Search

We previously demonstrated that the mTORC1/S6K1 pathway is activated by insulin and nutrient overload (e.g. amino acids (AA)), which leads to the inhibition of the PI3K/Akt pathway via the inhibitory serine phosphorylation of IRS-1, notably on serine 1101 (Ser-1101). However, even in the absence of AA, insulin can still promote IRS-1 Ser-1101 phosphorylation by other kinases that remain to be fully characterized. Here, we describe a new negative regulator of IRS-1, the p90 ribosomal S6 kinase (RSK). Computational analyses revealed that Ser-1101 within IRS-1 falls into the consensus motif of RSK. Moreover, recombinant RSK phosphorylated IRS-1 C-terminal fragment on Ser-1101, which was prevented by mutations of this site or when a kinase-inactive mutant of RSK was used. Using antibodies directed toward the phosphorylation sites located in the activation segment of RSK (Ser-221 or Ser-380), we found that insulin activates RSK in L6 myocytes in the absence of AA overload. Inhibition of RSK using either the pharmacological inhibitor BI-D1870 or after adenoviral expression of a dominant negative RSK1 mutant (RSK1-DN) showed that RSK selectively phosphorylates IRS-1 on Ser-1101. Accordingly, expression of the RSK1-DN mutant in L6 myocytes and FAO hepatic cells improved insulin action on glucose uptake and glucose production, respectively. Furthermore, RSK1 inhibition prevented insulin resistance in L6 myocytes chronically exposed to high glucose and high insulin. These results show that RSK is a novel regulator of insulin signaling and glucose metabolism and a potential mediator of insulin resistance, notably through the negative phosphorylation of IRS-1 on Ser-1101.

Peroxisome proliferator-activated receptors (PPAR) decrease the production of cytokine and inducible nitric-oxide synthase (iNOS) expression, which are associated with aging-related inflammation and insulin resistance. Recently, the involvement of the induction of heme oxygenase-1 (HO-1) in regulating inflammation has been suggested, but the exact mechanisms for reducing inflammation by HO-1 remains unclear. We found that overexpression of HO-1 and [Ru(CO)(3)Cl(2)](2), a carbon monoxide (CO)-releasing compound, increased not only ERK5 kinase activity, but also its transcriptional activity measured by luciferase assay with the transfection of the Gal4-ERK5 reporter gene. This transcriptional activity is required for coactivation of PPARdelta by ERK5 in C2C12 cells. [Ru(CO)(3)Cl(2)](2) activated PPARdelta transcriptional activity via the MEK5/ERK5 signaling pathway. The inhibition of NF-kappaB activity by ERK5 activation was reversed by a dominant negative form of PPARdelta suggesting that ERK5/PPARdelta activation is required for the anti-inflammatory effects of CO and HO-1. Based on these data, we propose a new mechanism by which CO and HO-1 mediate anti-inflammatory effects via activating ERK5/PPARdelta, and ERK5 mediates CO and HO-1-induced PPARdelta activation via its interaction with PPARdelta.

Protein kinase C (PKC)-induced phosphorylation of cardiac troponin I (cTnI) depresses the acto-myosin interaction and may be important during the progression of heart failure. Although both PKCbetaII and PKCepsilon can phosphorylate cTnI, only PKCbeta expression and activity are elevated in failing human myocardium during end-stage heart failure. Furthermore, although increased cTnI phosphorylation was observed in mice with cardiac-specific PKCbeta II overexpression, no differences were observed in cTnI phosphorylation status between wild type and cardiac-specific PKCepsilon overexpression mice. A potentially important downstream effector of PKCs is p90 ribosomal S6 kinase (p90RSK), which plays an important role in cell growth by activating several transcription factors as well as Na+/H+ exchanger. Since both Ser23 and Ser24 of cTnI are contained in putative consensus sequences of p90RSK phosphorylation sites, we hypothesized that p90RSK is downstream from PKCbeta II and can be a cTnI (Ser(23/24)) kinase. p90RSK, but not ERK1/2 activation, was increased in PKCbetaII overexpression mice but not in PKCepsilon overexpression mice. p90RSK could phosphorylate cTnI in vitro with high substrate affinity but not cardiac troponin T (cTnT). To confirm the role of p90RSK in cTnI phosphorylation in vivo, we generated adenovirus containing a dominant negative form of p90RSK (Ad-DN-p90RSK). We found that the inhibition of p90RSK prevented H2O2-mediated cTnI (Ser(23/24)) phosphorylation but not ERK1/2 and PKCalpha/betaII activation. Next, we generated cardiac-specific p90RSK transgenic mice and observed that cTnI (Ser(23/24)) phosphorylation was significantly increased. LY333,531, a specific PKCbeta inhibitor, inhibited both p90RSK and cTnI (Ser(23/24)) phosphorylation by H2O2. Taken together, our data support a new redox-sensitive mechanism regulating cTnI phosphorylation in cardiomyocytes.

Endothelial cell (EC) migration contributes to reendothelialization after angioplasty or rupture of atherosclerotic plaques. Extracellular signal-regulated kinase (ERK)1/2 translocates to the nucleus and activates transcription factors such as Ets-like transcription factor-1 and early growth response factor-1 (Egr-1) during reendothelialization. Because ERK1/2 does not possess a nuclear localization signal (NLS), its mechanism of translocation and accumulation in the nucleus remains unclear. Because Gab1 has a putative NLS in its N-terminal region, and Gab1 associates with phosphorylated ERK1/2, we hypothesized that Gab1 participates in ERK1/2 and Egr-1 nuclear accumulation. Using regenerating EC as a model system, we found that endogenous growth factor receptor-bound protein 2-associated binder-1 (Gab1) translocates into the nucleus in migrating EC. Wild-type red fluorescent protein-tagged Gab1 could be observed in both nucleus and cytoplasm, whereas the putative NLS deletion mutant (deltaNLS-Gab1) specifically localized in the cytoplasm. In addition, reduction of Gab1 expression by antisense Gab1 oligos or overexpression of deltaNLS-Gab1 inhibited serum-induced ERK1/2 and Egr-1 nuclear accumulation, suggesting a functional role for the NLS of Gab1 and a role for Gab1-ERK1/2 interactions in ERK1/2-Egr-1 nuclear accumulation. To investigate whether Gab1-ERK1/2 interaction is critical for ERK1/2 and Egr-1 nuclear accumulation, we created a dominant-negative Gab1 construct that consisted of the c-Met binding domain (amino acids 442-536) of Gab1. We found that overexpression of the c-Met binding domain of Gab1 disrupted serum-induced Gab1-ERK1 interaction and inhibited ERK1 and Egr-1 nuclear accumulation. These data suggest that Gab1-ERK1/2 binding and their nuclear translocation play a crucial role in Egr-1 nuclear accumulation.

Dok-like adapter molecules represent an expanding family of pleckstrin homology (PH) and phosphotyrosine-binding (PTB) domain-containing tyrosine kinase substrates with negative regulatory functions in hematopoietic cell signaling. In a search for nonhematopoietic counterparts to Dok molecules, we identified and characterized Dok-4, a recently cloned member of the family. dok-4 mRNA was strongly expressed in nonhematopoietic organs, particularly the intestine, kidney, and lung, whereas both mRNA and protein were expressed at high levels in cells of epithelial origin. In Caco-2 human colon cancer cells, endogenous Dok-4 underwent tyrosine phosphorylation in response to pervanadate stimulation. In transfected COS cells, Dok-4 was a substrate for the cytosolic tyrosine kinases Src and Fyn as well as for Jak2. Dok-4 could also be phosphorylated by the receptor tyrosine kinase Ret but not by platelet-derived growth factor receptor-beta or IGF-IR. In both mammalian cells and yeast, Dok-4 was constitutively localized at the membrane in a manner that required both its PH and PTB domains. The PH and PTB domains of Dok-4 were also required for tyrosine phosphorylation of Dok-4 by Fyn and Ret. Finally, wild type Dok-4 strongly inhibited activation of Elk-1 induced by either Ret or Fyn. The attenuation of this inhibitory effect by deletion of the PH domain and its restoration by the addition of a myristoylation signal suggested an important role for constitutive membrane localization of Dok-4. In summary, Dok-4 is a constitutively membrane-localized adapter molecule that may function as an inhibitor of tyrosine kinase signaling in epithelial cells.

Big mitogen-activated protein kinase 1/extracellular-regulated kinase 5 (BMK1/ERK5) is regulated sequentially by a series of upstream MAP kinase kinases (MEKs) in a signaling cascade. MEKs activate their downstream MAPK by phosphorylation of threonine and tyrosine in the T- X-Y motif. MEK5 is the upstream BMK1 kinase and exists as naturally occurring splice variants, MEK5alpha and MEK5beta. The full-length MEK5 (MEK5alpha) is 89 amino acids longer than MEK5beta at the N terminus, but the precise functional difference between the two splice variants is not known. Dual phosphorylation site mutation of MEK5alpha (Ser-311 --Asp and Thr- 315 --Asp; MEK5alpha(S311D/T315D)) activated BMK1, but the corresponding dual phosphorylation sites mutant of MEK5beta could not induce BMK1 kinase activation or nuclear translocation. Furthermore, MEK5beta inhibited epidermal growth factor-induced BMK1 activation and MEK5alpha(S311D/T315D)-induced MEF2 transcriptional activity. Both MEK5alpha and MEK5beta individually co-immunoprecipitated with BMK1, but the presence of MEK5beta prevented association of MEK5alpha with BMK1 suggesting a mechanistic basis for the dominant-negative behavior of MEK5beta on BMK1 activation. The ratio of MEK5alpha to MEK5beta expression was higher in cancer cell lines, and overexpression of MEK5beta-inhibited serum-induced DNA synthesis. These data suggest that alternative splicing of MEK5alpha and MEK5beta may play a critical role in BMK1 activation and subsequent cell proliferation.

The gap junction protein, Cx43, plays a pivotal role in coupling cells electrically and metabolically, and the putative phosphorylation sites that modulate its function are reflected as changes in gap junction communication. Growth factor stimulation has been correlated with a decrease in gap junction communication and a parallel activation of ERK1/2; the inhibition of epidermal growth factor (EGF)-induced Cx43 gap junction uncoupling was observed by using the MEK1/2 inhibitor, PD98059. Because 1) BMK1/ERK5, another MAPK family member also activated by growth factors, possesses a phosphorylation motif similar to ERK1/2, and 2) it has been reported that PD98059 can inhibit not only MEK1/2-ERK1/2 but also MEK5-BMK1 activation, we investigated whether BMK1 can regulate EGF-induced Cx43 gap junction uncoupling and phosphorylation, comparing this to the role of ERK1/2 on Cx43 function and phosphorylation induced by EGF. Selective activation or inactivation of ERK1/2 by using a constitutively active form or a dominant negative form of MEK1 did not regulate Cx43 gap junction coupling. In contrast, we found that BMK1, selectively activated by constitutively active MEK5alpha, induced gap junction uncoupling, and the inhibition of BMK1 activation by transfection of dominant negative BMK1 prevented EGF-induced gap junction uncoupling. Activated BMK1 selectively phosphorylates Cx43 on Ser-255 in vitro and in vivo, but not on S279/S282, which are reported as the consensus phosphorylation sites for MAPK. Furthermore, by co-immunoprecipitation, we found that BMK1 directly associates with Cx43 in vivo. These data indicate that BMK1 is more important than ERK1/2 in EGF-mediated Cx43 gap junction uncoupling by association and Cx43 Ser- 255 phosphorylation.

Grb2-associated binder-1 (Gab1) is an adapter protein related to the insulin receptor substrate family. It is a substrate for the insulin receptor as well as the epidermal growth factor (EGF) receptor and other receptor-tyrosine kinases. To investigate the role of Gab1 in signaling pathways downstream of growth factor receptors, we stimulated rat aortic vascular smooth muscle cells (VSMC) with EGF and platelet-derived growth factor (PDGF). Gab1 was tyrosine-phosphorylated by EGF and PDGF within 1 min. AG1478 (an EGF receptor kinase-specific inhibitor) failed to block PDGF-induced Gab1 tyrosine phosphorylation, suggesting that transactivated EGF receptor is not responsible for this signaling event. Because Gab1 associates with phospholipase Cgamma (PLCgamma), we studied the role of the PLCgamma pathway in Gab1 tyrosine phosphorylation. Gab1 tyrosine phosphorylation by PDGF was impaired in Chinese hamster ovary cells expressing mutant PDGFbeta receptor (Y977F/Y989F: lacking the binding site for PLCgamma). Pretreatment of VSMC with (a specific PLCgamma inhibitor) inhibited Gab1 tyrosine phosphorylation as well, indicating the importance of the PLCgamma pathway. Gab1 was tyrosine-phosphorylated by phorbol ester to the same extent as PDGF stimulation. Studies using antisense protein kinase C (PKC) oligonucleotides and specific inhibitors showed that PKCalpha and PKCepsilon are required for Gab1 tyrosine phosphorylation. Binding of Gab1 to the protein-tyrosine phosphatase SHP2 and phosphatidylinositol 3-kinase was significantly decreased by PLCgamma and/or PKC inhibition, suggesting the importance of the PLCgamma/PKC-dependent Gab1 tyrosine phosphorylation for the interaction with other signaling molecules. Because PDGF-mediated ERK activation is enhanced in Chinese hamster ovary cells that overexpress Gab1, Gab1 serves as an important link between PKC and ERK activation by PDGFbeta receptors in VSMC.

c-Jun NH(2)-terminal kinase (JNK) is activated by a number of cellular stimuli such as inflammatory cytokines and environmental stresses. Reactive oxygen species also cause activation of JNK; however, the signaling cascade that leads to JNK activation remains to be elucidated. Because recent reports showed that expression of Cas, a putative Src substrate, stimulates JNK activation, we hypothesized that the Src kinase family and Cas would be involved in JNK activation by reactive oxygen species. An essential role for both Src and Cas was demonstrated. First, the specific Src family tyrosine kinase inhibitor, PP2, inhibited JNK activation by H(2)O(2) in a concentration-dependent manner but had no effect on extracellular signal-regulated kinases 1 and 2 and p38 activation. Second, JNK activation in response to H(2)O(2) was completely inhibited in cells derived from transgenic mice deficient in Src but not Fyn. Third, expression of a dominant negative mutant of Cas prevented H(2)O(2)-mediated JNK activation but had no effect on extracellular signal-regulated kinases 1 and 2 and p38 activation. Finally, the importance of Src was further supported by the inhibition of both H(2)O(2)-mediated Cas tyrosine phosphorylation and Cas.Crk complex formation in Src-/- but not Fyn-/- cells. These results demonstrate an essential role for Src and Cas in H(2)O(2)-mediated activation of JNK and suggest a new redox-sensitive pathway for JNK activation mediated by Src.

Mitogen-activated protein (MAP) kinases are a multigene family activated by many extracellular stimuli. There are three groups of MAP kinases based on their dual phosphorylation motifs, TEY, TPY, and TGY, which are termed extracellular signal-regulated protein kinases (ERK1/2), c-Jun N-terminal kinases, and p38, respectively. A new MAP kinase family member termed Big MAP kinase 1 (BMK1) or ERK5 was recently cloned. BMK1 has a TEY sequence similar to ERK1/2 but has unique COOH-terminal and loop-12 domains. To define BMK1 regulation, its activation in cultured rat vascular smooth muscle cells was characterized. Angiotensin II, phorbol ester, platelet-derived growth factor, and tumor necrosis factor-alpha were the strongest stimuli for ERK1/2 but were weak activators of BMK1. In contrast, H2O2 caused concentration-dependent activation of BMK1 but not ERK1/2. Sorbitol activated both BMK1 and ERK1/2. BMK1 activation by H2O2 was calcium-dependent and appeared ubiquitous as shown by stimulation in human skin fibroblasts, human vascular smooth muscle cells, and human umbilical vein endothelial cells. These findings demonstrate that activation of BMK1 is different from ERK1/2 and suggest an important role for BMK1 as a redox-sensitive kinase.