Differential regulation of Raf-1 and B-Raf and Ras-dependent activation of mitogen-activated protein kinase by cyclic AMP in PC12 cells.

Growth factor stimulation of the mitogen-activated protein (MAP) kinase pathway in fibroblasts is inhibited by cyclic AMP (cAMP) as a result of inhibition of Raf-1. In contrast, cAMP inhibits neither nerve growth factor-induced MAP kinase activation nor differentiation in PC12 pheochromocytoma cells. Instead, in PC12 cells cAMP activates MAP kinase. Since one of the major differences between the Ras/Raf/MAP kinase cascades of these cell types is the expression of B-Raf in PC12 cells, we compared the effects of cAMP on Raf-1 and B-Raf. In PC12 cells maintained in serum-containing medium, B-Raf was refractory to inhibition by cAMP, whereas Raf-1 was effectively inhibited. In contrast, both B-Raf and Raf-1 were inhibited by cAMP in serum-starved PC12 cells. The effect of cAMP is thus dependent upon growth conditions, with B-Raf being resistant to cAMP inhibition in the presence of serum. These results were extended by studies of Rat-1 fibroblasts into which B-Raf had been introduced by transfection. As in PC12 cells, B-Raf was resistant to inhibition by cAMP in the presence of serum, whereas Raf-1 was effectively inhibited. In addition, the expression of B-Raf rendered Rat-1 cells resistant to the inhibitory effects of cAMP on both growth factor-induced activation of MAP kinase and mitogenesis. These results indicate that Raf-1 and B-Raf are differentially sensitive to inhibition by cAMP and that B-Raf expression can contribute to cell type-specific differences in the regulation of the MAP kinase pathway. In contrast to the situation in PC12 cells, cAMP by itself did not stimulate MAP kinase in B-Raf-expressing Rat-1 cells. The activation of MAP kinase by cAMP in PC12 cells was inhibited by the expression of a dominant negative Ras mutant, indicating that cAMP acts on a target upstream of Ras. Thus, it appears that a signaling component upstream of Ras is also require for cAMP stimulation of MAP kinase in PC12 cells.     

Acetylcholine muscarinic m1 receptor regulation of cyclic AMP synthesis controls growth factor stimulation of Raf activity.

Acetylcholine muscarinic m2 receptors (m2R) couple to heterotrimeric Gi proteins and activate the Ras/Raf/mitogen-activated protein kinase pathway and phosphatidylinositol 3-kinase in Rat 1a cells. In contrast to the m2R, stimulation of the acetylcholine muscarinic m1 receptor (m1R) does not activate the Ras/Raf/mitogen-activated protein kinase regulatory pathway in Rat 1a cells but rather causes a pronounced inhibition of epidermal growth factor and platelet-derived growth factor receptor activation of Raf. In Rat 1a cells, m1R stimulation of phospholipase C beta and the marked rise in intracellular calcium stimulated cyclic AMP (cAMP) synthesis, resulting in the activation of protein kinase A. Stimulation of protein kinase A inhibited Raf activation in response to growth factors. Platelet-derived growth factor receptor stimulation of phosphatidylinositol 3-kinase activity was not affected by either m1R stimulation or protein kinase A activation in response to forskolin-stimulated cAMP synthesis. GTP loading of Ras in response to growth factors was unaffected by protein kinase A activation but was partially inhibited by carbachol stimulation of the m1R. Therefore, protein kinase A action at the Ras/Raf activation interface selectively inhibited only one branch of the signal transduction network initiated by tyrosine kinases. Specific adenylyl cyclases responding to different signals, including calcium, with enhanced cAMP synthesis will regulate Raf activation in response to Ras.GTP. Taken together, the data indicate that G protein-coupled receptors can positively and negatively regulate the responsiveness of tyrosine kinase-stimulated mitogenic response pathways.     

Inhibition of platelet-derived growth factor- and epidermal growth factor-mediated mitogenesis and signaling in 3T3 cells expressing delta Raf-1:ER, an estradiol-regulated form of Raf-1.

We have recently described the properties of delta Raf-1:ER, a fusion protein consisting of an oncogenic form of human Raf-1 and the hormone binding domain of the human estrogen receptor. In this study, we demonstrate that activation of delta Raf-1:ER in quiescent 3T3 cells (C2 cells), while sufficient to promote morphological oncogenic transformation, was insufficient to promote the entry of cells into DNA synthesis. Indeed, activation of delta Raf-1:ER potently inhibited the mitogenic response of cells to platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) treatment. Addition of beta-estradiol to quiescent C2 cells led to rapid, sustained activation of delta Raf-1:ER and MEK but only two- to threefold activation of p42 mitogen-activating protein (MAP) kinase activity. Addition of PDGF or EGF to quiescent C2 cells in which delta Raf-1:ER was inactive led to rapid activation of Raf-1, MEK, and p42 MAP kinase activities, and entry of the cells into DNA synthesis. In contrast, when delta Raf-1:ER was activated in quiescent C2 cells prior to factor addition, there was a significant inhibition of certain aspects of the signaling response to subsequent treatment with PDGF or EGF. The expression and activation of PDGF receptors and the phosphorylation of p70S6K in response to PDGF treatment were unaffected by prior activation of delta Raf-1:ER. In contrast, PDGF-mediated activation of Raf-1 and p42 MAP kinases was significantly inhibited compared with that of controls. Interestingly, the mitogenic and signaling responses of quiescent C2 cells to stimulation with fetal bovine serum or phorbol myristate acetate were unaffected by prior activation of delta Raf-1:ER. It seems likely that at least two mechanisms contribute to the effects of delta Raf-1:ER in these cells. First, activation of delta Raf-1:ER appeared to uncouple the activation of Raf-1 from the activation of the PDGF receptor at the cell surface. This may be due to the fact that mSOS1 is constitutively phosphorylated as a consequence of the activation of delta Raf-1:ER. Second, quiescent C2 cells expressing activated delta Raf-1:ER appear to contain an inhibitor of the MAP kinase pathway that, because of its apparent sensitivity to sodium orthovanadate, may be a phosphotyrosine phosphatase. It is likely that the inhibitory effects of delta Raf-1:ER observed in these cells are a manifestation of the activation of some of the feedback inhibition pathways that normally modulate a cell's response to growth factors. 3T3 cells expressing delta Raf-1:ER will be a useful tool in unraveling the role of Raf-1 kinase activity in the regulation of such pathways.     

Cyclic AMP Inhibits Activation of Mitogen-Activated Protein Kinase and Cell Proliferation in Response to Growth Factors in Cultured Rat Cortical Astrocytes

Abstract: The cyclic AMP (cAMP)-induced inhibitory effect on cell proliferation was examined through inhibition of mitogen-activated protein kinase (MAP kinase) activation in cultured rat cortical astrocytes. Basic fibroblast growth factor (bFGF) at 10 ng/ml maximally stimulated MAP kinase activity, which peaks during 10 min and prolonged for 24 h. Likewise, DNA synthesis was maximally potentiated with 10 ng/ml bFGF and correlated with MAP kinase activity in a dose-dependent manner. Dibutyryl cAMP (dbcAMP) at 1 m M and isoproterenol at 10 µ M inhibited MAP kinase activation and DNA synthesis potentiation with bFGF and platelet-derived growth factor to the control level in cultured astrocytes and C6 glioma cells. The stimulation with bFGF caused a prominent translocation of MAP kinase from the cytosol to the nucleus after 1 h in astrocytes. Treatment of the cells with dbcAMP and isoproterenol completely prevented the translocation of MAP kinase. In experiments with ³²P-labeled cultured astrocytes, phosphorylation of Raf-1 was apparently stimulated with bFGF. Treatment with dbcAMP or isoproterenol had a greatly inhibitory effect on the stimulation of Raf-1 phosphorylation with bFGF. Consistent with the effect on Raf-1 phosphorylation, dbcAMP and isoproterenol completely prevented bFGF-induced phosphorylation of MAP kinase kinases, target proteins of Raf-1. Our observations suggest that cAMP-induced suppression of cell growth in astrocytes is due to the inhibitory effect on activation of MAP kinase and its translocation to the nucleus and that the site of the cAMP action is located at Raf-1 or the upstream site of Raf-1.     

Raf kinase inhibitory protein regulates Raf-1 but not B-Raf kinase activation

Raf kinase inhibitory protein (RKIP; also known as phosphatidylethanolamine-binding protein or PEBP) is a modulator of the Raf/MAPK signaling cascade and a suppressor of metastatic cancer. Here, we show that RKIP inhibits MAPK by regulating Raf-1 activation; specifically, RKIP acts subsequent to Raf-1 membrane recruitment, prevents association of Raf-1 and p21-activated kinase (PAK), and blocks phosphorylation of the Raf-1 kinase domain by PAK and Src family kinases. Mutation of the PAK and Src phosphorylation sites on Raf-1 to aspartate, a phosphate mimic, prevented RKIP association with or inhibition of Raf-1 signaling. Interestingly, although RKIP can interact with B-Raf, RKIP depletion had no effect on activation of B-Raf. Because c-Raf-1 and B-Raf are both required for maximal MAPK stimulation by epidermal growth factor in neuronal and epithelial cell lines, we determined whether RKIP significantly affects MAPK signaling. In fact, RKIP depletion increased not only the amplitude but also the sensitivity of MAPK and DNA synthesis to epidermal growth factor stimulation by up to an order of magnitude. These results indicate that selective modulation of c-Raf-1 but not B-Raf activation by RKIP can limit the dynamic range of the MAPK signaling response to growth factors and may play a critical role in growth and development.     

Mitogen-activated protein kinase activation is insufficient for growth factor receptor-mediated PC12 cell differentiation.

When expressed in PC12 cells, the platelet-derived growth factor beta receptor (beta PDGF-R) mediates cell differentiation. Mutational analysis of the beta PDGF-R indicated that persistent receptor stimulation of the Ras/Raf/mitogen-activated protein (MAP) kinase pathway alone was insufficient to sustain PC12 cell differentiation. PDGF receptor activation of signal pathways involving p60c-src or the persistent regulation of phospholipase C gamma was required for PC12 cell differentiation. beta PDGF-R regulation of phosphatidylinositol 3-kinase, the GTPase-activating protein of Ras, and the tyrosine phosphatase, Syp, was not required for PC12 cell differentiation. In contrast to overexpression of oncoproteins involved in regulating the MAP kinase pathway, growth factor receptor-mediated differentiation of PC12 cells requires the integration of other signals with the Ras/Raf/MAP kinase pathway.     

Cell type-specific regulation of B-Raf kinase by cAMP and 14-3-3 proteins

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.     

Nerve growth factor-stimulated B-Raf catalytic activity is refractory to inhibition by cAMP-dependent protein kinase

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.     

Characterization of Ser338 phosphorylation for Raf-1 activation

Raf kinases are essential for regulating cell proliferation, survival, and tumorigenesis. However, the mechanisms by which Raf is activated are still incompletely understood. Phosphorylation plays a critical role in Raf activation in response to mitogens. The present study characterizes phosphorylation of Ser338, a crucial event for Raf-1 activation. Here we report that mutation of Lys375 to Met diminishes phosphorylation of Ser338 on both wild type Raf-1 in cells treated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA) and a constitutively active mutant in which Tyr340/Tyr341 are replaced by 2 aspartic acids, a conserved substitution present in natural B-Raf. The loss of Ser338 phosphorylation in these Raf mutants is not engendered by a mutation-induced conformational change, inasmuch as mutation of another site (Ser471 to Ala) in the activation segment also abolishes Ser338 phosphorylation, whereas both the kinase-dead mutants of Raf-1 are phosphorylated well by active Pak1. Furthermore, our data demonstrate that EGF-stimulated phosphorylation of Ser338 is inhibited by Sorafenib, a Raf kinase inhibitor, but not by the MEK inhibitor U0126. Interestingly, a kinase-dead mutation and Sorafenib also markedly reduce phosphorylation of Ser445 on B-Raf, a site equivalent to Raf-1 Ser338. Finally, our data reveal that Ser338 is phosphorylated on inactive Raf-1 by an active mutant of Raf-1 when they are dimerized in cells and that artificial dimerization of Raf-1 causes Ser338 phosphorylation, accompanied by activation of ERK1/2. Altogether, our data suggest that Ser338 on Raf-1 is autophosphorylated in response to mitogens.     

RKIP regulates MAP kinase signaling in cells with defective B-Raf activity

MAP kinase (MAPK) signaling results from activation of Raf kinases in response to external or internal stimuli. Here, we demonstrate that Raf kinase inhibitory protein (RKIP) regulates the activation of MAPK when B-Raf signaling is defective. We used multiple models including mouse embryonic fibroblasts (MEFs) and primary keratinocytes from RKIP- or Raf-deficient mice as well as allografts in mice to investigate the mechanism. Loss of B-Raf protein or activity significantly reduces MAPK activation in these cells. We show that RKIP depletion can rescue the compromised ERK activation and promote proliferation, and this rescue occurs through a Raf-1 dependent mechanism. These results provide formal evidence that RKIP is a bona fide regulator of Raf-1. We propose a new model in which RKIP plays a key role in regulating the ability of cells to signal through Raf-1 to ERK in B-Raf compromised cells.     

Activation of B-Raf and regulation of the mitogen-activated protein kinase pathway by the G(o) alpha chain

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

Ras mediates the cAMP-dependent activation of extracellular signal-regulated kinases (ERKs) in melanocytes

In melanocytes and melanoma cells, cAMP activates extracellular signal-regulated kinases (ERKs) and MEK-1 by an unknown mechanism. We demonstrate that B-Raf is activated by cAMP in melanocytes. A dominant-negative mutant of B-Raf, but not of Raf-1, blocked the cAMP-induced activation of ERK, indicating that B-Raf is the MEK-1 upstream regulator mediating this cAMP effect. Studies using Clostridium sordelii lethal toxin and Clostridium difficile toxin B have suggested that Rap-1 or Ras might transduce cAMP action. We show that Ras, but not Rap-1, is activated cell-specifically and mediates the cAMP-dependent activation of ERKs, while Rap-1 is not involved in this process in melanocytes. Our results suggest a novel, cell-specific mechanism involving Ras small GTPase and B-Raf kinase as mediators of ERK activation by cAMP. Also, in melanocytes, Ras or ERK activation by cAMP is not mediated through protein kinase A activation. Neither the Ras exchange factor, Son of sevenless (SOS), nor the cAMP-responsive Rap-1 exchange factor, Epac, participate in the cAMP-dependent activation of Ras. These findings suggest the existence of a melanocyte-specific Ras exchange factor directly regulated by cAMP.     

Identification of Raf-1 S471 as a novel phosphorylation site critical for Raf-1 and B-Raf kinase activities and for MEK binding

The Ras-Raf-MAPK cascade is a key growth-signaling pathway and its uncontrolled activation results in cell transformation. Although the general features of the signal transmission along the cascade are reasonably defined, the mechanisms underlying Raf activation remain incompletely understood. Here, we show that Raf-1 dephosphorylation, primarily at epidermal growth factor (EGF)-induced sites, abolishes Raf-1 kinase activity. Using mass spectrometry, we identified five novel in vivo Raf-1 phosphorylation sites, one of which, S471, is located in subdomain VIB of Raf-1 kinase domain. Mutational analyses demonstrated that Raf-1 S471 is critical for Raf-1 kinase activity and for its interaction with mitogen-activated protein kinase kinase (MEK). Similarly, mutation of the corresponding B-Raf site, S578, resulted in an inactive kinase, suggesting that the same Raf-1 and B-Raf phosphorylation is needed for Raf kinase activation. Importantly, the naturally occurring, cancer-associated B-Raf activating mutation V599E suppressed the S578A mutation, suggesting that introducing a charged residue at this region eliminates the need for an activating phosphorylation. Our results demonstrate an essential role of specific EGF-induced Raf-1 phosphorylation sites in Raf-1 activation, identify Raf-1 S471 as a novel phosphorylation site critical for Raf-1 and B-Raf kinase activities, and point to the possibility that the V599E mutation activates B-Raf by mimicking a phosphorylation at the S578 site.     

Acetylcholine muscarinic receptor regulation of the Ras/Raf/MAP kinase pathway

Acetylcholine muscarinic m1 receptors and m2 receptors are predominantly coupled to the heterotrimeric G proteins Gq, 11 and Gi, respectively. Stimulation of the m1 and m2 receptors in different cell types activate the Ras/Raf/MAP kinase pathway. The ability of the m1 receptor to activate the MAP kinase pathway is dependent on the isoforms of adenylyl cyclase expressed in specific cell types. Specific adenylyl cyclases respond to different signals, including calcium and protein kinase C, with increased cAMP synthesis resulting in protein kinase A activation. Stimulation of protein kinase A inhibits Raf and subsequent MAP kinase activation by G protein-coupled receptors and growth factor receptor tyrosine kinases. G protein-coupled receptors can positively and negatively regulate the responsiveness of tyrosine kinase-stimulated response pathways.     

Disruption of the 14-3-3 binding site within the B-Raf kinase domain uncouples catalytic activity from PC12 cell differentiation

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.     

Activation of Raf-1 signaling by protein kinase C through a mechanism involving Raf kinase inhibitory protein

Protein kinase C (PKC) regulates activation of the Raf-1 signaling cascade by growth factors, but the mechanism by which this occurs has not been elucidated. Here we report that one mechanism involves dissociation of Raf kinase inhibitory protein (RKIP) from Raf-1. Classic and atypical but not novel PKC isoforms phosphorylate RKIP at serine 153 (Ser-153). RKIP Ser-153 phosphorylation by PKC either in vitro or in response to 12-O-tetradecanoylphorbol-13-acetate or epidermal growth factor causes release of RKIP from Raf-1, whereas mutant RKIP (S153V or S153E) remains bound. Increased expression of PKC can rescue inhibition of the mitogen-activated protein (MAP) kinase signaling cascade by wild-type but not mutant S153V RKIP. Taken together, these results constitute the first model showing how phosphorylation by PKC relieves a key inhibitor of the Raf/MAP kinase signaling cascade and may represent a general mechanism for the regulation of MAP kinase pathways.     

MEK-1 phosphorylation by MEK kinase, Raf, and mitogen-activated protein kinase: analysis of phosphopeptides and regulation of activity.

MEK-1 is a dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK). MEK-1 is in turn activated by phosphorylation. Raf and MAPK/extracellular signal-regulated kinase kinase (MEKK) independently phosphorylate and activate MEK-1. Recombinant MEK-1 is also capable of autoactivation. Purified recombinant wild type MEK-1 and a mutant kinase inactive MEK-1 were used as substrates for MEKK, Raf, and autophosphorylation. MEK-1 phosphorylation catalyzed by Raf, MEKK, or autophosphorylation resulted in activation of MEK-1 kinase activity measured by phosphorylation of a mutant kinase inactive MAPK. Phosphoamino acid analysis and peptide mapping identified similar MEK-1 tryptic phosphopeptides after phosphorylation by MEK kinase, Raf, or MEK-1 autophosphorylation. MEK-1 is phosphorylated by MAPK at sites different from that for Raf and MEKK. Phosphorylation of MEK-1 by MAPK does not affect MEK-1 kinase activity. Several phosphorylation sites present in MEK-1 immunoprecipitated from 32P-labeled cells after stimulation with epidermal growth factor were common to the in vitro phosphorylated enzyme. The major site of MAPK phosphorylation in MEK-1 is threonine 292. Mutation of threonine 292 to alanine eliminates 90% of MAPK catalyzed phosphorylation of MEK-1 but does not influence MEK-1 activity. The results demonstrate that MEKK and Raf regulate MEK-1 activity by phosphorylation of common residues and thus, two independent protein kinases converge at MEK-1 to regulate the activity of MAPK.     

Ras-dependent ERK activation by the human G(s)-coupled serotonin receptors 5-HT4(b) and 5-HT7(a)

Receptor tyrosine kinases activate mitogen-activated protein (MAP) kinases through Ras, Raf-1, and MEK. Receptor tyrosine kinases can be transactivated by G protein-coupled receptors coupling to G(i) and G(q). The human G protein-coupled serotonin receptors 5-HT(4(b)) and 5-HT(7(a)) couple to G(s) and elevate intracellular cAMP. Certain G(s)-coupled receptors have been shown to activate MAP kinases through a protein kinase A- and Rap1-dependent pathway. We report the activation of the extracellular signal-regulated kinases (ERKs) 1 and 2 (p44 and p42 MAP kinase) through the human serotonin receptors 5-HT(4(b)) and 5-HT(7(a)) in COS-7 and human embryonic kidney HEK293 cells. In transfected HEK293 cells, 5-HT-induced activation of ERK1/2 is sensitive to H89, which indicates a role for protein kinase A. The observed activation of ERK1/2 does not require transactivation of epidermal growth factor receptors. Furthermore, 5-HT induced activation of both Ras and Rap1. Whereas the presence of Rap1GAP1 did not influence the 5-HT-mediated activation of ERK1/2, the activation of ERK1/2 was abolished in the presence of dominant negative Ras (RasN17). ERK1/2 activation was reduced in the presence of "dominant negative" Raf1 (RafS621A) and slightly reduced by dominant negative B-Raf, indicating the involvement of one or more Raf isoforms. These findings suggest that activation of ERK1/2 through the human G(s)-coupled serotonin receptors 5-HT(4(b)) and 5-HT(7(a)) in HEK293 cells is dependent on Ras, but independent of Rap1.     

Protein Kinase Cδ Mediates Neurogenic but Not Mitogenic Activation of Mitogen-Activated Protein Kinase in Neuronal Cells

In several neuronal cell systems, fibroblast-derived growth factor (FGF) and nerve growth factor (NGF) act as neurogenic agents, whereas epidermal growth factor (EGF) acts as a mitogen. The mechanisms responsible for these different cellular fates are unclear. We report here that although FGF, NGF, and EGF all activate mitogen-activated protein (MAP) kinase (extracellular signal-related kinase [ERK]) in rat hippocampal (H19-7) and pheochromocytoma (PC12) cells, the activation of ERK by the neurogenic agents FGF and NGF is dependent upon protein kinase Cdelta (PKCdelta), whereas ERK activation in response to the mitogenic EGF is independent of PKCdelta. Antisense PKCdelta oligonucleotides or the PKCdelta-specific inhibitor rottlerin inhibited FGF- and NGF-induced, but not EGF-induced, ERK activation. In contrast, EGF-induced ERK activation was inhibited by the phosphatidylinositol-3-kinase inhibitor wortmannin, which had no effect upon FGF-induced ERK activation. Rottlerin also inhibited the activation of MAP kinase kinase (MEK) in response to activated Raf, but had no effect upon c-Raf activity or ERK activation by activated MEK. These results indicate that PKCdelta functions either downstream from or in parallel with c-Raf, but upstream of MEK. Inhibition of PKCdelta also blocked neurite outgrowth induced by FGF and NGF in PC12 cells and by activated Raf in H19-7 cells, indicating a role for PKCdelta in the neurogenic effects of FGF, NGF, and Raf. Interestingly, the PKCdelta requirement is apparently cell type specific, since FGF-induced ERK activation was independent of PKCdelta in NIH 3T3 murine fibroblasts, in which FGF is a mitogen. These data demonstrate that PKCdelta contributes to growth factor specificity and response in neuronal cells and may also promote cell-type-specific differences in growth factor signaling.     

Regulation and role of Raf-1/B-Raf heterodimerization

The Ras-Raf-MEK-extracellular signal-regulated kinase (ERK) pathway participates in the control of many fundamental cellular processes including proliferation, survival, and differentiation. The pathway is deregulated in up to 30% of human cancers, often due to mutations in Ras and the B-Raf isoform. Raf-1 and B-Raf can form heterodimers, and this may be important for cellular transformation. Here, we have analyzed the biochemical and biological properties of Raf-1/B-Raf heterodimers. Isolated Raf-1/B-Raf heterodimers possessed a highly increased kinase activity compared to the respective homodimers or monomers. Heterodimers between wild-type Raf-1 and B-Raf mutants with low or no kinase activity still displayed elevated kinase activity, as did heterodimers between wild-type B-Raf and kinase-negative Raf-1. In contrast, heterodimers containing both kinase-negative Raf-1 and kinase-negative B-Raf were completely inactive, suggesting that the kinase activity of the heterodimer specifically originates from Raf and that either kinase-competent Raf isoform is sufficient to confer high catalytic activity to the heterodimer. In cell lines, Raf-1/B-Raf heterodimers were found at low levels. Heterodimerization was enhanced by 14-3-3 proteins and by mitogens independently of ERK. However, ERK-induced phosphorylation of B-Raf on T753 promoted the disassembly of Raf heterodimers, and the mutation of T753 prolonged growth factor-induced heterodimerization. The B-Raf T753A mutant enhanced differentiation of PC12 cells, which was previously shown to be dependent on sustained ERK signaling. Fine mapping of the interaction sites by peptide arrays suggested a complex mode of interaction involving multiple contact sites with a main Raf-1 binding site in B-Raf encompassing T753. In summary, our data suggest that Raf-1/B-Raf heterodimerization occurs as part of the physiological activation process and that the heterodimer has distinct biochemical properties that may be important for the regulation of some biological processes.