Glucagon and p21 ras enhance the phosphorylation of the same 38-kilodalton membrane protein from rat liver cells.

We had reported earlier the enhanced phosphorylation of a 38-kilodalton protein (p38) in rat liver plasma membrane by ras proteins. Now we show that glucagon increased the phosphorylation of the same protein. The nature and site(s) of phosphorylation were the same as those for the ras proteins. Both ATP and GTP could donate phosphate for the phosphorylation of p38. The stimulation of p38 phosphorylation by glucagon was guanine nucleotide dependent. This observation, together with our data on the stimulation of p38 phosphorylation by AIF4-, suggest the involvement of G proteins in the reaction. We also showed that glucagon stimulates the phosphorylation of p38 in vivo.     

p38alpha antagonizes p38gamma activity through c-Jun-dependent ubiquitin-proteasome pathways in regulating Ras transformation and stress response

p38 MAPK family consists of four isoform proteins (alpha, beta, gamma, and delta) that are activated by the same stimuli, but the information about how these proteins act together to yield a biological response is missing. Here we show a feed-forward mechanism by which p38alpha may regulate Ras transformation and stress response through depleting its family member p38gamma protein via c-Jun-dependent ubiquitin-proteasome pathways. Analyses of MAPK kinase 6 (MKK6)-p38 fusion proteins showed that constitutively active p38alpha (MKK6-p38alpha) and p38gamma (MKK6-p38gamma) stimulates and inhibits c-Jun phosphorylation respectively, leading to a distinct AP-1 regulation. Depending on cell type and/or stimuli, p38alpha phosphorylation results in either Ras-transformation inhibition or a cell-death escalation that invariably couples with a decrease in p38gamma protein expression. p38gamma, on the other hand, increases Ras-dependent growth or inhibits stress induced cell-death independent of phosphorylation. In cells expressing both proteins, p38alpha phosphorylation decreases p38gamma protein expression, whereas its inhibition increases cellular p38gamma concentrations, indicating an active role of p38alpha phosphorylation in negatively regulating p38gamma protein expression. Mechanistic analyses show that p38alpha requires c-Jun activation to deplete p38gamma proteins by ubiquitin-proteasome pathways. These results suggest that p38alpha may, upon phosphorylation, act as a gatekeeper of the p38 MAPK family to yield a coordinative biological response through disrupting its antagonistic p38gamma family protein.     

Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation

Components of the ras signaling pathway contribute to activation of cellular p53. In MCF-7 cells, p38 kinase activated p53 more effectively than other members of the ras pathway. p53 and p38 kinase exist in the same physical complex, and co-expression of p38 stabilized p53 protein. In vitro, p38 kinase phosphorylated p53 at Ser33 and Ser46, a newly identified site. Mutation of these sites decreased p53-mediated and UV-induced apoptosis, and the reduction correlated with total abrogation of UV-induced phosphorylation on Ser37 and a significant decrease in Ser15 phosphorylation in mutant p53 containing alanine at Ser33 and Ser46. Inhibition of p38 activation after UV irradiation decreased phosphorylation of Ser33, Ser37 and Ser15, and also markedly reduced UV-induced apoptosis in a p53-dependent manner. These results suggest that p38 kinase plays a prominent role in an integrated regulation of N-terminal phosphorylation that regulates p53-mediated apoptosis after UV radiation.     

p38 MAP kinase mediates apoptosis through phosphorylation of BimEL at Ser-65

The stress-activated c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein (MAP) kinase (p38) regulate apoptosis induced by several forms of cellular insults. Potential targets for these kinases include members of the Bcl-2 family proteins, which mediate apoptosis generated through the mitochondria-initiated, intrinsic cell death pathway. Indeed, the activities of several Bcl-2 family proteins, both pro- and anti-apoptotic, are controlled by JNK phosphorylation. For example, the pro-apoptotic activity of Bim(EL), a member of the Bcl-2 family, is stimulated by JNK phosphorylation at Ser-65. In contrast, there is no reported evidence that p38-induced apoptosis is due to direct phosphorylation of Bcl-2 family proteins. Here we report evidence that sodium arsenite-induced apoptosis in PC12 cells may be due to direct phosphorylation of Bim(EL) at Ser-65 by p38. This conclusion is supported by data showing that ectopic expression of a wild type, but not a non-phosphorylatable S65A mutant of Bim(EL), potentiates sodium arsenite-induced apoptosis and by experiments showing direct phosphorylation of Bim(EL) at Ser-65 by p38 in vitro. Furthermore, sodium arsenite induced Bim(EL) phosphorylation at Ser-65, which was blocked by p38 inhibition. This study provides the first example whereby p38 induces apoptosis by phosphorylating a member of the Bcl-2 family and illustrates that phosphorylation of Bim(EL) on Ser-65 may be a common regulatory point for cell death induced by both JNK and p38 pathways.     

Multiple activation mechanisms of p38alpha mitogen-activated protein kinase

The p38alpha MAPK participates in a variety of biological processes. Activation of p38alpha is mediated by phosphorylation on specific regulatory tyrosine and threonine sites, and the three dual kinases, MAPK kinase 3 (MKK3), MKK4, and MKK6, are known to be the upstream activators of p38alpha. In addition to activation by upstream kinases, p38alpha can autoactivate when interacting with transforming growth factor-beta-activated protein kinase 1-binding protein 1 (TAB1). Here we used MKK3 and MKK6 double knock-out (MKK3/6 DKO) and MKK4/7 DKO mouse embryonic fibroblast (MEF) cells to examine activation mechanisms of p38alpha. We confirmed that the MKK3/6 pathway is a primary mechanism for p38alpha phosphorylation in MEF cells, and we also showed the presence of other p38alpha activation pathways. We show that TAB1-mediated p38alpha phosphorylation in MEF cells did not need MKK3/4/6, and it accounted for a small portion of the total p38alpha phosphorylation that was induced by hyperosmolarity and anisomycin. We observed that a portion of peroxynitrite-induced phospho-p38alpha is associated with an approximately 85-kDa disulfide complex in wild-type MEF cells. Peroxynitrite-induced phosphorylation of p38alpha in the approximately 85-kDa complex is independent from MKK3/6 because only phospho-p38alpha not associated with the disulfide complex was diminished in MKK3/6 DKO cells. In addition, our data suggest interference among different pathways because TAB1 had an inhibitory effect on p38alpha phosphorylation in the peroxynitrite-induced approximately 85-kDa complex. Mutagenesis analysis of the cysteines in p38alpha revealed that no disulfide bond forms between p38alpha and other proteins in the approximately 85-kDa complex, suggesting it is a p38alpha binding partner(s) that forms disulfide bonds, which enable it to bind to p38alpha. Therefore, multiple mechanisms of p38alpha activation exist that can influence each other, be simultaneously activated by a given stimulus, and/or be selectively used by different stimuli in a cell type-specific manner.     

Nerve growth factor stimulates the tyrosine phosphorylation of a 38-kDa protein that specifically associates with the src homology domain of phospholipase C-gamma 1.

The cellular actions of nerve growth factor (NGF) involve changes in protein phosphorylation, initiated by the binding and subsequent activation of its tyrosine kinase receptor, the trk protooncogene (pp140c-trk). Upon exposure to NGF, a 38-kDa tyrosine-phosphorylated protein (pp38) is identified in both PC-12 pheochromocytoma cells and NIH3T3 cells transfected with the full-length human pp140c-trk cDNA (3T3-c-trk) that is specifically coimmunoprecipitated with pp140c-trk or phosphatidylinositol-phospholipase C (PLC)-gamma 1. In both PC-12 and 3T3-c-trk cells, NGF rapidly stimulates the association of pp140c-trk and pp38 with a fusion protein containing the src homology (SH) domains of PLC gamma 1. This phosphorylation and subsequent association are specific for NGF, since epidermal growth factor, platelet-derived growth factor, and insulin do not stimulate the tyrosine phosphorylation of these proteins or their association with the PLC gamma 1 SH domains, although the receptors for these growth factors do undergo tyrosine phosphorylation and association with the PLC-gamma 1 fusion protein under these conditions. Furthermore, the NGF-dependent pp38-SH binding is specific for the SH2 domains of PLC-gamma 1, since the phosphoprotein does not bind to fusion proteins containing SH domains of ras GTPase-activating protein or the p85 subunit of phosphatidylinositol 3 kinase. Both amino- and carboxyl-terminal SH2 domains of PLC-gamma 1 are necessary for the association of pp38 with PLC-gamma 1, although each SH2 domain is sufficient for the association of pp140c-trk with PLC-gamma 1. In both PC-12 and 3T3-c-trk cells, the phosphorylation and association of pp38 with PLC gamma 1 is rapid, occurring maximally at 1 min and declining thereafter. Moreover, this effect of NGF is dose-dependent over a physiological concentration of the growth factor. The specificity and rapidity of pp38 phosphorylation and its association with PLC-gamma 1 suggest that it may be an important component in signal transduction for NGF.     

Sequential activation of the MEK-extracellular signal-regulated kinase and MKK3/6-p38 mitogen-activated protein kinase pathways mediates oncogenic ras-induced premature senescence

In primary mammalian cells, oncogenic ras induces premature senescence, depending on an active MEK-extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. It has been unclear how activation of the mitogenic MEK-ERK pathway by ras can confer growth inhibition. In this study, we have found that the stress-activated MAPK, p38, is also activated during the onset of ras-induced senescence in primary human fibroblasts. Constitutive activation of p38 by active MKK3 or MKK6 induces senescence. Oncogenic ras fails to provoke senescence when p38 activity is inhibited, suggesting that p38 activation is essential for ras-induced senescence. Furthermore, we have demonstrated that p38 activity is stimulated by ras as a result of an activated MEK-ERK pathway. Following activation of MEK and ERK, expression of oncogenic ras leads to the accumulation of active MKK3/6 and p38 activation in a MEK-dependent fashion and subsequently induces senescence. Active MEK1 induces the same set of changes and provokes senescence relying on active p38. Therefore, oncogenic ras provokes premature senescence by sequentially activating the MEK-ERK and MKK3/6-p38 pathways in normal, primary cells. These studies have defined the molecular events within the ras signaling cascade that lead to premature senescence and, thus, have provided new insights into how ras confers oncogenic transformation in primary cells.     

Smooth muscle actin determines mechanical force-induced p38 activation

The mitogen-activated protein kinase p38 is activated by mechanical force, but the cellular elements that mediate force-induced p38 phosphorylation are not defined. As alpha-smooth muscle actin (SMA) is an actin isoform associated with force generation in fibroblasts, we asked if SMA participates in the activation of p38 by force. Tensile forces (0.65 pn/mum(2)) generated by magnetic fields were applied to collagen-coated magnetite beads bound to Rat-2 cells. Immunoblotting showed that p38alpha was the predominant p38 isoform. Analysis of bead-associated proteins demonstrated that SMA enrichment of collagen receptor complexes required the alpha2beta1 integrin. SMA was present almost entirely as filaments. Swinholide depolymerized SMA filaments and blocked force-induced p38 phosphorylation and force-induced increases of SMA. Knockdown of SMA (70% reduction) using RNA interference did not affect beta-actin but inhibited force-induced p38 phosphorylation by 50%. Inhibition of Rho kinase blocked SMA filament assembly, force-induced increases of SMA, and force-induced p38 activation. Force application increased SMA content and enhanced the association of phosphorylated p38 with SMA filaments. Blockade of p38 phosphorylation by SB203586 abrogated force-induced increases of SMA. In cells transfected with SMA promoter-beta-galactosidase fusion constructs, co-transfection with constitutively active p38 or MKK6 increased SMA promoter activity by 2.5-3-fold. Dominant negative p38 blocked force-induced activation of the SMA promoter. In SMA negative cells, there was no force-induced p38 phosphorylation. We conclude that force-induced p38 phosphorylation is dependent on an SMA filament-dependent pathway that uses a feed-forward amplification loop to synergize force-induced SMA expression with p38 activation.     

Human caco-2 motility redistributes FAK and paxillin and activates p38 MAPK in a matrix-dependent manner

The signals involved in restitution during mucosal healing are poorly understood. We compared focal adhesion kinase (FAK) and paxillin protein and phosphorylation, extracellular signal-regulated kinase (ERK) 1, ERK2, and p38 activation, as well as FAK and paxillin organization in static and migrating human intestinal Caco-2 cells on matrix proteins and anionically derivatized polystyrene dishes (tissue culture plastic). We also studied effects of FAK, ERK, and p38 blockade in a monolayer-wounding model. Compared with static cells, cells migrating across matrix proteins matrix-dependently decreased membrane/cytoskeletal FAK and paxillin and cytosolic FAK. Tyrosine phosphorylated FAK and paxillin changed proportionately to FAK and paxillin protein. Conversely, cells migrating on plastic increased FAK and paxillin protein and phosphorylation. Migration matrix-dependently activated p38 and inactivated ERK1 and ERK2. Total p38, ERK1, and ERK2 did not change. Caco-2 motility was inhibited by transfection of FRNK (the COOH-terminal region of FAK) and PD-98059, a mitogen-activated protein kinase-ERK kinase inhibitor, but not by SB-203580, a p38 inhibitor, suggesting that FAK and ERK modulate Caco-2 migration. In contrast to adhesion-induced phosphorylation, matrix may regulate motile intestinal epithelial cells by altering amounts and distribution of focal adhesion plaque proteins available for phosphorylation as well as by p38 activation and ERK inactivation. Motility across plastic differs from migration across matrix.     

Bcl-2 Phosphorylation by p38 MAPK: identification of target sites and biologic consequences

The antiapoptotic role of Bcl-2 can be regulated by its phosphorylation in serine and threonine residues located in a nonstructured loop that links BH3 and BH4 domains. p38 MAPK has been identified as one of the kinases able to mediate such phosphorylation, through direct interaction with Bcl-2 protein in the mitochondrial compartment. In this study, we identify, by using mass spectrometry techniques and specific anti-phosphopeptide antibodies, Ser(87) and Thr(56) as the Bcl-2 residues phosphorylated by p38 MAPK and show that phosphorylation of these residues is always associated with a decrease in the antiapoptotic potential of Bcl-2 protein. Furthermore, we obtained evidence that p38 MAPK-induced Bcl-2 phosphorylation plays a key role in the early events following serum deprivation in embryonic fibroblasts. Both cytochrome c release and caspase activation triggered by p38 MAPK activation and Bcl-2 phosphorylation are absent in embryonic fibroblasts from p38alpha knock-out mice (p38alpha(-/-) MEF), whereas they occur within 12 h of serum withdrawal in p38alpha(+/+) MEF; moreover, they can be prevented by p38 MAPK inhibitors and are not associated with the synthesis of the proapoptotic proteins Bax and Fas. Thus, Bcl-2 phosphorylation by activated p38 MAPK is a key event in the early induction of apoptosis under conditions of cellular stress.     

Sodium nitroprusside activates p38 mitogen activated protein kinase through a cGMP/PKG independent mechanism

The objective of this study was to understand the mechanism of action of nitric oxide (NO) in the heart by determining whether nitric oxide (NO) released from sodium nitroprusside (SNP) induces p38 mitogen activated protein kinase (p38 MAPK) phosphorylation and whether this is mediated through a cyclic GMP (cGMP)/protein kinase G (PKG) pathway. p38 MAPK activation was examined by Western blotting of whole cell lysates of embryonic chick cardiomyocytes with antibodies specific to the native or phosphorylated forms of p38 MAPK. SNP, 1 mM, which released significant amounts of NO as determined by Griess reaction, induced p38 MAPK phosphorylation that was apparent within 10 min, was significantly (p<0.05) greater than control at 60 min and remained higher than initial levels up to the 4 h end point of the experiment. This could not be attributed to hydrogen peroxide release from SNP as catalase did not affect SNP-induced p38 MAPK phosphorylation. SB202190, a relatively selective inhibitor of p38 MAPK, mainly p38alpha MAPK, inhibited SNP-induced p38 MAPK phosphorylation. SNP-induced p38 MAPK phosphorylation was not altered by pre-treatment with the PKG inhibitor KT 5823 or by ODQ a potent and selective inhibitor of NO-sensitive guanylyl cyclase. p38 MAPK phosphorylation was not induced by the cell permeable cGMP analogue, 8-Br-cGMP. In summary, considering that new therapeutic strategies aimed at NO and p38 MAPK are being considered for myocardial injury and heart failure, these data demonstrate that SNP induces p38 MAPK phosphorylation through a pathway that is independent of NO-induced activation of cGMP/PKG pathways and suggest that non cGMP/PKG regulatory proteins leading to p38 MAPK phosphorylation merit further investigation to address this therapeutic target.     

Regulatory mechanisms for ras proteins.

The proteins encoded by the ras proto-oncogenes play critical roles in normal cellular growth, differentiation and development in addition to their potential for malignant transformation. Several proteins that are involved in the control of the activity of p21ras have now been characterised. p120GAP stimulates the GTPase activity of p21ras and hence acts as a negative regulator of ras proteins. It may be controlled by tyrosine phosphorylation or association with tyrosine phosphorylated proteins. The neurofibromatosis type 1 (NF 1) gene also encodes a potential GTPase activating protein which is likely to be subject to a different control mechanism. Guanosine nucleotide exchange factors for p21ras have now been identified: these may be positive regulators of ras protein function. It appears that p21ras is subject to rapid regulation by several distinct mechanisms which are likely to vary in different cell types; the ras proteins are thereby able to act as very sensitive cellular monitors of the extracellular environment.     

Regulation of PRAK subcellular location by p38 MAP kinases

The p38 mitogen-activated protein kinase (MAPK) pathway plays an important role in cellular responses to inflammatory stimuli and environmental stress. p38 regulated/activated protein kinase (PRAK, also known as mitogen-activated protein kinase activated protein kinase 5 [MAPKAPK5]) functions downstream of p38alpha and p38beta in mediating the signaling of the p38 pathway. Immunostaining revealed that endogenous PRAK was predominantly localized in the cytoplasm. Interestingly, ectopically expressed PRAK was localized in the nucleus and can be redistributed by coexpression of p38alpha or p38beta to the locations of p38alpha and p38beta. Mutations in the docking groove on p38alpha/p38beta, or the p38-docking site in PRAK, disrupted the PRAK-p38 interaction and impaired the ability of p38alpha and p38beta to redistribute ectopically expressed PRAK, indicating that the location of PRAK could be controlled by its docking interaction with p38alpha and p38beta. Although the majority of PRAK molecules were detected in the cytoplasm, PRAK is consistently shuttling between the cytoplasm and the nucleus. A sequence analysis of PRAK shows that PRAK contains both a putative nuclear export sequence (NES) and a nuclear localization sequence (NLS). The shuttling of PRAK requires NES and NLS motifs in PRAK and can be regulated through cellular activation induced by stress stimuli. The nuclear content of PRAK was reduced after stimulation, which resulted from a decrease in the nuclear import of PRAK and an increase in the nuclear export of PRAK. The nuclear import of PRAK is independent from p38 activation, but the nuclear export requires p38-mediated phosphorylation of PRAK. Thus, the subcellular distribution of PRAK is determined by multiple factors including its own NES and NLS, docking interactions between PRAK and docking proteins, phosphorylation of PRAK, and cellular activation status. The p38 MAPKs not only regulate PRAK activity and PRAK activation-related translocation, but also dock PRAK to selected subcellular locations in resting cells.     

PRAK is essential for ras-induced senescence and tumor suppression

Like apoptosis, oncogene-induced senescence is a barrier to tumor development. However, relatively little is known about the signaling pathways mediating the senescence response. p38-regulated/activated protein kinase (PRAK) is a p38 MAPK substrate whose physiological functions are poorly understood. Here we describe a role for PRAK in tumor suppression by demonstrating that PRAK mediates senescence upon activation by p38 in response to oncogenic ras. PRAK deficiency in mice enhances DMBA-induced skin carcinogenesis, coinciding with compromised senescence induction. In primary cells, inactivation of PRAK prevents senescence and promotes oncogenic transformation. Furthermore, we show that PRAK activates p53 by direct phosphorylation. We propose that phosphorylation of p53 by PRAK following activation of p38 MAPK by ras plays an important role in ras-induced senescence and tumor suppression.     

Platelet-derived growth factor stimulation of GTPase-activating protein tyrosine phosphorylation in control and c-H-ras-expressing NIH 3T3 cells correlates with p21ras activation.

Platelet-derived growth factor (PDGF) stimulation of NIH 3T3 cells leads to the rapid tyrosine phosphorylation of the GTPase-activating protein (GAP) and an associated 64- to 62-kDa tyrosine-phosphorylated protein (p64/62). To assess the functions of these proteins, we evaluated their phosphorylation state in normal NIH 3T3 cells as well as in cells transformed by oncogenically activated v-H-ras or overexpression of c-H-ras genes. No significant GAP tyrosine phosphorylation was observed in unstimulated cultures, while PDGF-BB induced rapid tyrosine phosphorylation of GAP in all cell lines analyzed. In NIH 3T3 cells, we found that PDGF stimulation led to the recovery of between 37 and 52% of GAP molecules by immunoprecipitation with monoclonal antiphosphotyrosine antibodies. Furthermore, PDGF exposure led to a rapid and sustained increase in the levels of p21ras bound to GTP, with kinetics similar to those observed for GAP tyrosine phosphorylation. The PDGF-induced increases in GTP-bound p21ras in NIH 3T3 cells were comparable to the steady-state level observed in serum-starved c-H-ras-overexpressing transformants, conditions in which these cells maintained high rates of DNA synthesis. These results imply that the level of p21ras activation following PDGF stimulation of NIH 3T3 cells is sufficient to support mitogenic stimulation. Addition of PDGF to c-H-ras-overexpressing cells also resulted in a rapid and sustained increase in GTP-bound p21ras. In these cells GAP, but not p64/62, showed increased tyrosine phosphorylation, with kinetics similar to those observed for increased GTP-bound p21ras. All of these findings support a role for GAP tyrosine phosphorylation in p21ras activation and mitogenic signaling.     

LZAP inhibits p38 MAPK (p38) phosphorylation and activity by facilitating p38 association with the wild-type p53 induced phosphatase 1 (WIP1)

LZAP (Cdk5rap3, C53) is a putative tumor suppressor that inhibits RelA, Chk1 and Chk2 and activates p53. LZAP is lost in a portion of human head and neck squamous cell carcinoma and experimental loss of LZAP expression is associated with enhanced invasion, xenograft tumor growth and angiogenesis. p38 MAPK can increase or decrease proliferation and cell death depending on cellular context. LZAP has no known enzymatic activity, implying that its biological functions are likely mediated by its protein-protein interactions. To gain further insight into LZAP activities, we searched for LZAP-associated proteins (LAPs). Here we show that the LZAP binds p38, alters p38 cellular localization, and inhibits basal and cytokine-stimulated p38 activity. Expression of LZAP inhibits p38 phosphorylation in a dose-dependent fashion while loss of LZAP enhances phosphorylation and activation with resultant phosphorylation of p38 downstream targets. Mechanistically, the ability of LZAP to alter p38 phosphorylation depended, at least partially, on the p38 phosphatase, Wip1. Expression of LZAP increased both LZAP and Wip1 binding to p38. Taken together, these data suggest that LZAP activity includes inhibition of p38 phosphorylation and activation.     

Phosphoproteomic analysis of thrombin- and p38 MAPK-regulated signaling networks in endothelial cells

Endothelial dysfunction is a hallmark of inflammation and is mediated by inflammatory factors that signal through G protein–coupled receptors including protease-activated receptor-1 (PAR1). PAR1, a receptor for thrombin, signals via the small GTPase RhoA and myosin light chain intermediates to facilitate endothelial barrier permeability. PAR1 also induces endothelial barrier disruption through a p38 mitogen-activated protein kinase–dependent pathway, which does not integrate into the RhoA/MLC pathway; however, the PAR1-p38 signaling pathways that promote endothelial dysfunction remain poorly defined. To identify effectors of this pathway, we performed a global phosphoproteome analysis of thrombin signaling regulated by p38 in human cultured endothelial cells using multiplexed quantitative mass spectrometry. We identified 5491 unique phosphopeptides and 2317 phosphoproteins, four distinct dynamic phosphoproteome profiles of thrombin-p38 signaling, and an enrichment of biological functions associated with endothelial dysfunction, including modulators of endothelial barrier disruption and a subset of kinases predicted to regulate p38-dependent thrombin signaling. Using available antibodies to detect identified phosphosites of key p38-regulated proteins, we discovered that inhibition of p38 activity and siRNA-targeted depletion of the p38α isoform increased basal phosphorylation of extracellular signal–regulated protein kinase 1/2, resulting in amplified thrombin-stimulated extracellular signal–regulated protein kinase 1/2 phosphorylation that was dependent on PAR1. We also discovered a role for p38 in the phosphorylation of α-catenin, a component of adherens junctions, suggesting that this phosphorylation may function as an important regulatory process. Taken together, these studies define a rich array of thrombin- and p38-regulated candidate proteins that may serve important roles in endothelial dysfunction.     

The p38 SAPK pathway is required for Ha-ras induced in vitro invasion of NIH3T3 cells

Constitutive activation of the ras oncoprotein plays a critical role in cancer invasion and metastasis. Particularly, ras-related protease expression such as the serine protease urokinase plasminogen activator (u-PA) has been implicated in mediating cancer cell invasion. Previous studies have shown that ras-mediated u-PA expression is regulated through the mitogen- (MAPK) and stress-activated protein kinase (SAPK) signal transduction pathways extracellular signal-regulated kinase (ERK) and c-Jun-activating kinase (JNK). We therefore asked the question, if ras-related cell invasion might additionally require the third MAPK/SAPK signal transduction cascade, p38. Indeed, we found that ras induces invasion based on the activation of certain p38 protein kinase isoforms, in particular, p38alpha. Moreover, ras activation through transient or stable expression of a Ha-rasEJ mutant induced the expression of u-PA. This was found to be a consequence of an increase of u-PA m-RNA, which was paralleled by only a modest activation of the u-PA promoter. In conclusion, we provide evidence for the requirement of a novel ras-p38alpha-u-PA pathway for ras-dependent cellular invasion.