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.
IL-10 has proved to be a key cytokine in regulating inflammatory responses by controlling the production and function of various other cytokines. The suppressor of cytokine signaling (SOCS) gene products are a family of cytoplasmic molecules that are essential mediators for negatively regulating cytokine signaling. It has been previously shown that IL-10 induced SOCS3 expression and that forced constitutive expression of SOCS3 inhibits IL-10/STAT3 activation and LPS-induced macrophage activation. In this report, we show that, in addition to SOCS3 expression, IL-10 induces SOCS1 up-regulation in all cell lines tested, including Ba/F3 pro-B cells, MC/9 mast cells, M1 leukemia cells, U3A human fibroblasts, and primary mouse CD4+ T cells. Induction of SOCS molecules is dependent on STAT3 activation by IL-10R1. Cell lines constitutively overexpressing SOCS proteins demonstrated that SOCS1 and SOCS3, but not SOCS2, are able to partially inhibit IL-10-mediated STAT3 activation and proliferative responses. Pretreatment of M1 cells with IFN-γ resulted in SOCS1 induction and a reduction of IL-10-mediated STAT3 activation and cell growth inhibition. IL-10-induced SOCS is associated with the inhibition of IFN-γ signaling in various cell types, and this inhibition is independent of C-terminal serine residues of the IL-10R, previously shown to be required for other anti-inflammatory responses. Thus, the present results show that both SOCS1 and SOCS3 are induced by IL-10 and may be important inhibitors of both IL-10 and IFN-γ signaling. IL-10-induced SOCS1 may directly inhibit IL-10 IFN-γ signaling, while inhibition of other proinflammatory cytokine responses may use additional IL-10R1-mediated mechanisms.
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.The extracellular signal-regulated protein kinases (ERKs) are ubiquitous protein kinases that function downstream of the ras oncogene and are involved in many cellular responses, including adhesion and migration (43). Ras is activated in response to multiple extracellular stimuli and in turn regulates multiple downstream signaling pathways (7), including the Raf protein kinases (58, 60). Raf proteins phosphorylate and activate MEKs (mitogen-activated protein [MAP] kinase or ERKs) (16,30,37,62), which phosphorylate and activate ERK1 and ERK2 on a TEY sequence in the ERK catalytic domain (27,44).Propagation of the Ras/Raf/MEK/ERK signal is modulated by a variety of inputs that must be integrated to achieve a full signaling response. Cell adhesion is required to couple Ras activation to MEK and ERK activation, as stimulation of suspended cells with growth factors leads to Ras and possibly Raf activation; however, activation of MEK and ERK fails to occur (38, 47). Signal transduction through the Ras-ERK pathway can be enhanced by the Rho family GTPases Rac and Cdc42 through their effector p21-activated kinase (PAK) (12,23,24), and Rac and Raf can synergize to promote cellular transformation (31). Expression of constitutively active Rac or Cdc42 activates the Jun N-terminal kinases (JNKs) and p38 kinases but not the ERKs (14). However, Rac and Cdc42 are able to synergize with Raf to stimulate ERK activation through mechanisms involving PAK1 phosphorylation of the MEK1 prolinerich sequence (PRS) (12,23,54) and PAK3 phosphorylation of Raf-1 (11, 32). PAK1 has been reported to enhance the phosphorylation of T292 and S298 of MEK1 in cells, although only S2...
Signal transduction occurs by the reversible assembly of oligomeric protein complexes that include both enzymatic proteins and proteins without known enzymatic activity. These nonenzymatic components can serve as scaffolds or anchors and regulate the efficiency, specificity, and localization of the signaling pathway. Here we report the identification of MORG1 (mitogen-activated protein kinase organizer 1), a member of the WD-40 protein family that was isolated as a binding partner of the extracellular signalregulated kinase (ERK) pathway scaffold protein MP1. MORG1 specifically associates with several components of the ERK pathway, including MP1, Raf-1, MEK, and ERK, and stabilizes their assembly into an oligomeric complex. MORG1 facilitates ERK activation when cells are stimulated with lysophosphatidic acid, phorbol 12-myristate 13-acetate, or serum, but not in response to epidermal growth factor. Suppression of MORG1 by short interfering RNA leads to a marked reduction in ERK activity when cells are stimulated with serum. We propose that MORG1 is a component of a modular scaffold system that participates in the regulation of agonist-specific ERK signaling.
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