Non-redundant role of Shc in Erk activation by cytoskeletal reorganization

We have shown previously that cytoskeletal reorganization (CSR) induced by pharmacological reagents such as colchicine or cytochalasins can up-regulate the urokinase-type plasminogen activator (uPA) gene via the Ras/Erk signaling pathway. In this present study using the small interfering RNA technique, we have found that ShcA adapter proteins play a rather active role in CSR-induced Erk activation, contrary to their mostly redundant role in other signaling pathways, e.g. growth factor-induced Erk activation, where Grb2 can bind directly to the receptor tyrosine kinase and activate Erk in the absence of ShcA. ShcA knockdown abolished CSR-induced activation of both Erk and the uPA promoter. Expression of small interfering RNA-escaping silent mutants of p52 or p46 but not p66 ShcA isoform efficiently rescued CSR-induced Erk activation. Moreover, we have shown that phosphorylation of either Tyr-239/Tyr-240 or Tyr-313 in p52(ShcA) can mediate CSR-induced Erk activation equally well. In a quest for molecules upstream of ShcA in this signaling, we found that CSR-induced ShcA tyrosine phosphorylation, its association with Grb2, Erk activation, and uPA gene expression were all dependent on Rho kinase, p38 mitogen-activated protein kinase, and Src. In summary, we have found a novel, non-redundant role for ShcA in contrast to its redundant role in many other signaling pathways.     

CrkL plays a role in SDF-1-induced activation of the Raf-1/MEK/Erk pathway through Ras and Rac to mediate chemotactic signaling in hematopoietic cells

Intracellular signaling mechanisms regulating SDF-1-induced chemotaxis of hematopoietic cells have remained elusive. Here we demonstrate that overexpression of the adaptor molecule CrkL enhances SDF-1-induced chemotaxis of hematopoietic BaF3 and 32Dcl3 cells. Overexpression of CrkL also enhanced SDF-1-induced activation of the Raf-1/MEK/Erk signaling pathway as well as that of the small GTPases Ras, Rap1, and Rac, while a dominant negative mutant of Ras or Rac suppressed CrkL-enhanced Erk activation. SDF-1 stimulation induced tyrosine phosphorylation of CrkL, which was inhibited by the Src family kinase inhibitor PP1 or by dominant negative mutants of Lyn, thus indicating that Lyn mediated SDF-1-induced phosphorylation of CrkL. However, inhibition of the Lyn kinase activity failed to affect SDF-1-induced activation of the small GTPases and Erk. On the other hand, SDF-1-induced activation of the Erk signaling pathway as well as chemotaxis was inhibited by overexpression of a CrkL mutant lacking the N-terminal SH3 domain, which mediates interaction with various signaling molecules including guanine nucleotide exchange factors for the Ras and Rho family GTPases. SDF-1-induced chemotaxis was also inhibited by the dominant negative Ras or Rac mutant as well as by the MEK inhibitor PD98059. These results indicate that CrkL mediates SDF-1-induced activation of the Raf-1/MEK/Erk signaling pathway through Ras as well as Rac in hematopoietic cells and, thereby, plays important roles in the induction of chemotactic response.     

Activation of integrin β1-focal adhesion kinase-RasGTP pathway plays a critical role in TGF beta1-induced podocyte injury

The depletion of glomerular podocytes is the key mechanism of glomerulosclerosis and progressive renal failure. Transforming growth factor-β (TGFβ) is a central mediator of signaling networks that control a diverse set of cellular processes, such as cell proliferation, differentiation, and apoptosis. Though many key events in TGFβ1 signaling have been documented at cellular and molecular level in podocytes, the complete effects of TGFβ1 on podocyte integrity are still elusive. In this study, the function of adhesion protein integrin β1, focal adhesion kinase (FAK), and a small GTPase Ras was explored in TGFβ1-induced podocyte injury. In cultured mouse podocyte, caspase 3-positive cells were counted by flow cytometry to evaluate podocyte damage at different time points after TGFβ1 treatment. Immunoblotting assay showed that integrin β1, FAK, Src kinase, and an adaptor protein Grb2 were activated rapidly after TGFβ1 stimulation. Active Ras Pull-Down assay revealed that the active Ras (GTP-bound Ras) level was upregulated in TGFβ1-treated cell. Immunoprecipitation results displayed that TGFβ1 enhanced the complex formation of integrin β1, FAK and Src kinase, as well as FAK, Grb2 and Ras. The FAK inhibitor TAE226 and the specific knockdown of Grb2 remarkably alleviated TGFβ1-induced podocyte apoptosis. The activation of p38MAPK and Erk1/2, and the nuclear translocation of NFκB(p65) were increased evidently in TGFβ1-treated cell, which could be dramatically prohibited by the application of the p38MAPK inhibitor SB202190 and the Ras inhibitor FPT Inhibitor III. The Src kinase inhibitor PP2 obviously prevented the activation of FAK and Ras, as well as the translocation of NFκB(p65) from cytoplasm to nuclei. The PP2, FPT Inhibitor III, and SB202190 significantly decreased TGFβ1-induced podocyte apoptosis. Taken together, these data demonstrated that the activation of integrin β1/Src/FAK and Grb2/RasGTP should be responsible for TGFβ1-induced podocyte damage through the p38MAPK and Erk1/2-mediated nuclear translocation of NFκB(p65).     

Csk homologous kinase (CHK), unlike Csk, enhances MAPK activation via Ras-mediated signaling in a Src-independent manner

Substantial evidence exists supporting the notion that Csk and CHK, two negative regulatory kinases of the Src tyrosine kinase family, play distinct roles during development of the nervous system. One of the differences relies on the effects of both kinases on the MAPK transduction pathway. Specifically, CHK was shown to enhance MAPK signaling, while the role of Csk was unclear. In this work, we compared the effect of CHK versus Csk on MAPK signaling and elucidated the signaling pathway mediated by CHK leading to the activation of Erk1/2. Exogenous expression of wild-type CHK, but not Csk or a dead-kinase mutant of CHK, resulted in enhanced Erk1/2 phosphorylation in PC12 cells. CHK inhibited Src activity following stimulation of the cells with NGF. However, stimulation of Erk1/2 activation by CHK was independent of the NGF stimulation or the inhibition of Src kinase by CHK. CHK induced a complex formation between SHP-2 and Grb2, subsequently leading to the increased activity of Ras as well as Erk1/2 activation via the Raf/MEK1/2 pathway. Down-regulation of the expression of endogenous CHK by RNAi in PC12 cells led to a significant decrease in MAPK activation following NGF stimulation. Stimulation of CHK-overexpressing PC12 cells with EGF induced neurite outgrowth in the majority of cells. Taken together, this study describes for the first time the Src-independent actions of CHK and provides novel insights into CHK function in neural cells.     

Cytoskeletal reorganization and TPA differently modify AP-1 to induce the urokinase-type plasminogen activator gene in LLC-PK1 cells.

Urokinase-type plasminogen activator (uPA) is an extracellular protease and expressed in various cells that exhibit dynamic changes in cell morphology, suggesting a link between cytoskeletal reorganization (CSR) and uPA expression. CSR can be induced by pharmacological agents, such as by colchicine for microtubule cytoskeleton and by cytochalasin for microfilament cytoskeleton. Using these agents, we previously showed that CSR induced the uPA gene in LLC-PK1 cells independently of the protein kinase C and cAMP-dependent protein kinase. Here we show that the induction of the uPA gene by CSR is mediated by the activation of c-Jun which interacts with an AP-1-like site located 2 kb upstream of the uPA gene. 12-O-tetradecanoylphorbol 13-acetate (TPA) induces the uPA gene through the same elements, but additionally utilizes an adjacent PEA3 element and induces c-fos. Furthermore, CSR induces a greater accumulation and a more pronounced phosphorylation of c-Jun than TPA induction. AP-1 is a positive regulator of growth and oncogenesis, and CSR is an integral part of these processes. Our results provide a view how CSR and AP-1 could be coupled in these processes. We also show that TPA and CSR act synergistically, suggesting a model where an initial activation signal could be amplified by CSR.     

BCR/ABL activates Rap1 and B-Raf to stimulate the MEK/Erk signaling pathway in hematopoietic cells

The BCR/ABL fusion tyrosine kinase activates various intracellular signaling pathways, thus causing chronic myeloid leukemia (CML). Here we demonstrate that the inducible expression of BCR/ABL in a murine hematopoietic cell line, TonB210, leads to the activation of the Ras family small GTPase Rap1, which is inhibited by the ABL kinase inhibitor imatinib. The Rap1 activity in a CML cell line, K562, was also inhibited by imatinib. Inhibition of Rap1 activation by a dominant negative mutant of Rap1, Rap1-N17, or SPA-1 inhibited the BCR/ABL-induced activation of Elk-1. BCR/ABL also activated in a kinase activity-dependent manner the B-Raf kinase, which is an effector molecule of Rap1 and a potent activator of the MEK/Erk/Elk-1 signaling pathway. Together, these data suggest that, in addition to the well-established Ras/Raf-1 pathway, BCR/ABL activates the alternative signaling pathway involving Rap1 and B-Raf to activate Erk, which may play important roles in leukemogenesis.     

Fak/Src signaling in human intestinal epithelial cell survival and anoikis: differentiation state-specific uncoupling with the PI3-K/Akt-1 and MEK/Erk pathways

Human intestinal epithelial cell survival and anoikis are distinctively regulated according to the state of differentiation. In the present study, we analyzed the roles of focal adhesion kinase (Fak)/Src signaling to the PI3-K/Akt-1 and mitogen-activated protein kinase (MEK)/extracellular regulated kinases (Erk) pathways, within the context of such differentiation-state distinctions. Anoikis was induced by inhibition of beta1 integrins (antibody blocking), inhibition of Fak (pharmacologic inhibition or overexpression of dominant negative mutants), or by maintaining cells in suspension. Activation parameters of Fak, Src, Akt-1, and Erk1/2 were analyzed. Activities of Src, Akt-1, or Erk1/2 were also blocked by pharmacological inhibition or by overexpression of dominant-negative mutants. We report that: (1) the loss or inhibition of beta1 integrin binding activity causes anoikis and results in a down-activation of Fak, Src, Akt-1, and Erk1/2 in both undifferentiated, and differentiated cells; (2) the inhibition of Fak likewise causes anoikis and a down-activation of Src, Akt-1, and Erk1/2, regardless of the differentiation state; (3) Src, PI3-K/Akt-1, and MEK/Erk contribute to the survival of differentiated cells, whereas MEK/Erk does not play a role in the survival of undifferentiated ones; (4) the inhibition/loss of beta1 integrin binding and/or Fak activity results in a loss of Src engagement with Fak, regardless of the state of differentiation; and (5) Src contributes to the activation of both the PI3-K/Akt-1 and MEK/Erk pathways in undifferentiated cells, but does not influence PI3-K/Akt-1 in differentiated ones. Hence, Fak/Src signaling to the PI3-K/Akt-1 and MEK/Erk pathways undergoes a differentiation state-specific uncoupling which ultimately reflects upon the selective engagement of these same pathways in the mediation of intestinal epithelial cell survival.     

OipA plays a role in Helicobacter pylori-induced focal adhesion kinase activation and cytoskeletal re-organization

The initial signalling events leading to Helicobacter pylori infection associated changes in motility, cytoskeletal reorganization and elongation of gastric epithelial cells remain poorly understood. Because focal adhesion kinase (FAK) is known to play important roles in regulating actin cytoskeletal organization and cell motility we examined the effect of H. pylori in gastric epithelial cells co-cultured with H. pylori or its isogenic cag pathogenicity island (PAI) or oipA mutants. H. pylori induced FAK phosphorylation at distinct tyrosine residues in a dose- and time-dependent manner. Autophosphorylation of FAK Y397 was followed by phosphorylation of Src Y418 and resulted in phosphorylation of the five remaining FAK tyrosine sites. Phosphorylated FAK and Src activated Erk and induced actin stress fibre formation. FAK knock-down by FAK-siRNA inhibited H. pylori- mediated Erk phosphorylation and abolished stress fibre formation. Infection with oipA mutants reduced phosphorylation of Y397, Y576, Y577, Y861 and Y925, inhibited stress fibre formation and altered cell morphology. cag PAI mutants reduced phosphorylation of only FAK Y407 and had less effect on stress fibre formation than oipA mutants. We propose that activation of FAK and Src are responsible for H. pylori -induced induction of signalling pathways resulting in the changes in cell phenotype important for pathogenesis.     

Role of atypical protein kinase C in estradiol-triggered G1/S progression of MCF-7 cells

Expression of a dominant negative atypical protein kinase C (aPKC), PKCzeta, prevents nuclear translocation of extracellular regulated kinase 2 (ERK-2), p27 nuclear reduction, and DNA synthesis induced by estradiol in human mammary cancer-derived MCF-7 cells. aPKC action upstream of these events has been analyzed. In hormone-stimulated NIH 3T3 and Cos cells ectopically expressing human estrogen receptor alpha (hERalpha), aPKC is activated by phosphatidylinositol 3-kinase (PI 3-kinase) and, in turn, controls the Ras/MEK-1/ERK cascade. In MCF-7 and Cos cells stimulated by hormone, PI 3-kinase activates PKCzeta by Thr410 phosphorylation. Serine phosphorylation of PKCzeta is simultaneously induced. PKCzeta activation leads to recruitment of Ras to a multimolecular complex that also includes hERalpha, Src, PI 3-kinase, and aPKC. We propose that PKCzeta pushes Ras and the signaling complex close together in such a way that it facilitates the Src-dependent Ras activation. This activation is crucial for the interplay between estradiol-triggered signaling and cell cycle machinery.     

Phosphospecific antibodies reveal focal adhesion kinase activation loop phosphorylation in nascent and mature focal adhesions and requirement for the autophosphorylation site.

Focal adhesion kinase (FAK) is a key signaling molecule regulating cellular responses to integrin-mediated adhesion. Integrin engagement promotes FAK phosphorylation at multiple sites to achieve full FAK activation. Phosphorylation of FAK Tyr-397 creates a binding site for Src-family kinases, and phosphorylation of FAK Tyr-576/Tyr-577 in the kinase domain activation loop enhances catalytic activity. Using novel phosphospecific antibody reagents, we show that FAK activation loop phosphorylation is significantly elevated in cells expressing activated Src and is an early event following cell adhesion to fibronectin. In both cases, this regulation is largely dependent on Tyr-397. We also show that the FAK activation loop tyrosines are required for maximal Tyr-397 phosphorylation. Finally, immunostaining analyses revealed that tyrosine-phosphorylated forms of FAK are present in both newly forming and mature focal adhesions. Our findings support a model for reciprocal activation of FAK and Src-family kinases and suggest that FAK/Src signaling may occur during both focal adhesion assembly and turnover.     

Polyamine-dependent activation of Rac1 is stimulated by focal adhesion-mediated Tiam1 activation

Integrin receptors cluster on the cell surface and bind to extra cellular matrix (ECM) proteins triggering the formation of focal contacts and the activation of various signal transduction pathways that affect the morphology, motility, gene expression and survival of adherent cells. Polyamine depletion prevents the increase in autophosphorylation of focal adhesion kinase (FAK) and Src during attachment. Rac activity also shows a steady decline, and its upstream guanine nucleotide exchange factor (GEF), Tiam1 also shows a reduction in total protein level when cells are depleted of polyamines. When Tiam1 and Rac1 interaction was inhibited by NSC-23766, there was not only a decrease in Rac1 activity as expected but also a decrease in FAK auto-phosphorylation. Inhibition of Src activity by PP2 also reduced FAK auto-phosphorylation, which implies that Src modulates FAK autophosphorylation. From the data obtained in this study we conclude that FAK and Src are rapidly activated upon fibronectin mediated signaling leading to Tiam1-mediated Rac1 activation and that intracellular polyamines influence the signaling strength by modulating interaction of Src with Tiam1 using focal adhesion kinase as a scaffolding site.     

Regulation of the mitogen-activated protein kinase signaling pathway by SHP2.

Gab1-SHP2 association is required for Erk mitogen-activated protein kinase activation by several growth factors. Gab1-SHP2 interaction activates SHP2. However, an activated SHP2 still needs to associate with Gab1 to mediate Erk activation. It was unclear whether SHP2 is required to dephosphorylate a negative phosphorylation site on Gab1 or whether SHP2 needs the Gab1 pleckstrin homology (PH) domain to target it to the plasma membrane. We found that expression of a fusion protein consisting of the Gab1 PH domain and an active SHP2 (Gab1PH-SHP2DeltaN) induced constitutive Mek1 and Erk2 activation. Linking the active SHP2DeltaN to the PDK1 PH domain or the FRS2beta myristoylation sequence also induced Mek1 activation. Mek1 activation by Gab1PH-SHP2DeltaN was inhibited by an Src inhibitor and by Csk. Significantly, Gab1PH-SHP2DeltaN induced Src activation. Gab1PH-SHP2DeltaN expression activated Ras, and the Gab1PH-SHP2DeltaN-induced Mek1 activation was blocked by RasN17. These findings suggest that Gab1PH-SHP2DeltaN activated a signaling step upstream of Src and Ras. The SHP2 tyrosine phosphatase activity is essential for the function of the fusion protein. Together, these data show that the Gab1 sequence, besides the PH domain and SHP2 binding sites, is dispensable for Erk activation, suggesting that the primary role of Gab1 association with an activated SHP2 is to target it to the membrane.     

Mechanism of 17-beta-estradiol-induced Erk1/2 activation in breast cancer cells. A role for HER2 AND PKC-delta

Activation of mitogen-activated protein kinase (Erk/MAPK) is a critical signal transduction event for estrogen (E(2))-mediated cell proliferation. Recent studies from our group and others have shown that persistent activation of Erk plays a major role in cell migration and tumor progression. The signaling mechanism(s) responsible for persistent Erk activation are not fully characterized, however. In this study, we have shown that E(2) induces a slow but persistent activation of Erk in MCF-7 breast carcinoma cells. The E(2)-induced Erk activation is dependent on new protein synthesis, suggesting that E(2)-induced growth factors play a major role in Erk activation. When MCF-7 cells were treated with E(2) in the presence of an anti-HER-2 monoclonal antibody (herceptin), 60-70% of E(2)-induced Erk activation is blocked. In addition, when untreated MCF-7 cells were exposed to conditioned medium from E(2)-treated cells, Erk activity was significantly enhanced. Furthermore Erk activity was blocked by an antibody against HER-2 or by heregulin (HRG) depletion from the conditioned medium through immunoprecipitation. In contrast, epidermal growth factor receptor (Ab528) antibody only blocked 10-20% of E(2)-induced Erk activation, suggesting that E(2)-induced Erk activation is predominantly mediated through the secretion of HRG and activation of HER-2 by an autoctine/paracrine mechanism. Inhibition of PKC-delta-mediated signaling by a dominant negative mutant or the relatively specific PKC-delta inhibitor rottlerin blocked most of the E(2)-induced Erk activation but had no effect on TGF alpha-induced Erk activation. By contrast inhibition of Ras, by inhibition of farnesyl transferase (Ftase-1) or dominant negative (N17)-Ras, significantly inhibited both E(2)- and TGF alpha-induced Erk activation. This evaluation of downstream signaling revealed that E(2)-induced Erk activation is mediated by a HRG/HER-2/PKC-delta/Ras pathway that could be crucial for E(2)-dependent growth-promoting effects in early stages of tumor progression.     

Three-dimensional matrix induces sustained activation of ERK1/2 via Src/Ras/Raf signaling pathway

Research in cell signaling often depends on tissue culture, but the artificial substrates used to grow cells in vitro are likely to distort the conclusions, particularly when adhesion-mediated signaling events are investigated. Studies of signal transduction pathways operating in cells grown in three-dimensional (3D) matrices provide a better system, giving a closer insight of the cell signaling in vivo. We compared the steady-state levels of ERK1/2 activity in primary human fibroblasts, induced by cell-derived 3D fibronectin matrix or fibronectin, coated on flat surfaces. 3D environment caused ERK1/2 stimulation concomitant with a 2.5-fold increase in Ras GTP loading and Src activation. Under these conditions FAK autophosphorylation was suppressed. Treatment with Src inhibitor PP2 abolished these effects indicating that 3D fibronectin matrix activated ERK1/2 through Src/Ras/Raf pathway, bypassing FAK. These observations suggest that within in vivo-like conditions Src may have a leading role in the induction of sustained ERK1/2 activation.     

Polyamine-dependent activation of Rac1 is stimulated by focal adhesion-mediated Tiam1 activation

Integrin receptors cluster on the cell surface and bind to extra cellular matrix (ECM) proteins triggering the formation of focal contacts and the activation of various signal transduction pathways that affect the morphology, motility, gene expression and survival of adherent cells. Polyamine depletion prevents the increase in autophosphorylation of focal adhesion kinase (FAK) and Src during attachment. Rac activity also shows a steady decline, and its upstream guanine nucleotide exchange factor (GEF), Tiam1 also shows a reduction in total protein level when cells are depleted of polyamines. When Tiam1 and Rac1 interaction was inhibited by NSC-23766, there was not only a decrease in Rac1 activity as expected but also a decrease in FAK auto-phosphorylation. Inhibition of Src activity by PP2 also reduced FAK autophosphorylation, which implies that Src modulates FAK autophosphorylation. From the data obtained in this study we conclude that FAK and Src are rapidly activated upon fibronectin mediated signaling leading to Tiam1-mediated Rac1 activation and that intracellular polyamines influence the signaling strength by modulating interaction of Src with Tiam1 using focal adhesion kinase as a scaffolding site.Key words: fibronectin, DFMO, polyamines, FAK, Src