Na/H exchange regulatory factor 1, a novel AKT-associating protein, regulates extracellular signal-regulated kinase signaling through a B-Raf-mediated pathway

Na/H exchange regulatory factor 1 (NHERF1) is a scaffolding protein that regulates signaling and trafficking of several G protein-coupled receptors (GPCRs), including the parathyroid hormone receptor (PTH1R). GPCRs activate extracellular signal-regulated kinase (ERK)1/2 through different mechanisms. Here, we characterized NHERF1 regulation of PTH1R-stimulated ERK1/2. Parathyroid hormone (PTH) stimulated ERK1/2 phosphorylation by a protein kinase A (PKA)-dependent, but protein kinase C-, cyclic adenosine 5'-monophosphate-, and Rap1-independent pathway in Chinese hamster ovary cells stably transfected with the PTH1R and engineered to express NHERF1 under the control of tetracycline. NHERF1 blocked PTH-induced ERK1/2 phosphorylation downstream of PKA. This suggested that NHERF1 inhibitory effects on ERK1/2 occur at a postreceptor locus. Forskolin activated ERK1/2, and this effect was blocked by NHERF1. NHERF1 interacted with AKT and inhibited ERK1/2 activation by decreasing the stimulatory effect of 14-3-3 binding to B-Raf, while increasing the inhibitory influence of AKT negative regulation on ERK1/2 activation. This novel regulatory mechanism provides a new model by which cytoplasmic adapter proteins modulate ERK1/2 activation through a receptor-independent mechanism involving B-Raf.     

ERK5 Activation by Gq-Coupled Muscarinic Receptors Is Independent of Receptor Internalization and β-Arrestin Recruitment

G-protein-coupled receptors (GPCRs) are known to activate both G protein- and β-arrestin-dependent signalling cascades. The initiation of mitogen-activated protein kinase (MAPK) pathways is a key downstream event in the control of cellular functions including proliferation, differentiation, migration and apoptosis. Both G proteins and β-arrestins have been reported to mediate context-specific activation of ERK1/2, p38 and JNK MAPKs. Recently, the activation of ERK5 MAPK by Gq-coupled receptors has been described to involve a direct interaction between Gαq and two novel effectors, PKCζ and MEK5. However, the possible contribution of β-arrestin towards this pathway has not yet been addressed. In the present work we sought to investigate the role of receptor internalization processes and β-arrestin recruitment in the activation of ERK5 by Gq-coupled GPCRs. Our results show that ERK5 activation is independent of M1 or M3 muscarinic receptor internalization. Furthermore, we demonstrate that phosphorylation-deficient muscarinic M1 and M3 receptors are still able to fully activate the ERK5 pathway, despite their reported inability to recruit β-arrestins. Indeed, the overexpression of Gαq, but not that of β-arrestin1 or β-arrestin2, was found to potently enhance ERK5 activation by GPCRs, whereas silencing of β-arrestin2 expression did not affect the activation of this pathway. Finally, we show that a β-arrestin-biased mutant form of angiotensin II (SII; Sar1-Ile4-Ile8 AngII) failed to promote ERK5 phosphorylation in primary cardiac fibroblasts, as compared to the natural ligand. Overall, this study shows that the activation of ERK5 MAPK by model Gq-coupled GPCRs does not depend on receptor internalization, β-arrestin recruitment or receptor phosphorylation but rather is dependent on Gαq-signalling.     

G protein-coupled receptor kinase 2 (GRK2) is a Rho-activated scaffold protein for the ERK MAP kinase cascade

The G protein-coupled receptor kinases (GRKs) are best known for their role in phosphorylating and desensitising G protein-coupled receptors (GPCRs). The GRKs also regulate signalling downstream of other families of receptors and have a number of non-receptor substrates and binding partners. Here we identify RhoA_GTP and Raf1 as novel binding partners of GRK2 and report a previously unsuspected function for this kinase. GRK2 is a RhoA effector that serves as a RhoA-activated scaffold protein for the ERK MAP kinase cascade. The ability of GRK2 to bind to Raf1, MEK1 and ERK2 is dependent on RhoA_GTP binding to the catalytic domain of the kinase. Exogenous GRK2 has previously been shown to increase ERK activation downstream of the epidermal growth factor receptor (EGFR). Here we find that GRK2-mediated ERK activation downstream of the EGFR is Rho-dependent and that treatment with EGF promotes RhoA_GTP binding and ERK scaffolding by GRK2. Depletion of GRK2 expression by RNAi reveals that GRK2 is required for EGF-induced, Rho- and ERK-dependent thymidine incorporation in vascular smooth muscle cells (VSMCs). We therefore hypothesise that Rho-dependent ERK MAPK scaffolding by GRK2 downstream of the EGFR may have an important role in the vasculature, where increased levels of both GRK2 and RhoA have been associated with hypertension.     

The V2 vasopressin receptor stimulates ERK1/2 activity independently of heterotrimeric G protein signalling

The V2 vasopressin receptor (V2R) activates the mitogen activated protein kinases (MAPK) ERK1/2 through a mechanism involving the scaffolding protein beta arrestin. Here we report that this activating pathway is independent of G alpha s, G alpha i, G alpha q or G betagamma and that the V2R-mediated activation of G alpha s inhibits ERK1/2 activity in a cAMP/PKA-dependent manner. In the HEK293 cells studied, the beta arrestin-promoted activation was found to dominate over the PKA-mediated inhibition of the pathway, leading to a strong vasopressin-stimulated ERK1/2 activation. Despite the strong MAPK activation and in contrast with other GPCR, V2R did not induce any significant increase in DNA synthesis, consistent with the notion that the stable interaction between V2R and beta arrestin prevents signal propagation to the nucleus. Beta arrestin was found to be essential for the ERK1/2 activation, indicating that the recruitment of the scaffolding protein is necessary and sufficient to initiate the signal in the absence of any other stimulatory cues. Based on the use of selective pharmacological inhibitors, dominant negative mutants and siRNA, we conclude that the beta arrestin-dependent activation of ERK1/2 by the V2R involves c-Src and a metalloproteinase-dependent trans-activation event. These findings demonstrate that beta arrestin is a genuine signalling initiator that can, on its own, engage a MAPK activation machinery upon stimulation of a GPCR by its natural ligand.     

The Mos/MAP kinase pathway stabilizes c-Fos by phosphorylation and augments its transforming activity in NIH 3T3 cells.

The c-mos proto-oncogene product, Mos, is a serine/threonine kinase that can activate ERK1 and 2 mitogen-activated protein (MAP) kinases by direct phosphorylation of MAPK/ERK kinase (MEK). ERK activation is essential for oncogenic transformation of NIH 3T3 cells by Mos. In this study, we examined how mitogenic and oncogenic signalling from the Mos/MEK/ERK pathway reaches the nucleus to activate downstream target genes. We show that c-Fos (the c-fos protooncogene product), which is an intrinsically unstable nuclear protein, is metabolically highly stabilized, and greatly enhances the transforming efficiency of NIH 3T3 cells, by Mos. This stabilization of c-Fos required Mos-induced phosphorylation of its C-terminal region on Ser362 and Ser374, and double replacements of these serines with acidic (Asp) residues markedly increased the stability and transforming efficiency of c-Fos even in the absence of Mos. Moreover, activation of the ERK pathway was necessary and sufficient for the c-Fos phosphorylation and stabilization by Mos. These results indicate that c-Fos undergoes stabilization, and mediates at least partly the oncogenic signalling, by the Mos/MEK/ERK pathway. The present findings also suggest that, in general, the ERK pathway may regulate the cell fate and function by affecting the metabolic stability of c-Fos.     

Modulation of arsenic-induced epidermal growth factor receptor pathway signalling by resveratrol

Arsenic (As) is both a human carcinogen and an effective anticancer drug. These aspects of arsenic toxicity develop as a consequence of arsenic-induced oxidative stress and modifications to signal pathway activity which alter gene expression. Resveratrol (RVL) a food antioxidant found in grapes and other fruits, exhibits anti-carcinogenic properties by reducing oxidative stress and restoring signal pathway control. This study investigated the impact of RVL on arsenite [As(III)]-induced cell signalling in HaCaT keratinocytes by assaying phosphorylation status of epidermal growth factor receptor (EGFR) signalling intermediates and measuring changes in expression of Phase II and DNA repair biomarkers. As(III) exposure produced dose-dependent toxicity which was associated with increased activation of EGFR pathway intermediates, cSrc, Rac1 and extracellular signal-regulated kinases 1 and 2 (ERK1/2). Arsenic-mediated ERK1/2 activation negatively regulated DNA polymerase beta expression and up regulated heme-oxygenase-1 at toxic concentrations. RVL treatment modulated As(III)-mediated ERK1/2 activation by shifting the balance of cSrc regulatory domain phosphorylation. These effects significantly altered the response of the EGFR pathway to growth factor-induced stimulation. Our research provides evidence that treatment with pharmacologically relevant doses of RVL influences cellular responses to As(III), largely due to RVL-mediated changes to Src and ERK1/2 activation.     

cAMP antagonizes p21ras-directed activation of extracellular signal-regulated kinase 2 and phosphorylation of mSos nucleotide exchange factor.

In fibroblasts, stimulation of receptor tyrosine kinases results in the activation of the extracellular signal-regulated kinase 2 (ERK2). The major signalling pathway employed by these receptors involves the activation of p21ras and raf-1 kinase. Here we show that in NIH3T3 and rat-1 fibroblasts, elevation of the intracellular cAMP level results in the inhibition of ERK2 activation induced by PDGF, EGF and insulin treatment. Analysis of various signalling intermediates shows that cAMP interferes at a site downstream of p21ras, but upstream of raf-1 kinase. Inhibition by cAMP depends on both the cAMP concentration and the absolute amount of p21ras molecules bound to GTP, suggesting a mechanism of competitive inhibition. Also TPA-induced, p21ras-independent, activation of raf-1 kinase and ERK2 is inhibited by cAMP. We have used the inhibitory effect of cAMP to investigate whether phosphorylation of mSos, a p21ras nucleotide exchange factor, is dependent on the activity of the raf-1 kinase/ERK2 pathway. We found that phosphorylation of mSos, as monitored by a mobility shift, is delayed with respect to p21ras and ERK2 activation and is inhibited by cAMP in a similar cell type- and concentration-dependent manner as the inactivation of ERK2. These results provide evidence for a model of p21ras-directed signalling towards ERK2 that feeds back on mSos by regulating its phosphorylation status and that can be negatively modulated by protein kinase A and positively modulated by protein kinase C action.     

PKC isoenzymes differentially modulate the effect of thrombin on MAPK-dependent RPE proliferation

Thrombin signalling through PAR (protease-activated receptor)-1 is involved in cellular processes, such as proliferation, differentiation and cell survival. Following traumatic injury to the eye, thrombin signalling may participate in disorders, such as PVR (proliferative vitreoretinopathy), a human eye disease characterized by the uncontrolled proliferation, transdifferentiation and migration of otherwise quiescent RPE (retinal pigment epithelium) cells. PARs activate the Ras/Raf/MEK/ERK MAPK pathway (where ERK is extracellular-signal-regulated kinase, MAPK is mitogen-activated protein kinase and MEK is MAPK/ERK kinase) through the activation of G(alpha) and G(betagamma) heterotrimeric G-proteins, and the downstream stimulation of the PLC (phospholipase C)-beta/PKC (protein kinase C) and PI3K (phosphoinositide 3-kinase) signalling axis. In the present study, we examined the molecular signalling involved in thrombin-induced RPE cell proliferation, using rat RPE cells in culture as a model system for PVR pathogenesis. Our results showed that thrombin activation of PAR-1 induces RPE cell proliferation through Ras-independent activation of the Raf/MEK/ERK1/2 MAPK signalling cascade. Pharmacological analysis revealed that the activation of 'conventional' PKC isoforms is essential for proliferation, although thrombin-induced phosphorylation of ERK1/2 requires the activation of atypical PKCzeta by PI3K. Consistently, thrombin-induced ERK1/2 activation and RPE cell proliferation were prevented completely by PI3K or PKCzeta inhibition. These results suggest that thrombin induces RPE cell proliferation by joint activation of PLC-dependent and atypical PKC isoforms and the Ras-independent downstream stimulation of the Raf/MEK/ERK1/2 MAPK cascade. The present study is the first report demonstrating directly thrombin-induced ERK phosphorylation in the RPE, and the involvement of atypical PKCzeta in this process.     

Transactivation of the epidermal growth factor receptor mediates parathyroid hormone and prostaglandin F2 alpha-stimulated mitogen-activated protein kinase activation in cultured transgenic murine osteoblasts

Recent data suggest that G protein-coupled receptors (GPCRs), including those for PTH and prostaglandins (PGs), contribute to the proliferation and differentiation of osteoblasts in vivo. To understand how these signals are transduced, we studied activation of the ERK1/2 MAPK cascade in cultures of differentiating TMOb murine osteoblasts. In TMOb cells, stimulation of endogenous Gs/Gq-coupled PTH receptors, Gq-coupled PGF2 alpha receptors, and Gi/Gq-coupled lysophosphatidic acid receptors, but not Gs-coupled PGE2 receptors, caused a rapid 5- to 10-fold increase in ERK1/2 phosphorylation. GPCR-stimulated ERK1/2 activation coincided with increased tyrosine phosphorylation of epidermal growth factor (EGF) receptors and was blocked by the EGF receptor inhibitor, tyrphostin AG1478, and the metalloprotease inhibitor, batimastat, suggesting that the response involved transactivation of EGF receptors through the proteolytic release of an EGF receptor ligand. To further examine the mechanism of PTH-stimulated EGF receptor transactivation, we employed COS-7 cells expressing the rat PTH receptor. Here, stimulation with PTH(1-34) caused proteolysis of hemagglutinin epitope-tagged heparin binding-EGF, increased tyrosine autophosphorylation of EGF receptors, and AG1478-sensitive ERK1/2 activation. When PTH receptor-expressing COS-7 cells were placed in a mixed culture with cells lacking the PTH receptor but expressing a green fluorescent protein-tagged ERK2, stimulation with PTH(1-34) induced phosphorylation of green fluorescent protein-ERK2 that was abolished by either batimastat or tyrphostin AG1478. These data suggest that autocrine/paracrine cross-talk between EGF receptors and Gi- or Gq/11-coupled GPCRs represents the predominant mechanism of GPCR-mediated activation of ERK1/2 in cultured TMOb osteoblasts.     

Tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin does not require extracellular signal-regulated kinase activation in Swiss 3T3 cells stimulated by bombesin or platelet-derived growth factor

The experiments presented here were designed to examine the contribution of the extracellular signal-regulated mitogen-activated protein kinases (ERKs) to the tyrosine phosphorylation of the focal adhesion proteins p125(Fak), p130(Cas), and paxillin induced by G protein-coupled receptors (GPCRs) and tyrosine kinase receptors in Swiss 3T3 cells. Stimulation of these cells with bombesin, lysophosphatidic acid (LPA), endothelin, and platelet-derived growth factor (PDGF) led to a marked increase in the tyrosine phosphorylation of these focal adhesion proteins and in ERK activation. Exposure of the cells to two structurally unrelated mitogen-activated protein kinase or ERK kinase (MEK) inhibitors, PD98059 and U0126, completely abrogated ERK activation but did not prevent tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin. Furthermore, different dose-response relationships were obtained for tyrosine phosphorylation of focal adhesion proteins and for ERK activation in response to PDGF. Putative upstream events in the activation of focal adhesion proteins including actin cytoskeletal reorganization and myosin light chain (MLC) phosphorylation were also not prevented by inhibition of ERK activation. Thus, our results demonstrate that the activation of the ERK pathway is not necessary for the increase of the tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin induced by either GPCRs or tyrosine kinase receptors in Swiss 3T3 cells.     

Truncated MEK1 is required for transient activation of MAPK signalling in G2 phase cells

The primary endpoint of signalling through the canonical Raf–MEK–ERK MAP kinase cascade is ERK activation. Here we report a novel signalling outcome for this pathway. Activation of the MAP kinase pathway by growth factors or phorbol esters during G2 phase results in only transient activations of ERK and p90RSK, then suppression to below control levels. A small peak of ERK and p90RSK activation in early G2 phase cells was identified, and inhibition of this delayed entry into mitosis. The previously identified, proteolytically cleaved form of MEK1 termed tMEK (truncated MEK1), is also induced with G2 phase MAPK pathway activation. We demonstrate that addition of recombinant mutants of MEK1 with an N-terminal truncation similar to that of tMEK also inhibited ERK and p90RSK activations and delayed progression into mitosis. Only catalytically inactive forms of tMEK were capable of these effects, but surprisingly, phosphorylation on the activating Ser218/222 sites was also required. A lack of MEK1 or ability to accumulate tMEK resulted in the absence of the feedback inhibition of ERK and p90RSK activations. tMEK is a novel output from the canonical MAP kinase signalling pathway, acting in a MAPK signalling-regulated dominant negative manner to inhibit ERK and p90RSK activations, acting as a dampening mechanism to reduce the magnitude or duration of MAPK pathway signalling in G2/M phase.     

Rapid and transient activation of the ERK MAPK signalling pathway by macrophage migration inhibitory factor (MIF) and dependence on JAB1/CSN5 and Src kinase activity

Macrophage migration inhibitory factor (MIF) is a 12.5 kD polypeptide that serves as a critical regulator of cell functions such as gene expression, proliferation or apoptosis. However, the signal transduction pathways through which MIF takes part in cellular regulation are only incompletely understood. MIF leads to CD74-dependent "sustained" activation of ERK1/2 MAPK, but MIF's role in "transient" ERK activation and the involved upstream pathways are unknown. Here we report that the transient ERK pathway was markedly activated by MIF. This effect involved the phosphorylation and activation of Raf-1, MEK, ERK, and Elk-1. Of note, rapid and transient ERK phosphorylation by MIF was measurable in MIF-deficient cells, suggesting that MIF acted in a non-autocrine fashion. Applying the inhibitor genistein, a tyrosine kinase (TPK) activity was identified as a critical upstream signalling event in MIF-induced transient ERK signalling. Experiments using the Src kinase inhibitor PP2 indicated that the involved TPK was a Src-type tyrosine kinase. A role for an upstream Src kinase was proven by applying Src-deficient cells which did not exhibit transient ERK activation upon treatment with MIF, but in which MIF-induced ERK signalling could be restored by re-expressing Src. Intriguingly, JAB1/CSN5, a signalosome component, cellular binding protein of MIF and regulator of cell proliferation and survival, had a marked, yet dual, effect on MIF-induced ERK signalling. JAB1 overexpression inhibited sustained, but not transient, ERK phosphorylation. By contrast, JAB1-knock-down by siRNA revealed that minimum JAB1 levels were necessary for transient activation of ERK by MIF. In conclusion, MIF rapidly and transiently activates the ERK pathway, an effect that has not been recognized previously. This signalling pathway involves the upstream activation of a Src-type kinase and is co-regulated by the cellular MIF binding protein JAB1/CSN5. Our study thus has unravelled a novel MIF-driven signalling pathway and an intricate regulatory system involving extra- and possibly intracellular MIF, and which likely critically participates in controlling cell proliferation and survival.     

Tyrosine kinase-independent activation of extracellular-regulated kinase (ERK) 1/2 by the insulin-like growth factor-1 receptor

The extracellular-regulated kinase (ERK1/2) is a key conduit for transduction of signals from growth factor receptors to the nucleus. Previous work has shown that ERK1/2 activation in response to IGF-1 may require the participation of G proteins, but the role of the receptor tyrosine kinase in this process has not been clearly resolved. This investigation of IGF-1 receptor function was therefore designed to examine the contribution of the receptor tyrosine kinase to ERK1/2 activation. Phosphorylation of ERK1/2 in smooth muscle cells following treatment with IGF-1 was not blocked by pretreatment with AG1024 or picropodophylin, inhibitors of the IGF-1 receptor tyrosine kinase. Likewise, IGF-1 activated ERK1/2 in cells expressing a kinase-dead mutant of the IGF-1 receptor. ERK1/2 activation was unaffected by the phosphatidylinositol 3-kinase inhibitor LY-294002, but was sensitive to inhibitors of Src kinase, phospholipase C and Gβγ subunit signalling. Treatment with αIR-3, a neutralizing monoclonal antibody, also stimulated ERK1/2 phosphorylation without concomitant activation of the receptor tyrosine kinase. Phosphoprotein mapping of IGF-1 and αIR-3 treated cells confirmed that antibody-induced ERK1/2 phosphorylation occurred in the absence of tyrosine kinase phosphorylation, and enabled extension of these findings to p38 MAPK. These results suggest that stimulation of ERK1/2 phosphorylation by IGF-1 does not require activation of the receptor tyrosine kinase.     

The role of beta-arrestins in the formyl peptide receptor-like 1 internalization and signaling

The N-formyl peptide receptor-like 1 (FPRL1) is a G protein-coupled receptor (GPCR) that transmits intracellular signals in response to a variety of agonists, many of them being clearly implicated in human pathology. beta-arrestins are adaptor proteins that uncouple GPCRs from G protein and regulate receptor internalization. They can also function as signal transducers through the scaffolding of signaling molecules, such as components of the extracellular signal-regulated kinase (ERK) cascade. We investigated the role of beta-arrestins in ligand-induced FPRL1 internalization and signaling. In HEK293 cells expressing FPRL1, fluorescence microscopy revealed that agonist-stimulated FPRL1 remained co-localized with beta-arrestins during endocytosis. Internalization of FPRL1, expressed in a mouse embryonic fibroblast (MEF) cell line lacking endogenous beta-arrestins, was highly compromised. This distinguishes FPRL1 from the prototypical formyl peptide receptor FPR that is efficiently internalized in the absence of beta-arrestins. In both HEK293 and MEF cells, FPRL1-mediated ERK1/2 activation was a rapid and transient event. The kinetics and extent of ERK1/2 activation were not significantly modified by beta-arrestin overexpression. The pattern of FPRL1-mediated ERK1/2 activation was similar whether cells express or not beta-arrestins. Furthermore, treatment of the FPRL1 expressing cells with pertussis toxin inhibited ERK1/2 activation in MEF and in HEK293 cells. These results led us to conclude that activation of ERK1/2 mediated by FPRL1 occurs primarily through G protein signaling. Since beta-arrestin-mediated signaling has been observed essentially for receptors coupled to G proteins other than G(i), this may be a characteristic of G(i) protein-coupled chemoattractant receptors.     

Stimulation by ghrelin of p42/p44 mitogen-activated protein kinase through the GHS-R1a receptor: role of G-proteins and beta-arrestins

Results presented in this study indicate that in human embryonic kidney 293 cells (HEK 293), the ghrelin receptor growth hormone secretagogue receptor type 1a (GHS-R1a) activates the extracellular signal-related kinases 1 and 2 (ERK 1/2) via three pathways. One pathway is mediated by the beta-arrestins 1 and 2, and requires entry of the receptor into a multiprotein complex with the beta-arrestins, Src, Raf-1, and ERK 1/2. A second pathway is G(q/11)-dependent and involves a Ca(2+)-dependent PKC (PKCalpha/beta) and Src. A third pathway is G(i)-dependent and involves phosphoinositide 3-kinase (PI3K), PKCepsilon, and Src. Our current study reveals that G(i/o)- and G(q/11)-proteins are crucially involved in the beta-arrestin-mediated ERK 1/2 activation. These results thus support the view that the beta-arrestins act as both scaffolding proteins and signal transducers in ERK 1/2 activation, as reported for other receptors. The different pathways of ERK 1/2 activation suggest that binding to GHS-R1a activates ERK 1/2 pools at different locations within the cell, and thus probably with different physiological consequences.     

G protein-coupled receptor-mediated ERK1/2 phosphorylation: towards a generic sensor of GPCR activation

The development of new analytical methods, aimed at profiling G protein-coupled receptor (GPCR) ligands, regardless of the G protein-coupling pattern of their respective receptor, remains a key goal in drug discovery. Considerable evidence has recently revived the central role that could be played by extracellular-signal-regulated kinase (ERK), the cornerstone protein kinase of the first tyrosine kinase receptor-mediated pathway identified, in response to the activation of various types of GPCRs. Here we reveal a conceptual study in which the potential of ERK phosphorylation is evaluated as a generic readout in response to three different receptors activating three main classes of G proteins: Galphas, Galphai and Galphaq. GPCR-mediated ERK phosphorylation was compared with different readouts such as GTPgammaS, CAMP, or Ca2 +. We propose the measurement of GPCR-activated ERK phosphorylation as an alternative assay to better understand the molecular pharmacology of ligands of promiscuous GPCRs.     

Targeting of PYK2 to focal adhesions as a cellular mechanism for convergence between integrins and G protein-coupled receptor signaling cascades

The non-receptor tyrosine kinase PYK2 appears to function at a point of convergence of integrins and certain G protein-coupled receptor (GPCR) signaling cascades. In this study, we provide evidence that translocation of PYK2 to focal adhesions is triggered both by cell adhesion to extracellular matrix proteins and by activation of the histamine GPCR. By using different mutants of PYK2 as green fluorescent fusion proteins, we show that the translocation of PYK2 to focal adhesions is not dependent on its catalytic activity but rather is mediated by its carboxyl-terminal domain. Translocation of PYK2 to focal adhesions was attributed to enhanced tyrosine phosphorylation of PYK2 and its association with the focal adhesion proteins paxillin and p130(Cas). Translocation of PYK2 to focal adhesions, as well as its tyrosine phosphorylation in response to histamine treatment, was abolished in the presence of protein kinase C inhibitors or cytochalasin D treatment, whereas activation of protein kinase C by phorbol ester resulted in focal adhesion targeting of PYK2 and its tyrosine phosphorylation in an integrin-clustering dependent manner. Overexpression of a wild-type PYK2 enhanced ERK activation in response to histamine, whereas a kinase-deficient mutant substantially inhibited this response. Furthermore, inhibition of PYK2 translocation to focal adhesions abolished ERK activation in response to histamine treatment. These results suggest that PYK2 apparently links between GPCRs and focal adhesion-dependent ERK activation and can provide the molecular basis underlying PYK2 function at a point of convergence between signaling pathways triggered by extracellular matrix proteins and certain GPCR agonists.     

Coordinating ERK/MAPK signalling through scaffolds and inhibitors

The pathway from Ras through Raf and MEK (MAPK and ERK kinase) to ERK/MAPK (extracellular signal-regulated kinase/mitogen-activated protein kinase) regulates many fundamental cellular processes. Recently, a number of scaffolding proteins and endogenous inhibitors have been identified, and their important roles in regulating signalling through this pathway are now emerging. Some scaffolds augment the signal flux, but also mediate crosstalk with other pathways; certain adaptors target MEK-ERK/MAPK complexes to subcellular localizations; others provide regulated inhibition. Computational modelling indicates that, together, these modulators can determine the dynamic biological behaviour of the pathway.