Insulin activates the Raf-1 protein kinase

Several growth factors and mitogens have been shown to activate the proto-oncogene product Raf-1 protein kinase in murine fibroblasts, apparently through a direct agonist-stimulated tyrosine phosphorylation of the Raf-1 protein. We investigated the possibility that insulin could also activate the Raf-1 kinase, since its receptor also contains an intrinsic insulin-activated protein tyrosine kinase activity. In several cell lines expressing relatively large numbers of insulin receptors, insulin rapidly stimulated the phosphorylation of immunoreactive Raf-1 protein. In H35 cells, a line of well differentiated rat hepatoma cells, the effect of insulin was maximal by 6 min and at 7 nM insulin and occurred normally in cells virtually completely depleted of protein kinase C activity. The insulin-stimulated increase in Raf-1 protein phosphorylation occurred concurrently with a 3-fold increase in Raf-1 protein kinase activity. However, phosphoamino acid analysis showed that only phosphoserine and a trace of phosphothreonine were present in the Raf-1 protein after insulin stimulation of the cells. This was true even when investigated at shorter times (4 min) after insulin stimulation and despite the use of phosphotyrosine phosphatase inhibitors. We conclude that insulin can rapidly activate the Raf-1 kinase in some insulin-sensitive cell types but that this activation probably occurs through a mechanism distinct from direct phosphorylation of the Raf-1 protein by the insulin receptor protein tyrosine kinase.     

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.     

Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase.

The Raf protein kinases function downstream of Ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC epsilon inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC epsilon stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC epsilon also stimulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKC alpha and PKC epsilon overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerol-regulated PKCs function as redundant activators of Raf-1 in vivo.     

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.     

Phosphorylation of Raf-1 by p21-activated kinase 1 and Src regulates Raf-1 autoinhibition

Exposure of cells to mitogens or growth factors stimulates Raf-1 activity through a complex mechanism that involves binding to active Ras, phosphorylation on multiple residues, and protein-protein interactions. Recently it was shown that the amino terminus of Raf-1 contains an autoregulatory domain that can inhibit its activity in Xenopus oocytes. In the present work we show that expression of the Raf-1 autoinhibitory domain blocks extracellular signal-regulated kinase 2 activation by the Raf-1 catalytic domain in mammalian cells. We also show that phosphorylation of Raf-1 on serine 338 by PAK1 and tyrosines 340 and 341 by Src relieves autoinhibition and that this occurs through a specific decrease in the binding of the Raf-1 regulatory domain to its catalytic domain. In addition, we demonstrate that phosphorylation of threonine 491 and serine 494, two phosphorylation sites in the catalytic domain that are required for Raf-1 activation, is unlikely to regulate autoinhibition. These results demonstrate that the autoinhibitory domain of Raf-1 is functional in mammalian cells and that its interaction with the Raf-1 catalytic domain is regulated by phosphorylation of serine 338 and tyrosines 340 and 341.     

Cilia movement regulates expression of the Raf-1 kinase inhibitor protein

Renal epithelial cell primary cilia act as mechanosensors in response to changes in luminal fluid flow. To determine the role of cilia bending in the mechanosensory function of cilia, we performed proteomic analysis of collecting duct cell lines with or without cilia that were kept stationary or rotated to stimulate cilia bending. Expression of the Raf-1 kinase inhibitor protein (RKIP), an inhibitor of the MAPK pathway, was significantly elevated in rotated cilia (+) cells. This was compared with RKIP levels in cilia (-) cells that were stationary or rotated as well as in cilia (+) cells that were stationary. This result was confirmed in cilia knockout adult mice that had lower renal RKIP levels compared with adult mice with cilia. Downstream of RKIP, expression of phosphorylated ERK was decreased only in cells that had cilia and were subjected to constant cilia bending. Furthermore, elevated RKIP levels were associated with reduced cell proliferation. Blockade of PKC abrogated ciliary bending-induced increases in RKIP. In summary, we found that ciliary movement may help control the expression of the Raf-1 kinase inhibitor protein and thus maintain cell differentiation. In terms of polycystic kidney disease, loss of cilia and therefore sensitivity to flow may lead to reduced RKIP levels, activation of the MAPK pathway, and contribute to the formation of cysts.     

Raf-1-associated protein phosphatase 2A as a positive regulator of kinase activation

The Raf-1 kinase plays a key role in relaying proliferation signals elicited by mitogens or oncogenes. Raf-1 is regulated by complex and incompletely understood mechanisms including phosphorylation. A number of studies have indicated that phosphorylation of serines 259 and 621 can inhibit the Raf-1 kinase. We show that both serines are hypophosphorylated during early mitogenic stimulation and that hypophosphorylation correlates with peak Raf-1 activation. Concentrations of okadaic acid that selectively inhibit protein phosphatase 2A (PP2A) induce phosphorylation of these residues and prevent maximal activation of the Raf-1 kinase. This effect is mediated via phosphorylation of serine 259. The PP2A core heterodimer forms complexes with Raf-1 in vivo and in vitro. These data identify PP2A as a positive regulator of Raf-1 activation and are the first indication that PP2A may support the activation of an associated kinase.     

Solution structure and backbone dynamics of human Raf-1 kinase inhibitor protein

Human Raf-1 kinase inhibitor protein (hRKIP) is a small multi-functional protein of 187 residues. It contains a conserved pocket, which binds a wide range of ligands from various small molecules to distinct proteins. To provide a structural basis for the ligand diversity of RKIP, we herein determined the solution structure of hRKIP, and analyzed its structural dynamics. In solution, hRKIP mainly comprises two antiparallel β sheets, two α helices and two 3₁₀ helices. NMR dynamic analysis reveals that the overall structure of hRKIP is rigid, but its C-terminal helix which is close to the ligand-binding site is mobile. In addition, residues around the ligand-binding pocket exhibit significant conformational exchange on the μs–ms timescale. Conformational flexibility may allow the ligand-binding pocket and the C-terminal helix to adopt various conformations to interact with different substrates. This work may shed light on the underlying molecular mechanisms of how hRKIP recognizes and binds diverse substrate ligands.     

Mechanism of protein kinase B activation by cyclic AMP-dependent protein kinase.

Activation of protein kinase B (PKB) by growth factors and hormones has been demonstrated to proceed via phosphatidylinositol 3-kinase (PI3-kinase). In this report, we show that PKB can also be activated by PKA (cyclic AMP [cAMP]-dependent protein kinase) through a PI3-kinase-independent pathway. Although this activation required phosphorylation of PKB, PKB is not likely to be a physiological substrate of PKA since a mutation in the sole PKA consensus phosphorylation site of PKB did not abolish PKA-induced activation of PKB. In addition, mechanistically, this activation was different from that of growth factors since it did not require phosphorylation of the S473 residue, which is essential for full PKB activation induced by insulin. These data were supported by the fact that mutation of residue S473 of PKB to alanine did not prevent it from being activated by forskolin. Moreover, phosphopeptide maps of overexpressed PKB from COS cells showed differences between insulin- and forskolin-stimulated cells that pointed to distinct activation mechanisms of PKB depending on whether insulin or cAMP was used. We looked at events downstream of PKB and found that PKA activation of PKB led to the phosphorylation and inhibition of glycogen synthase kinase-3 (GSK-3) activity, a known in vivo substrate of PKB. Overexpression of a dominant negative PKB led to the loss of inhibition of GSK-3 in both insulin- and forskolin-treated cells, demonstrating that PKB was responsible for this inhibition in both cases. Finally, we show by confocal microscopy that forskolin, similar to insulin, was able to induce translocation of PKB to the plasma membrane. This process was inhibited by high concentrations of wortmannin (300 nM), suggesting that forskolin-induced PKB movement may require phospholipids, which are probably not generated by class I or class III PI3-kinase. However, high concentrations of wortmannin did not abolish PKB activation, which demonstrates that translocation per se is not important for PKA-induced PKB activation.     

Raf-1 is activated by the p38 mitogen-activated protein kinase inhibitor, SB203580

SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imi dazole) is widely used as a specific inhibitor of p38 mitogen-activated protein kinase (MAPK). Here, we report that SB203580 activates the serine/threonine kinase Raf-1 in quiescent smooth muscle cells in a dose-dependent fashion. The concentrations of SB203580 required lie above those necessary to inhibit p38 MAPK and we were unable to detect basal levels of active p38 MAPK. SB203580 does not directly activate Raf-1 in vitro, and fails to activate Ras, MEK, and ERK in intact cells. In vitro, however, SB203580-stimulated Raf-1 activates MEK1 in a coupled assay. We conclude that activation of Raf-1 by SB203580 is not mediated by an inhibition of p38 MAPK, is Ras-independent, and is uncoupled from MEK/ERK signaling.     

Raf-1 and B-Raf promote protein kinase C theta interaction with BAD

PKCtheta regulates the proliferation, survival and differentiation of T-cells. Here we show that PKCtheta interacts with Raf-1 and B-Raf kinases. Raf-1 enhanced the kinase activity of associated PKCtheta, while PKCtheta reduced the catalytic activity of associated Raf-1. In contrast, B-Raf binding did not affect PKCtheta kinase activity, and PKCtheta did not change B-Raf activity. Coexpression of mutationally activated Raf-1 in cells enhanced the phosphorylation of T538 in the PKCtheta activation loop. PKCtheta and Raf cooperated in terms of binding to BAD, a pro-apoptotic Bcl-2 family protein that is inactivated by phosphorylation. While neither Raf-1 nor B-Raf could phosphorylate BAD, they enhanced the ability of PKCtheta to interact with BAD and to phosphorylate BAD in vitro and in vivo, suggesting a new role for Raf proteins in T-cells by targeting PKCtheta to interact with and phosphorylate BAD.     

Pharmacophore identification of Raf-1 kinase inhibitors

A three-dimensional pharmacophore model was developed based on 25 currently available Raf-1 kinase inhibitors. The best pharmacophore hypothesis (Hypo1), consisting of four chemical features (one hydrogen-bond acceptor, one hydrogen-bond donor, and two hydrophobic groups), has a correlation coefficient of 0.972. The results of our study provide a valuable tool in designing new leads with desired biological activity by virtual screening.     

Cyclic AMP-dependent kinase regulates Raf-1 kinase mainly by phosphorylation of serine 259

The Raf-1 kinase activates the ERK (extracellular-signal-regulated kinase) pathway. The cyclic AMP (cAMP)-dependent protein kinase (PKA) can inhibit Raf-1 by direct phosphorylation. We have mapped all cAMP-induced phosphorylation sites in Raf-1, showing that serines 43, 259, and 621 are phosphorylated by PKA in vitro and induced by cAMP in vivo. Serine 43 phosphorylation decreased the binding to Ras in serum-starved but not in mitogen-stimulated cells. However, the kinase activity of a RafS43A mutant was fully inhibited by PKA. Mutation of serine 259 increased the basal Raf-1 activity and rendered it largely resistant to inhibition by PKA. cAMP increased Raf-1 serine 259 phosphorylation in a PKA-dependent manner with kinetics that correlated with ERK deactivation. PKA also decreased Raf-1 serine 338 phosphorylation of Raf-1, previously shown to be required for Raf-1 activation. Serine 338 phosphorylation of a RafS259A mutant was unaffected by PKA. Using RafS259 mutants we also demonstrate that Raf-1 is the sole target for PKA inhibition of ERK and ERK-induced gene expression, and that Raf-1 inhibition is mediated mainly through serine 259 phosphorylation.     

Mechanism of calmodulin inhibition of cAMP-dependent protein kinase activation of phosphorylation kinase

The activation of phosphorylase kinase (EC 2.7.1.38; ATP:phosphorylase b phosphotransferase) by the catalytic subunit of cAMP-dependent protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) is inhibited by calmodulin. The mechanism of that inhibition has been studied by kinetic measurements of the interactions of the three proteins. The binding constant for calmodulin with phosphorylase kinase was found to be 90 nM when measured by fluorescence polarization spectroscopy. Glycerol gradient centrifugation studies indicated that 1 mol of calmodulin was bound to each phosphorylase kinase. Phosphorylation of the phosphorylase kinase did not reduce the amount of calmodulin bound. Kinetic studies of the activity of the catalytic subunit of cAMP-dependent protein kinase on phosphorylase kinase as a function of phosphorylase kinase and calmodulin concentrations were performed. The results of those studies were compared with mathematical models of four different modes of inhibition: competitive, noncompetitive, substrate depletion, and inhibition by a complex between phosphorylase kinase and calmodulin. The data conform best to the model in which the inhibitory species is a complex of phosphorylase kinase and calmodulin. The complex apparently competes with the substrate, phosphorylase kinase, which does not have exogenous calmodulin bound to it. In contrast, the phosphorylation of the synthetic phosphate acceptor peptide, Kemptide, is not inhibited by calmodulin.     

Rheb interacts with Raf-1 kinase and may function to integrate growth factor- and protein kinase A-dependent signals.

Rheb is a recently described member of the Ras family that was originally identified as an immediate-early gene in brain but is also widely expressed in other tissues. Here we demonstrate that Rheb interacts with and appears to regulate Raf-1 kinase, an essential component of the H-Ras signaling pathway. In direct contrast to H-Ras, however, the interaction of Rheb with Raf-1 is potentiated by growth factors in combination with agents that increase cyclic AMP (cAMP) levels. Protein kinase A-dependent phosphorylation of serine 43 within the regulatory domain of Raf-1 reciprocally potentiates its interaction with Rheb and decreases its interaction with H-Ras. A single amino acid in the G2 effector domain is critical for the differential properties of Rheb. Since Rheb is an immediate-early gene, our studies suggest that Rheb functions in concert with H-Ras to dynamically integrate cAMP and growth factor signaling.     

Calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates Raf-1 at serine 338 and mediates Ras-stimulated Raf-1 activation

The calcium/calmodulin-dependent kinase II (CaMKII) participates with Ras to Raf-1 activation, and it is necessary for activation of the extracellular signal-regulated kinase (ERK) by different factors in epithelial and mesenchimal cells. Raf-1 activation is a complex multistep process, and its maximal activation is achieved by phosphorylation at Y341 by Src and at S338 by other kinase/s. Although early data proposed the involvement of p21-activated kinase 3 (Pak3), the kinase phosphorylating S338 remains to be definitively identified. In this study, we verified the hypothesis that CaMKII phosphorylates Raf-1 at Ser338. To do so, we determined the role of CaMKII in Raf-1 and ERK activation by oncogenic Ras and other factors. Serum, fibronectin, Src^Y527 and Ras^V12 activated CaMKII and ERK, at different extents. The inhibition of CaMKII attenuated Raf-1 and ERK activation by all these factors. CaMKII was also necessary for the phosphorylation of Raf-1 at S338 by serum, fibronectin and Ras. Conversely, inhibition of Pak3 activation by blocking phosphatidylinositol 3-kinase was ineffective. The direct phosphorylation of S338 Raf-1 by CaMKII was demonstrated in vitro by interaction of purified kinases. These results demonstrate that Ras activates CaMKII, which, in turn, phosphorylates Raf-1 at S338 and participates in ERK activation upon different stimuli.     

Protein kinase A blocks Raf-1 activity by stimulating 14-3-3 binding and blocking Raf-1 interaction with Ras

Cyclic AMP (cAMP) blocks Raf-1 activation by stimulating its phosphorylation on serine 43 (Ser43), serine 233 (Ser233), and serine 259 (Ser259). We show here that phosphorylation of all three sites blocks Raf-1 binding to Ras.GTP in vivo and that cAMP stimulates binding of 14-3-3 proteins to Ser233 and Ser259. We also show that Raf-1 and protein kinase A (PKA) form a complex in vivo that is disrupted by cAMP and that ablation of PKA by use of small interfering RNA blocks phosphorylation by cAMP. The ability of PKA to block Raf-1 activation is ablated by the PKA inhibitor H89. These studies suggest that Raf-1 and cAMP form a signaling complex in cells. Upon activation of PKA, Raf-1 is phosphorylated and 14-3-3 binds, blocking Raf-1 recruitment to the plasma membrane and preventing its activation.