MEK1 activation by PAK: a novel mechanism

Extracellular signal-Regulated Kinase (ERK) controls a variety of cellular processes, including cell proliferation and cell motility. While oncogenic mutations in Ras and B-Raf result in deregulated ERK activity and proliferation and migration in some tumor cells, other tumors exhibit elevated ERK signaling in the absence of these mutations. Here we provide evidence that PAK can directly activate MEK1 by a mechanism distinct from conventional Ras/Raf mediated activation. We find that PAK phosphorylation of MEK1 serine 298 stimulates MEK1 autophosphorylation on the activation loop, and activation of MEK1 activity towards ERK in in vitro reconstitution experiments. Serines 218 and/or 222 in the MEK1 activation loop are required for PAK-stimulated MEK1 activity towards ERK. MEK2, which is a poor target for PAK phosphorylation in cells, is not activated in this manner. Tissue culture experiments verify that this mechanism is used in suspended fibroblasts expressing mutationally activated PAK1. We speculate that aberrant signaling through PAK may directly induce anchorage-independent MEK1 activation in tumor cells lacking oncogenic Ras or Raf mutations, and that this mechanism may contribute to localized MEK signaling in focal contacts and adhesions during cell adhesion or migration.     

Mitogen-activated protein kinase feedback phosphorylation regulates MEK1 complex formation and activation during cellular adhesion

Cell adhesion and spreading depend on activation of mitogen-activated kinase, which in turn is regulated both by growth factor and integrin signaling. Growth factors, such as epidermal growth factor, are capable of activating Ras and Raf, but integrin signaling is required to couple Raf to MEK and MEK to extracellular signal-regulated protein kinase (ERK). It was previously shown that Rac-p21-activated kinase (PAK) signaling regulated the physical association of MEK1 with ERK2 through phosphorylation sites in the proline-rich sequence (PRS) of MEK1. It was also shown that activation of MEK1 and ERK by integrins depends on PAK phosphorylation of S298 in the PRS. Here we report a novel MEK1-specific regulatory feedback mechanism that provides a means by which activated ERK can terminate continued PAK phosphorylation of MEK1. Activated ERK can phosphorylate T292 in the PRS, and this blocks the ability of PAK to phosphorylate S298 and of Rac-PAK signaling to enhance MEK1-ERK complex formation. Preventing ERK feedback phosphorylation on T292 during cellular adhesion prolonged phosphorylation of S298 by PAK and phosphorylation of S218 and S222, the MEK1 activating sites. We propose that activation of ERK during adhesion creates a feedback system in which ERK phosphorylates MEK1 on T292, and this in turn blocks additional S298 phosphorylation in response to integrin signaling.     

PAK1 is a novel MEK-independent raf target controlling expression of the IAP survivin in M-CSF-mediated osteoclast survival

As activation of the Ras/Raf/MEK/ERK pathway is a critical component of M-CSF-promoted osteoclast survival, determining specific mechanism by which M-CSF activates this signal transduction pathway is paramount towards advancing treatment of pathological conditions resulting in increased bone turnover. The p21 activated kinase PAK1 modulates activation of the Raf/MEK/ERK pathway by either directly activating Raf or priming MEK for activation by Raf. Therefore a role for PAK1 in M-CSF-mediated activation of the MEK/ERK pathway controlling osteoclast survival was assessed. Here we show that PAK1 is activated by M-CSF in a Ras-dependent mechanism that promotes osteoclast survival. Surprisingly, PAK1 did not modulate Raf activation or Raf-mediated MEK activation. M-CSF mediated activation of Raf was required for PAK1 activation and osteoclast survival promoted by PAK1. This survival response was MEK-independent as expression of constitutively active MEK did not rescue osteoclasts from apoptosis induced by blocking PAK1 function. Functionally, PAK1 promoted osteoclast survival by modulating expression of the IAP family member Survivin. M-CSF therefore functions to promote PAK1 activation as a novel MEK-independent Raf target to control Survivin-mediated osteoclast survival.     

Rac-PAK signaling stimulates extracellular signal-regulated kinase (ERK) activation by regulating formation of MEK1-ERK complexes

Utilizing mutants of extracellular signal-regulated kinase 2 (ERK2) that are defective for intrinsic mitogen-activated protein kinase or ERK kinase (MEK) binding, we have identified a convergent signaling pathway that facilitates regulated MEK-ERK association and ERK activation. ERK2-delta19-25 mutants defective in MEK binding could be phosphorylated in response to mitogens; however, signaling from the Raf-MEK pathway alone was insufficient to stimulate their phosphorylation in COS-1 cells. Phosphorylation of ERK2-delta19-25 but not of wild-type ERK2 in response to Ras V12 was greatly inhibited by dominant-negative Rac. Activated forms of Rac and Cdc42 could enhance the association of wild-type ERK2 with MEK1 but not with MEK2 in serum-starved adherent cells. This effect was p21-activated kinase (PAK) dependent and required the putative PAK phosphorylation sites T292 and S298 of MEK1. In detached cells placed in suspension, ERK2 was complexed with MEK2 but not with MEK1. However, upon replating of cells onto a fibronectin matrix, there was a substantial induction of MEK1-ERK2 association and ERK activation, both of which could be inhibited by dominant-negative PAK1. These data show that Rac facilitates the assembly of a mitogen-activated protein kinase signaling complex required for ERK activation and that this facilitative signaling pathway is active during adhesion to the extracellular matrix. These findings reveal a novel mechanism by which adhesion and growth factor signals are integrated during ERK activation.     

PAK1 phosphorylation of MEK1 regulates fibronectin-stimulated MAPK activation

Activation of the Ras-MAPK signal transduction pathway is necessary for biological responses both to growth factors and ECM. Here, we provide evidence that phosphorylation of S298 of MAPK kinase 1 (MEK1) by p21-activated kinase (PAK) is a site of convergence for integrin and growth factor signaling. We find that adhesion to fibronectin induces PAK1-dependent phosphorylation of MEK1 on S298 and that this phosphorylation is necessary for efficient activation of MEK1 and subsequent MAPK activation. The rapid and efficient activation of MEK and phosphorylation on S298 induced by cell adhesion to fibronectin is influenced by FAK and Src signaling and is paralleled by localization of phospho-S298 MEK1 and phospho-MAPK staining in peripheral membrane-proximal adhesion structures. We propose that FAK/Src-dependent, PAK1-mediated phosphorylation of MEK1 on S298 is central to the organization and localization of active Raf-MEK1-MAPK signaling complexes, and that formation of such complexes contributes to the adhesion dependence of growth factor signaling to MAPK.     

p21-Activated Kinase 1 (PAK1) Can Promote ERK Activation in a Kinase-independent Manner

PAK1 plays an important role in proliferation and tumorigenesis, at least partially by promoting ERK phosphorylation of C-RAF (Ser-338) or MEK1 (Ser-298). We observed how that overexpression of a kinase-dead mutant form of PAK1 increased phosphorylation of MEK1/2 (Ser-217/Ser-221) and ERK (Thr-202/Tyr-204), although phosphorylation of B-RAF (Ser-445) and C-RAF (Ser-338) remained unchanged. Furthermore, increased activation of the PAK1 activator Rac1 induced the formation of a triple complex of Rac1, PAK1, and MEK1 independent of the kinase activity of PAK1. These data suggest that PAK1 can stimulate MEK activity in a kinase-independent manner, probably by serving as a scaffold to facilitate interaction of C-RAF.     

The MEK1 scaffolding protein MP1 regulates cell spreading by integrating PAK1 and Rho signals

How the extracellular signal-regulated kinase (ERK) cascade regulates diverse cellular functions, including cell proliferation, survival, and motility, in a context-dependent manner remains poorly understood. Compelling evidence indicates that scaffolding molecules function in yeast to channel specific signals through common components to appropriate targets. Although a number of putative ERK scaffolding proteins have been identified in mammalian systems, none has been linked to a specific biological response. Here we show that the putative scaffold protein MEK partner 1 (MP1) and its partner p14 regulate PAK1-dependent ERK activation during adhesion and cell spreading but are not required for ERK activation by platelet-derived growth factor. MP1 associates with active but not inactive PAK1 and controls PAK1 phosphorylation of MEK1. Our data further show that MP1, p14, and MEK1 serve to inhibit Rho/Rho kinase functions necessary for the turnover of adhesion structures and cell spreading and reveal a signal-channeling function for a MEK1/ERK scaffold in orchestrating cytoskeletal rearrangements important for cell motility.     

Trihydrophobin 1 phosphorylation by c-Src regulates MAPK/ERK signaling and cell migration

c-Src activates Ras-MAPK/ERK signaling pathway and regulates cell migration, while trihydrophobin 1 (TH1) inhibits MAPK/ERK activation and cell migration through interaction with A-Raf and PAK1 and inhibiting their kinase activities. Here we show that c-Src interacts with TH1 by GST-pull down assay, coimmunoprecipitation and confocal microscopy assay. The interaction leads to phosphorylation of TH1 at Tyr-6 in vivo and in vitro. Phosphorylation of TH1 decreases its association with A-Raf and PAK1. Further study reveals that Tyr-6 phosphorylation of TH1 reduces its inhibition on MAPK/ERK signaling, enhances c-Src mediated cell migration. Moreover, induced tyrosine phosphorylation of TH1 has been found by EGF and estrogen treatments. Taken together, our findings demonstrate a novel mechanism for the comprehensive regulation of Ras/Raf/MEK/ERK signaling and cell migration involving tyrosine phosphorylation of TH1 by c-Src.     

Opposing roles of Ras/Raf oncogenes and the MEK1/ERK signaling module in regulation of expression and adhesive function of surface transglutaminase

Tissue transglutaminase (tTG) serves as a potent and ubiquitous integrin-associated adhesion co-receptor for fibronectin on the cell surface and affects several key integrin functions. Here we report that in fibroblasts, activated H-Ras and Raf-1 oncogenes decrease biosynthesis, association with beta1 integrins, and surface expression of tTG because of down-regulation of tTG mRNA. In turn, the reduction of surface tTG inhibits adhesion of H-Ras- and Raf-1-transformed cells on fibronectin and, in particular, on its tTG-binding fragment I(6)II(1,2)I(7-9), which does not interact directly with integrins. Analysis of Ras/Raf downstream signaling with specific pharmacological inhibitors reveals that the decrease in tTG expression is mediated by the p38 MAPK, c-Jun NH2-terminal kinase, and phosphatidylinositol 3-kinase pathways. In contrast, increased activation of the ERK pathway by constitutively active MEK1 stimulates tTG mRNA expression, biosynthesis, and surface expression of tTG, whereas MEK inhibitors or dominant negative MEK1 exert an opposite effect. This modulation of surface tTG by ERK signaling alters adhesion of cells on fibronectin and its fragment that binds tTG. Furthermore, transient stimulation of ERK signaling in untransformed fibroblasts by adhesion on fibronectin or growth factors elevates tTG biosynthesis, increases complex formation with beta1 integrins, and raises surface expression of tTG. Finally, ERK activation is required for growth factor-induced redistribution of tTG on the surface of adherent fibroblasts and co-clustering of beta1 integrins and tTG at cell-matrix adhesion contacts. Together, our data indicate that down-regulation of surface tTG by Ras and Raf oncogenes contributes to adhesive deficiency of transformed fibroblasts, whereas stimulation of biosynthesis and surface expression of tTG by the MEK1/ERK module promotes and sustains cell-matrix adhesion of untransformed cells. Contrasting effects of Ras/Raf oncogenes and their immediate downstream signaling module, MEK1/ERK, on tTG expression are consistent with adhesive function of surface tTG.     

Feedback regulation of beta-arrestin1 function by extracellular signal-regulated kinases.

The functions of beta-arrestin1 to facilitate clathrin-mediated endocytosis of the beta2-adrenergic receptor and to promote agonist-induced activation of extracellular signal-regulated kinases (ERK) are regulated by its phosphorylation/dephosphorylation at Ser-412. Cytoplasmic beta-arrestin1 is almost stoichiometrically phosphorylated at Ser-412. Dephosphorylation of beta-arrestin1 at the plasma membrane is required for targeting a signaling complex that includes the agonist-occupied receptors to the clathrin-coated pits. Here we demonstrate that beta-arrestin1 phosphorylation and function are modulated by an ERK-dependent negative feedback mechanism. ERK1 and ERK2 phosphorylate beta-arrestin1 at Ser-412 in vitro. Inhibition of ERK activity by a dominant-negative MEK1 mutant significantly attenuates beta-arrestin1 phosphorylation, thereby increasing the concentration of dephosphorylated beta-arrestin1. Under such conditions, beta-arrestin1-mediated beta2-adrenergic receptor internalization is enhanced as is its ability to bind clathrin. In contrast, if ERK-mediated phosphorylation is increased by transfection of a constitutively active MEK1 mutant, receptor internalization is inhibited. Our results suggest that dephosphorylated beta-arrestin1 mediates endocytosis-dependent ERK activation. Following activation, ERKs phosphorylate beta-arrestin1, thereby exerting an inhibitory feedback control of its function.     

The N-terminal ERK-binding site of MEK1 is required for efficient feedback phosphorylation by ERK2 in vitro and ERK activation in vivo

An ERK2-binding site at the N terminus of MEK1 was reported to mediate their stable association. We examined the importance of this binding site in the feedback phosphorylation of MEK1 on Thr(292) and Thr(386) by ERK2, the phosphorylation and activation of ERK2 by MEK1, and the interaction of MEK1 with ERK2 and Raf-1. Deletion of the binding site from MEK1 reduced its phosphorylation by ERK2, but had no effect on its phosphorylation by p21-activated protein kinase-1 (PAK1). A MEK1 N-terminal peptide containing the binding site inhibited MEK1 phosphorylation by ERK2. However, it did not affect MEK1 phosphorylation by p21-activated protein kinase or myelin basic protein phosphorylation by ERK2. Deletion of the N-terminal ERK-binding domain of MEK1 also reduced its ability to phosphorylate ERK2 in vitro, to co-immunoprecipitate with ERK2, and to stimulate ERK2 activation in transfected cells, but it did not alter the association with endogenous Raf-1. Using ERK2-p38 chimeras and an ERK2 deletion mutant, a MEK1-binding site of ERK2 was localized to its N terminus.     

Phosphorylation regulates KSR1 stability, ERK activation, and cell proliferation

Kinase suppressor of Ras (KSR) is a molecular scaffold that interacts with the components of the Raf/MEK/ERK kinase cascade and positively regulates ERK signaling. Phosphorylation of KSR1, particularly at Ser(392), is a critical regulator of KSR1 subcellular localization and ERK activation. We examined the role of phosphorylation of both Ser(392) and Thr(274) in regulating ERK activation and cell proliferation. We hypothesized that KSR1 phosphorylation is involved in generating signaling specificity through the Raf/MEK/ERK kinase cascade in response to stimulation by different growth factors. In fibroblasts, platelet-derived growth factor stimulation induces sustained ERK activation and promotes S-phase entry. Treatment with epidermal growth factor induces transient ERK activation but fails to drive cells into S phase. Mutation of Ser(392) and Thr(274) (KSR1.TVSA) promotes sustained ERK activation and cell cycle progression with either platelet-derived growth factor or epidermal growth factor treatment. KSR1(-/-) mouse embryo fibroblasts expressing KSR1.TVSA proliferate two times faster and grow to a higher density than cells expressing the same level of wild-type KSR1. In addition, KSR1.TVSA is more stable than wild-type KSR1. These data demonstrate that phosphorylation and stability of the molecular scaffold KSR1 are critical regulators of growth factor-specific responses that promote cell proliferation.     

MEK1-ERKs级联激活方式是调控单纯疱疹病毒Ⅱ型复制的重要机制

本研究通过阐明MEK1和MEK2亚型在单纯疱疹病毒Ⅱ型(herpes simplex virus type 2,HSV2)复制中介导的Raf/MEK/ERK(简称ERK)通路活化中的作用,以期进一步阐明该通路调控病毒复制的机制.研究中应用了MEK抑制剂U0126、针对MEK1和MEK2的特异性小干扰RNA(small ...     

CK2 Is a component of the KSR1 scaffold complex that contributes to Raf kinase activation

Kinase Suppressor of Ras (KSR) is a molecular scaffold that interacts with the core kinase components of the ERK cascade, Raf, MEK, and ERK and provides spatial and temporal regulation of Ras-dependent ERK cascade signaling. In this report, we identify the heterotetrameric protein kinase, casein kinase 2 (CK2), as a new KSR1-binding partner. Moreover, we find that the KSR1/CK2 interaction is required for KSR1 to maximally facilitate ERK cascade signaling and contributes to the regulation of Raf kinase activity. Binding of the CK2 holoenzyme is constitutive and requires the basic surface region of the KSR1 atypical C1 domain. Loss of CK2 binding does not alter the membrane translocation of KSR1 or its interaction with ERK cascade components; however, disruption of the KSR1/CK2 interaction or inhibition of CK2 activity significantly reduces the growth-factor-induced phosphorylation of C-Raf and B-Raf on the activating serine site in the negative-charge regulatory region (N-region). This decrease in Raf N-region phosphorylation further correlates with impaired Raf, MEK, and ERK activation. These findings identify CK2 as a novel component of the KSR1 scaffolding complex that facilitates ERK cascade signaling by functioning as a Raf family N-Region kinase.     

Mechanism of mitosis-specific activation of MEK1

Activation of cyclin B-Cdc2 is an absolute requirement for entry into mitosis, but other protein kinase pathways that also have mitotic functions are activated during G(2)/M progression. The MAPK cascade has well established roles in entry and exit from mitosis in Xenopus, but relatively little is known about the regulation and function of this pathway in mammalian mitosis. Here we report a detailed analysis of the activity of all components of the Ras/Raf/MEK/ERK pathway in HeLa cells during normal G(2)/M. The focus of this pathway is the dramatic activation of an endomembrane-associated MEK1 without the corresponding activation of the MEK substrate ERK. This is because of the uncoupling of MEK1 activation from ERK activation. The mechanism of this uncoupling involves the cyclin B-Cdc2-dependent proteolytic cleavage of the N-terminal ERK-binding domain of MEK1 and the phosphorylation of Thr(286). These results demonstrate that cyclin B-Cdc2 activity regulates signaling through the MAPK pathway in mitosis.     

PAK1 kinase is required for CXCL1-induced chemotaxis

The CXC subfamily of chemokines plays an important role in diverse processes, including inflammation, wound healing, growth regulation, angiogenesis, and tumorigenesis. The CXC chemokine CXCL1, or MGSA/GROalpha, is traditionally considered to be responsible for attracting leukocytes into sites of inflammation. To better understand the molecular mechanisms by which CXCL1 induces CXCR2-mediated chemotaxis, the signal transduction components involved in CXCL1-induced chemotaxis were examined. It is shown here that CXCL1 induces cdc42 and PAK1 activation in CXCR2-expressing HEK293 cells. Activation of the cdc42-PAK1 cascade is required for CXCL1-induced chemotaxis but not for CXCL1-induced intracellular Ca2+ mobilization. Moreover, CXCL1 activation of PAK1 is independent of ERK1/2 activation, a conclusion based on the observations that the inhibition of MEK-ERK activation by expression of dominant negative ERK or by the MEK inhibitor, PD98059, has no effect on CXCL1-induced PAK1 activation or CXCL1-induced chemotaxis.     

M-Ras induces Ral and JNK activation to regulate MEK/ERK-independent gene expression in MCF-7 breast cancer cells

Constitutive activation of M-Ras has previously been reported to cause morphologic and growth transformation of murine cells, suggesting that M-Ras plays a role in tumorigenesis. Cell transformation by M-Ras correlated with weak activation of the Raf/MEK/ERK pathway, although contributions from other downstream effectors were suggested. Recent studies indicate that signaling events distinct from the Raf/MEK/ERK cascade are critical for human tumorigenesis. However, it is unknown what signaling events M-Ras triggers in human cells. Using constitutively active M-Ras (Q71L) containing additional mutations within its effector-binding loop, we found that M-Ras induces MEK/ERK-dependent and -independent Elk1 activation as well as phosphatidylinositol 3 kinase (PI3K)/Akt and JNK/cJun activation in human MCF-7 breast cancer cells. Among several human cell lines examined, M-Ras-induced MEK/ERK-independent Elk1 activation was only detected in MCF-7 cells, and correlated with Rlf/M-Ras interaction and Ral/JNK activation. Supporting a role for M-Ras signaling in breast cancer, EGF activated M-Ras and promoted its interaction with endogenous Rlf. In addition, constitutive activation of M-Ras induced estrogen-independent growth of MCF-7 cells that was dependent on PI3K/Akt, MEK/ERK, and JNK activation. Thus, our studies demonstrate that M-Ras signaling activity differs between human cells, highlighting the importance of defining Ras protein signaling within each cell type, especially when designing treatments for Ras-induced cancer. These findings also demonstrate that M-Ras activity may be important for progression of EGFR-dependent tumors.     

Growth arrest signaling of the Raf/MEK/ERK pathway in cancer

The Raf/MEK/extraceUular signal-regulated kinase （ERK） pathway has a pivotal role in facilitating cell proliferation, and its deregulated activation is a central signature of many epithelial cancers. However paradoxically, sustained activity of Raf/MEK/ERK can also result in growth arrest in many different cell types. This anti-proliferative Raf/MEK/ERK signaling also has physiological significance, as exemplified by its potential as a tumor suppressive mechanism. Therefore, significant questions include in which cell types and by what mechanisms this pathway can mediate such an opposing context of signaling. Particularly, our understating of the role of ERK1 and ERK2, the focal points of pathway signaling, in growth arrest signaling is still limited. This review discusses these aspects of Raf/MEK/ ERK-mediated growth arrest signaling.     

Uncoupling Raf1 from MEK1/2 impairs only a subset of cellular responses to Raf activation

The Raf family of serine/threonine protein kinases is intimately involved in the transmission of cell regulatory signals controlling proliferation and differentiation. The best characterized Raf substrates are MEK1 and MEK2. The activation of MEK1/2 by Raf is required to mediate many of the cellular responses to Raf activation, suggesting that MEK1/2 are the dominant Raf effector proteins. However, accumulating evidence suggests that there are additional Raf substrates and that subsets of Raf-induced regulatory events are mediated independently of Raf activation of MEK1/2. To examine the possibility that there is bifurcation at the level of Raf in activation of MEK1/2-dependent and MEK1/2-independent cell regulatory events, we engineered a kinase-active Raf1 variant (RafBXB(T481A)) with an amino acid substitution that disrupts MEK1 binding. We find that disruption of MEK1/2 association uncouples Raf from activation of ERK1/2, induction of serum-response element-dependent gene expression, and induction of growth and morphological transformation. However, activation of NF-kappaB-dependent gene expression and induction of neurite differentiation were unimpaired. In addition, Raf-dependent activation of p90 ribosomal S6 kinase was only slightly impaired. These results support the hypothesis that Raf kinases utilize multiple downstream effectors to regulate distinct cellular activities.