polymerase chain reaction-directed mutagenesis was employed to generate a lysine to methionine mutation at amino acid 482 (K482M) within pXen B-rafcat to generate the kinase-deficient
B-rafcat K482M. The catalytic subunit of PKA in
SR (SR-PKAcat) (36) was utilized for expression of PKA
in PC12 cells. The kinase-deficient C-terminal domain of Raf-1,
rafcat K375M (pXen vNAF) has been described
previously (37). For in vitro transcription, pXen plasmids
were linearized with EcoRI and capped RNA synthesized with
SP6 RNA polymerase as described previously (20).
glycerophosphate, 1 mM
dithiothreitol, 50 µM ATP, 10 µCi of
[
-32P]ATP, 100 ng of kinase-negative MEK, and 30 ng of
PKA inhibitor (Sigma) for 10 min at 30 °C (20, 38). The reactions
were terminated by adding SDS gel loading buffer. Samples were loaded
on a 10% polyacrylamide gel (Novex) and subjected to
SDS-polyacrylamide gel electrophoresis. The proteins were then
transferred to nitrocellulose, and radiolabeled phosphate incorporation
into MEK was visualized by autoradiography and quantitated by
PhosphorImager analysis. Western blotting to determine Raf-GST
fusion protein levels was performed with a GST antibody at a 1:1000
dilution (Santa Cruz Biotechnology). Bound antibody was visualized by
ECL (Amersham Pharmacia Biotech) and exposure to hyperfilm (Amersham
Pharmacia Biotech).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (38K):
[in a new window]
Fig. 1.
The catalytic domain of B-Raf is
phosphorylated by PKA. The kinase-defective mutants of the Raf-1
and B-Raf catalytic domains were expressed as GST fusion proteins in
immature Xenopus oocytes. Protein lysates were prepared
16 h after microinjection of in vitro transcribed RNA,
and the fusion proteins were partially purified over
glutathione-Sepharose resin. Following incubation with PKA and
radiolabeled ATP, the samples were separated on SDS-polyacrylamide gel
electrophoresis, and the phosphoproteins were identified following
autoradiography. The positions of the phosphorylated catalytic domains
of Raf-1 (Raf-1cat(K375M))- and B-Raf
(B-Rafcat(K482M))-GST fusion proteins are
indicated by a closed arrowhead. Kinase-deficient mutants of
Raf-1 and B-Raf were used to prevent any contribution from
autophosphorylation. The GST moiety alone was not phosphorylated by PKA
(the migration of GST is indicated by an open
arrowhead).
We next determined whether the activity of the B-Raf catalytic domain
could be inhibited by PKA phosphorylation. The B-Rafcat-GST protein was
expressed in Xenopus oocytes and partially purified by
glutathione-Sepharose affinity chromatography. B-Rafcat-GST was
incubated in the absence or presence of PKA, as described for Fig. 1,
but with nonradioactively labeled ATP. The glutathione-Sepharose B-Rafcat-GST complex was then washed extensively, and the
phosphotransferase activity of the B-Rafcat-GST was determined using a
kinase-negative MEK substrate in the presence of radioactively labeled
ATP. Preincubation with PKA significantly reduced B-Rafcat activity
(Fig. 2, 44.7% inhibition ± 6.5 S.E., n = 3).
|
We next wished to determine whether the inhibitory effects of PKA on
B-Raf catalytic activity observed in vitro could be
reproduced following transfection of mammalian tissue culture cells.
For these experiments we utilized PC12 cells, which had previously been
utilized to demonstrate cross-talk between the PKA and B-Raf signaling
pathways (26, 31, 39, 40). PC12 cells were transfected with
B-rafcat-GST in the presence or absence of a co-transfected plasmid encoding the catalytic subunit of PKA (PKAcat). Transfection of
this PKAcat construct results in a high and persistent level of PKA activity in PC12 cells as measured with a CAMP response element-CAT reporter plasmid (data not shown). B-Rafcat-GST
was partially purified by glutathione-Sepharose affinity chromatography from PC12 cell lysates prepared 24 h after transfection. We
observed that co-transfection with PKAcat inhibited the
activity of B-Rafcat-GST in PC12 cells (Fig.
3A, +PKA). As in the in
vitro study (Fig. 2), we did not observe complete inhibition of
B-Rafcat activity by PKA (55.5% inhibition ± 4.4 S.E.M.,
n = 4). Immunoblot analysis of the same lysates
revealed that similar amounts of B-Raf protein were present in each
sample (Fig. 3B), indicating that expression of PKAcat did
not lead to translational inhibition of the co-transfected B-Rafcat-GST. Thus, the difference in catalytic activity reflects changes in the specific activity of the B-Rafcat-GST protein following PKA co-transfection. It would appear that PKA co-transfection results
in a mobility shift of a portion of the B-Rafcat-GST protein (Fig. 3B).
|
Given the inhibitory effect of PKA on the isolated B-Raf catalytic
domain in transfected PC12 cells, we wished to determine whether PKA
exerted an inhibitory influence on the activity of the full-length
B-Raf enzyme present in PC12 cells. To directly assess the effects of
PKA, we immunoprecipitated active B-Raf from NGF-stimulated PC12 cells.
The B-Raf immune complexes were incubated in the presence or absence of
PKA in vitro and subsequently assayed for B-Raf activity.
PKA treatment did not result in any inhibition of B-Raf activity (Fig.
4A). By contrast, PKA
treatment reduced PKC-activated Raf-1 activity (Fig. 4B,
38.2% inhibition ± 4.1 S.E., n = 3) as
previously reported (17, 19). We conclude that following NGF
stimulation, the catalytic activity of B-Raf is not subject to
inhibition by PKA. Moreover, rather than exerting an inhibitory
influence on catalytic activity, PKA actually stimulated B-Raf
activation. PC12 cells were transfected with full-length B-Raf-GST in
the presence or absence of co-transfected PKAcat. Immunoblot analysis
of the glutathione-Sepharose affinity-purified lysates indicated that
similar amounts of full-length B-Raf-GST were expressed in the absence
or presence of co-transfected PKAcat (Fig.
5A). In contrast to the
inhibitory effect of PKAcat upon the activity of the transfected B-Raf
catalytic domain (Fig. 3), PKA co-transfection stimulated the activity
of full-length B-Raf-GST approximately 3-fold (Fig. 5B).
This activation of B-Raf-GST by PKAcat is in agreement with a published
report that demonstrated that elevated levels of cAMP in PC12 cells led
to activation of B-Raf (26).
|
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
It has previously been shown that PKA can negatively regulate Raf-1 activity by two distinct mechanisms. First, PKA can prevent Raf-1 activation by phosphorylating the Raf-1 N-terminal regulatory domain and blocking Ras interactions. Second, PKA can phosphorylate the Raf-1 C-terminal catalytic domain and inhibit Raf-1 catalytic activity. The association of Ras with B-Raf is also inhibited by PKA (31), and PKA blocks Ras-dependent B-Raf activation. Similarly, based on sequence homology, it has been predicted that B-Raf catalytic activity would also be subject to inhibition by PKA (19). However, recent evidence has suggested that rather than play an obligate inhibitory function in the regulation of B-Raf, PKA can actually stimulate B-Raf activity in certain PC12 cell lines via the small G-protein, Rap1 (26, 33). To address these apparently contradictory roles for PKA in the control of B-Raf, we have investigated whether B-Raf catalytic activity is inhibited by PKA. We report here that contrary to the effects on Raf-1, PKA does not inhibit the catalytic activity of the full-length B-Raf protein (Fig. 4). Interestingly, an N-terminally truncated form of B-Raf was inhibited by PKA both in vitro and in transfected PC12 cells (Figs. 2 and 3).
These results suggest key differences in the mechanism by which PKA can modulate Raf isoform catalytic activity. The N-terminal regulatory domain of B-Raf may serve to mask the catalytic domain from PKA action, or the N-terminal domain may cause the catalytic domain to adopt a conformation that is not compatible with PKA inhibition in the context of the full-length B-Raf enzyme. Alternatively, PKA may exert both positive and negative effects on B-Raf, but the sum of these two effects of PKA favors B-Raf activity within the context of the full-length protein. The kinase activity of the Raf-1 isoform, on the other hand, is subject to inhibition by PKA either in the context of the full-length protein or when the catalytic domain is expressed alone (17, 19, 20, 41). The demonstration that cAMP agonists can inhibit growth factor-stimulated cellular proliferation (9-18) has prompted speculation that they may be of therapeutic use in the treatment of certain cancers (41). Indeed, a cell-permeable cAMP analogue has been shown to inhibit tumors in nude mice (42), revert v-raf transformation, and induce apoptosis in v-abl transformed cells (41). Our data suggest that cAMP analogues would not revert B-raf-mediated cellular transformation.
Our findings indicating that the catalytic activity of full-length B-Raf is not subject to inhibition by PKA are compatible with several recent observations concerning PKA and B-Raf cross-talk. It has been shown that elevated intracellular cAMP levels had differential effects on Raf-1 and B-Raf when the cells were maintained in serum-containing media (39). This would be consistent with cAMP-induced PKA inhibiting active Raf-1 but having little effect on active, full-length B-Raf. PKA has also been demonstrated to stimulate MAPK in certain cell types (26, 43, 44). This stimulation is mediated indirectly by PKA through the Rap1-dependent activation of B-Raf (26, 33). Unlike the Ras-dependent activation of B-Raf, Rap1-dependent activation is refractory to PKA.
These regulatory differences between Raf isoforms may tailor the cellular response following extracellular ligand stimulation. For example, although simultaneous cAMP elevation generally results in a block to growth factor-stimulated signaling, in PC12 cells (which express B-Raf) the combination results in the potentiation of growth factor-stimulated differentiation. The mechanistic basis of the isoform-specific resistance to inhibition by PKA has not hitherto been fully understood. In addition to Ras, Rap1-dependent B-Raf activation was necessary for both the phenotypic extension of neurites and the induction of differentiation-specific gene expression in PC12 cells (33). Moreover, the Rap1-dependent activation of differentiation was dependent upon endogenous PKA activity (32). Our findings would suggest that although Raf-1 activity is susceptible to PKA-mediated inhibition, Rap1-stimulated B-Raf catalytic activity is refractory to the inhibitory action of PKA. Thus, the differential regulation of Raf isoforms by PKA would allow certain cell types to interpret PKA signaling in a stimulatory rather than an inhibitory manner on MAPK-dependent processes. Indeed, we observe activation rather than inhibition of full-length B-Raf in response to PKA co-transfection of PC12 cells (Fig. 5). Taken together, the primary means of negatively regulating B-Raf activity via PKA may be through regulation of Ras-dependent B-Raf activation rather than through inhibition of catalytic activity. Indeed, elevated cAMP and PKA have been shown to block Ras binding to B-Raf (31). B-Raf activation by Rap1, on the other hand, is stimulated by PKA (26, 33). Because Ras and Rap1 have highly related effector domains and have been shown to bind to the same target proteins, further studies will be required to determine why Rap1 binding is refractory to PKA phosphorylation of B-Raf.
The results of our study are most compatible with the hypothesis that
following NGF-stimulated activation of B-Raf, the N-terminal regulatory
domain serves to protect the catalytic domain from the inhibitory
action of PKA. The idea that communication between the N- and
C-terminal domains of Raf proteins contributes to the overall
regulation of activity is not without precedent (45). Further, the
14-3-3 family of proteins has been shown to interact with both the
N-terminal and C-terminal domains of Raf-1, and it has been proposed
that these interactions may be dynamic in nature resulting in the
stabilization of active or inactive Raf-1 conformations (46-48).
Curiously, the N-terminal regulatory domain does not protect the
catalytic domain of Raf-1 from inhibition by PKA (17, 19, 41). The
regulatory domains of Raf-1 and B-Raf, even though sharing two regions
of high homology, are less well conserved than the respective kinase
domains. Thus, amino acid differences between the B-Raf and Raf-1
regulatory domains may specify the determinants that modulate the
susceptibility to PKA-mediated inhibition of catalytic activity.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Drs. Walter Kolch and Gary Johnson for cDNA constructs and Joseph Welk for excellent technical support. We thank David Straus, Jean van Seventer, and Amanda Charlesworth for critical reading of the manuscript.
| |
FOOTNOTES |
|---|
* This work was supported, in part, by Diabetes Research Training Grant P60 DK20595-18, the Charlotte Geyer Foundation, and National Institutes of Health Grant CA70846.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work is dedicated to the memory of Jack Porter, a gentleman and a scholar.
To whom correspondence should be addressed: 5841 S. Maryland Ave.,
The University of Chicago, Chicago, IL 60637. Tel.: 773-702-2676; Fax:
773-702-2681; E-mail: amacnico{at}medicine.bsd.uchicago.edu.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: MEK, mitogen-activated/extracellular signal-regulated protein kinase kinase; GST, glutathione S-transferase; MAPK, mitogen-activated protein kinase; NGF, nerve growth factor; PKA, cyclic AMP-dependent protein kinase; PKAcat, catalytic subunit of PKA; PKC, protein kinase C.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
This article has been cited by other articles:
|
|
 |
|
  I. Hmitou, S. Druillennec, A. Valluet, C. Peyssonnaux, and A. Eychene Differential Regulation of B-Raf Isoforms by Phosphorylation and Autoinhibitory Mechanisms Mol. Cell. Biol., January 1, 2007; 27(1): 31 - 43. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. W. Pearson, S. Earnest, and M. H. Cobb Cyclic AMP Selectively Uncouples Mitogen-Activated Protein Kinase Cascades from Activating Signals Mol. Cell. Biol., April 15, 2006; 26(8): 3039 - 3047. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. F. POWERS, K. H. SCHELLING, J. M. BRACHOLD, and A. S. TISCHLER Plasticity of Pheochromocytoma Cell Lines from Neurofibromatosis Knockout Mice Ann. N.Y. Acad. Sci., October 1, 2002; 971(1): 371 - 378. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Fujita, T. Meguro, R. Fukuyama, H. Nakamuta, and M. Koida New Signaling Pathway for Parathyroid Hormone and Cyclic AMP Action on Extracellular-regulated Kinase and Cell Proliferation in Bone Cells. CHECKPOINT OF MODULATION BY CYCLIC AMP J. Biol. Chem., June 14, 2002; 277(25): 22191 - 22200. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Schmitt and P. J. S. Stork Cyclic AMP-Mediated Inhibition of Cell Growth Requires the Small G Protein Rap1 Mol. Cell. Biol., June 1, 2001; 21(11): 3671 - 3683. [Abstract] [Full Text]
|
|
|
|
|
|
M. C. MacNicol, A. J. Muslin, and A. 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., February 11, 2000; 275(6): 3803 - 3809. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Schmitt and P. J. S. Stork beta 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., August 11, 2000; 275(33): 25342 - 25350. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Qiu, S. Zhuang, F. C. von Lintig, G. R. Boss, and R. B. Pilz Cell Type-specific Regulation of B-Raf Kinase by cAMP and 14-3-3 Proteins J. Biol. Chem., October 6, 2000; 275(41): 31921 - 31929. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. F. Sidovar, P. Kozlowski, J. W. Lee, M. A. Collins, Y. He, and L. M. Graves Phosphorylation of Serine 43 Is Not Required for Inhibition of c-Raf Kinase by the cAMP-dependent Protein Kinase J. Biol. Chem., September 8, 2000; 275(37): 28688 - 28694. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
PKA) or
presence (+PKA) of PKA in vitro, and the subsequent activity
of B-Rafcat-GST was measured using kinase-negative MEK
(KN-MEK). A GST immunoblot analysis of the same samples
indicates that an equal amount of B-Rafcat-GST fusion protein was
present in each assay. B, a histogram summarizing the
inhibitory effect of PKA on B-Rafcat activity from three independent
experiments. The error bar indicates the standard error of
the mean.
