A collapsin response mediator protein 2 isoform controls myosin II-mediated cell migration and matrix assembly by trapping ROCK II

Collapsin response mediator protein 2 (CRMP-2) is known as a regulator of neuronal polarity and differentiation through microtubule assembly and trafficking. Here, we show that CRMP-2 is ubiquitously expressed and a splice variant (CRMP-2L), which is expressed mainly in epithelial cells among nonneuronal cells, regulates myosin II-mediated cellular functions, including cell migration. While the CRMP-2 short form (CRMP-2S) is recognized as a substrate of the Rho-GTP downstream kinase ROCK in neuronal cells, a CRMP-2 complex containing 2L not only bound the catalytic domain of ROCK II through two binding domains but also trapped and inhibited the kinase. CRMP-2L protein levels profoundly affected haptotactic migration and the actin-myosin cytoskeleton of carcinoma cells as well as nontransformed epithelial cell migration in a ROCK activity-dependent manner. Moreover, the ectopic expression of CRMP-2L but not -2S inhibited fibronectin matrix assembly in fibroblasts. Underlying these responses, CRMP-2L regulated the kinase activity of ROCK II but not ROCK I, independent of GTP-RhoA levels. This study provides a new insight into CRMP-2 as a controller of myosin II-mediated cellular functions through the inhibition of ROCK II in nonneuronal cells.     

Mechanical regulation of glycogen synthase kinase 3β (GSK3β) in mesenchymal stem cells is dependent on Akt protein serine 473 phosphorylation via mTORC2 protein

Mechanical signals can inactivate glycogen synthase kinase 3β (GSK3β), resulting in stabilization of β-catenin. This signaling cascade is necessary for the inhibition of adipogenesis in mesenchymal stem cells (MSC) that is produced by a daily strain regimen. We investigated whether Akt is the mechanically activated kinase responsible for phosphorylation and inactivation of GSK3β in MSC. Mechanical strain (2% magnitude, 0.17 Hz) induced phosphorylation of Akt at Ser-473 and Thr-308 in parallel with phosphorylation of GSK3β at Ser-9. Inhibiting Akt (Akt1/2 kinase inhibitor treatment or Akt knockdown) prevented strain-induced phosphorylation of GSK3β at Ser-9. Inhibition of PI3K prevented Thr-308 phosphorylation, but strain-induced Ser-473 phosphorylation was measurable and induced phosphorylation of GSK3β, suggesting that Ser-473 phosphorylation is sufficient for the downstream mechanoresponse. As Rictor/mTORC2 (mammalian target of rapamycin complex 2) is known to transduce phosphorylation of Akt at Ser-473 by insulin, we investigated whether it contributes to strain-induced Ser-473 phosphorylation. Phosphorylation of Ser-473 by both mechanical and insulin treatment in MSC was prevented by the mTOR inhibitor KU0063794. When mTORC2 was blocked, mechanical GSK3β inactivation was prevented, whereas insulin inhibition of GSK3β was still measured in the absence of Ser-473 phosphorylation, presumably through phosphorylation of Akt at Thr-308. In sum, mechanical input initiates a signaling cascade that is uniquely dependent on mTORC2 activation and phosphorylation of Akt at Ser-473, an effect sufficient to cause inactivation of GSK3β. Thus, mechanical regulation of GSK3β downstream of Akt is dependent on phosphorylation of Akt at Ser-473 in a manner distinct from that of growth factors. As such, Akt reveals itself to be a pleiotropic signaling molecule whose downstream targets are differentially regulated depending upon the nature of the activating input.     

Phosphorylation of LCRMP-1 by GSK3β promotes filopoda formation, migration and invasion abilities in lung cancer cells

LCRMP-1, a novel isoform of CRMP-1, can promote cancer cell migration, invasion and associate with poor clinical outcome in patients with non-small-cell lung cancer (NSCLC). However, the underlying regulatory mechanisms of LCRMP-1 in cancer cell invasiveness still remain obscure. Here, we report that GSK3β can phosphorylate LCRMP-1 at Thr-628 in consensus sequences and this phosphorylation is crucial for function of LCRMP-1 to promote filopodia formation, migration and invasion in cancer cells. Impediment of Thr-628 phosphorylation attenuates the stimulatory effects of LCRMP-1 on filopodia forming, migration and invasion abilities in cancer cells; simultaneously, kinase-dead GSK3β diminishes regulation of LCRMP-1 on cancer cell invasion. Furthermore, we also found that patients with low-level Ser-9-phosphorylated GSK3β expression and high-level LCRMP-1 expression have worse overall survival than those with high-level inactive GSK3β expressions and low-level LCRMP-1 expressions (P<0.0001). Collectively, these results demonstrate that GSK3β-dependent phosphorylation of LCRMP-1 provides an important mechanism for regulation of LCRMP-1 on cancer cell invasiveness and clinical outcome.     

Phosphatidylinositide 3-kinase regulates angiotensin II-induced cytosolic phospholipase A2 activity and growth in vascular smooth muscle cells

Angiotensin (Ang) II via the AT(1) receptor acts as a mitogen in vascular smooth muscle cells (VSMC) through stimulation of multiple signaling mechanisms, including tyrosine kinases and mitogen-activated protein kinase (MAPK). In addition, cytosolic phospholipase A(2)(cPLA(2))-dependent release of arachidonic acid (AA) is linked to VSMC growth and we have reported that Ang II stimulates cPLA(2) activity via the AT(1) receptor. The coupling of Ang II to the activation of cPLA(2) appears to involve mechanisms both upstream and downstream of MAPK such that AA stimulates MAPK activity which phosphorylates cPLA(2) to further enhance AA release. However, the upstream mechanisms responsible for activation of cPLA(2) are not well-defined. One possibility includes phosphatidylinositide 3-kinase (PI3K), since PI3K has been reported to participate in the upstream signaling events linked to activation of MAPK. However, it is not known whether PI3K is involved in the Ang II-induced activation of cPLA(2) or if this mechanism is associated with the Ang II-mediated growth of VSMC. Therefore, we used cultured rat VSMC to examine the role of PI3K in the Ang II-dependent phosphorylation of cPLA(2), release of AA, and growth induced by Ang II. Exposure of VSMC to Ang II (100 nM) increased [(3)H]thymidine incorporation, cell number, and the release of [(3)H]AA. Also, using Western analysis, Ang II increased the phosphorylation of MAPK and cPLA(2) which were blocked by the MAPK kinase inhibitor PD98059 (10 microM/L). Similarly, the PI3K inhibitor LY294002 (10 microM/L) abolished the Ang II-mediated increase in MAPK phosphorylation, as well as phosphoserine-PLA(2). Further, inhibition of PI3K blocked the Ang II-induced release of AA and VSMC mitogenesis. However, exogenous AA was able to restore VSMC growth in the presence of LY294002, as well as reverse the inhibition of MAPK and cPLA(2) phosphorylation by LY294002. Thus, it appears from these data that Ang II stimulates the PI3K-sensitive release of AA which stimulates MAPK to phosphorylate cPLA(2) and enhance AA release. This mechanism may play an important role in the Ang II-induced growth of VSMC.     

Src homology domain 2-containing protein-tyrosine phosphatase-1 (SHP-1) binds and dephosphorylates G(alpha)-interacting, vesicle-associated protein (GIV)/Girdin and attenuates the GIV-phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway

GIV (Gα-interacting vesicle-associated protein, also known as Girdin) is a bona fide enhancer of PI3K-Akt signals during a diverse set of biological processes, e.g. wound healing, macrophage chemotaxis, tumor angiogenesis, and cancer invasion/metastasis. We recently demonstrated that tyrosine phosphorylation of GIV by receptor and non-receptor-tyrosine kinases is a key step that is required for GIV to directly bind and enhance PI3K activity. Here we report the discovery that Src homology 2-containing phosphatase-1 (SHP-1) is the major protein-tyrosine phosphatase that targets two critical phosphotyrosines within GIV and antagonizes phospho-GIV-dependent PI3K enhancement in mammalian cells. Using phosphorylation-dephosphorylation assays, we demonstrate that SHP-1 is the major and specific protein-tyrosine phosphatase that catalyzes the dephosphorylation of tyrosine-phosphorylated GIV in vitro and inhibits ligand-dependent tyrosine phosphorylation of GIV downstream of both growth factor receptors and GPCRs in cells. In vitro binding and co-immunoprecipitation assays demonstrate that SHP-1 and GIV interact directly and constitutively and that this interaction occurs between the SH2 domain of SHP-1 and the C terminus of GIV. Overexpression of SHP-1 inhibits tyrosine phosphorylation of GIV and formation of phospho-GIV-PI3K complexes, and specifically suppresses GIV-dependent activation of Akt. Consistently, depletion of SHP-1 enhances peak tyrosine phosphorylation of GIV, which coincides with an increase in peak Akt activity. We conclude that SHP-1 antagonizes the action of receptor and non-receptor-tyrosine kinases on GIV and down-regulates the phospho-GIV-PI3K-Akt axis of signaling.     

Deoxycholyltaurine rescues human colon cancer cells from apoptosis by activating EGFR-dependent PI3K/Akt signaling

Recent studies indicate that secondary bile acids promote colon cancer cell proliferation but their role in maintaining cell survival has not been explored. We found that deoxycholyltaurine (DCT) markedly attenuated both unstimulated and TNF-alpha-stimulated programmed cell death in colon cancer cells by a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. To examine the role of bile acids and PI3K signaling in maintaining colon cancer cell survival, we explored the role of signaling downstream of bile acid-induced activation of the epidermal growth factor receptor (EGFR) in regulating both apoptosis and proliferation of HT-29 and H508 human colon cancer cells. DCT caused dose- and time-dependent Akt (Ser(473)) phosphorylation, a commonly used marker of activated PI3K/Akt signaling. Both EGFR kinase and PI3K inhibitors attenuated DCT-induced Akt phosphorylation and Akt activation, as demonstrated by reduced phosphorylation of a GSK-3-paramyosin substrate. Transfection of HT-29 cells with kinase-dead EGFR (K721M) reduced DCT-induced Akt phosphorylation. In HT-29 cells, EGFR and PI3K inhibitors as well as transfection with dominant negative AKT attenuated DCT-induced cell proliferation. DCT-induced PI3K/Akt activation resulted in downstream phosphorylation of GSK-3 (Ser(21/9)) and BAD (Ser(136)), and nuclear translocation (activation) of NF-kappaB, thereby confirming that DCT-induced activation of PI3K/Akt signaling regulates both proproliferative and prosurvival signals. Collectively, these results indicate that DCT-induced activation of post-EGFR PI3K/Akt signaling stimulates both colon cancer cell survival and proliferation.     

Syk regulates multiple signaling pathways leading to CX3CL1 chemotaxis in macrophages

Several studies have clearly established the importance of the interaction between macrophages and CX3CL1 in the progression of disease. A previous study demonstrated that Syk was required for CX3CL1-mediated actin polymerization and chemotaxis. Here, we delineated the signaling cascade of Syk-mediated cell migration in response to CX3CL1. Inhibition of Syk in bone marrow-derived macrophages or reduction of Syk expression using siRNA in RAW/LR5 cells indicated that Syk was required for the activation of PI3K, Cdc42, and Rac1. Also, reduction in WASP or WAVE2 levels, common downstream effectors of Cdc42 or Rac1, resulted in impaired cell migration to CX3CL1. Syk indirectly regulated WASP tyrosine phosphorylation through Cdc42 activation. Altogether, our data identify that Syk mediated chemotaxis toward CX3CL1 by regulating both Rac1/WAVE2 and Cdc42/WASP pathways, whereas Src family kinases were required for proper WASP tyrosine phosphorylation.     

The p110delta isoform of PI 3-kinase negatively controls RhoA and PTEN

Inactivation of PI 3-kinase (PI3K) signalling is critical for tumour suppression by PTEN. This is thought to be a unidirectional relationship in which PTEN degrades the lipids produced by PI3K, thus controlling cell proliferation, survival and migration. We now show that this relationship is in fact bidirectional, whereby PI3K reciprocally controls PTEN. We report that the p110delta PI3K negatively regulates PTEN, through a pathway involving inhibition of RhoA. Inactivation of p110delta in macrophages led to reduced Akt and Rac1 activation, but paradoxically to increased RhoA and PTEN activity. Partial inactivation of p190RhoGAP and a reduced binding of cytoplasmic RhoA to the cyclin-dependent kinase inhibitor p27 both contributed to the increased RhoA-GTP levels upon p110delta inactivation. Pharmacological inhibition of ROCK, a downstream effector kinase of RhoA, restored all signalling and functional defects of p110delta inactivation, including Akt phosphorylation, chemotaxis and proliferation. This work identifies the RhoA/ROCK pathway as a major target of p110delta-mediated PI3K signalling, and establishes for the first time that PI3K controls itself, via a feedback loop involving PTEN.     

Muscarinic receptor agonists stimulate human colon cancer cell migration and invasion

Muscarinic receptors (CHRM) are overexpressed in colon cancer. To explore a role for muscarinic receptor signaling in colon cancer metastasis, we used human H508 and HT29 colon cancer cells that coexpress epidermal growth factor (ERBB) and CHRM3 receptors. In a wound closure model, following 8-h incubation of H508 cells with 100 μM ACh we observed a threefold increase in cell migration indistinguishable from the actions of epidermal growth factor (EGF). Atropine blocked the actions of ACh but not of EGF. In SNU-C4 colon cancer cells that express ERBB but not CHRM, EGF caused a threefold increase in migration; ACh had no effect. ACh-induced cell migration was attenuated by chemical inhibitors of ERBB1 activation, by anti-ERBB1 antibody, and by inhibitors of ERK and phosphatidylinositol 3-kinase (PI3K) signaling. Consistent with matrix metalloproteinase-7 (MMP7)-mediated release of an ERBB1 ligand, heparin binding epidermal growth factor-like growth factor (HBEGF), ACh-induced migration was inhibited by an MMP inhibitor and by anti-MMP7 and -HBEGF antibodies. ACh-induced cell migration was blocked by inhibiting RhoA and ROCK, key proteins that interact with the actin cytoskeleton. ACh-induced RhoA activation was attenuated by agents that inhibit ERBB1, ERK, and PI3K activation. Collectively, these findings indicate that ACh-induced cell migration is mediated by MMP7-mediated release of HBEGF, an ERBB ligand that activates ERBB1 and downstream ERK and PI3K signaling. In a cell invasion model, ACh-induced HT29 cell invasion was blocked by atropine. In concert with previous observations, these findings indicate that muscarinic receptor signaling plays a key role in colon cancer cell proliferation, survival, migration, and invasion.     

Discovery of thieno[2,3-d]pyrimidin-4(3H)-one derivatives as a new class of ROCK inhibitors

Herein, we report the discovery of a series of thieno[2,3-d]pyrimidin-4(3H)-one derivatives as a new class of ROCK inhibitors. Structure-activity relationship studies of these compounds led to the identification of the most potent compound, 3-(3-methoxybenzyl)-6-(1H-pyrrolo[2,3-b]pyridin-4-yl)thieno[2,3-d]pyrimidin-4(3H)-one (8k), which showed IC₅₀ values of 0.004 μM and 0.001 μM against ROCK Ⅰ and ROCK Ⅱ, respectively. In vitro, 8k significantly reduced the phosphorylation level of ROCK downstream signaling protein and induce changes in cell morphology and migration. Overall, this study provides a promising lead compound for drug discovery targeting ROCKs.     

Phosphatidylinositol 3-Kinase (PI3K) Activity Bound to Insulin-like Growth Factor-I (IGF-I) Receptor, which Is Continuously Sustained by IGF-I Stimulation, Is Required for IGF-I-induced Cell Proliferation

Continuous stimulation of cells with insulin-like growth factors (IGFs) in G(1) phase is a well established requirement for IGF-induced cell proliferation; however, the molecular components of this prolonged signaling pathway that is essential for cell cycle progression from G(1) to S phase are unclear. IGF-I activates IGF-I receptor (IGF-IR) tyrosine kinase, followed by phosphorylation of substrates such as insulin receptor substrates (IRS) leading to binding of signaling molecules containing SH2 domains, including phosphatidylinositol 3-kinase (PI3K) to IRS and activation of the downstream signaling pathways. In this study, we found prolonged (>9 h) association of PI3K with IGF-IR induced by IGF-I stimulation. PI3K activity was present in this complex in thyrocytes and fibroblasts, although tyrosine phosphorylation of IRS was not yet evident after 9 h of IGF-I stimulation. IGF-I withdrawal in mid-G(1) phase impaired the association of PI3K with IGF-IR and suppressed DNA synthesis the same as when PI3K inhibitor was added. Furthermore, we demonstrated that Tyr(1316)-X-X-Met of IGF-IR functioned as a PI3K binding sequence when this tyrosine is phosphorylated. We then analyzed IGF signaling and proliferation of IGF-IR(-/-) fibroblasts expressing exogenous mutant IGF-IR in which Tyr(1316) was substituted with Phe (Y1316F). In these cells, IGF-I stimulation induced tyrosine phosphorylation of IGF-IR and IRS-1/2, but mutated IGF-IR failed to bind PI3K and to induce maximal phosphorylation of GSK3β and cell proliferation in response to IGF-I. Based on these results, we concluded that PI3K activity bound to IGF-IR, which is continuously sustained by IGF-I stimulation, is required for IGF-I-induced cell proliferation.     

Angiotensin II induces focal adhesion kinase/paxillin phosphorylation and cell migration in human umbilical vein endothelial cells

In the present study, we demonstrated that Ang II provokes a transitory enhancement of focal adhesion kinase (FAK) and paxillin phosphorylation in human umbilical endothelial cells (HUVEC). Moreover, Ang II induces a time- and dose-dependent augmentation in cell migration, but does not affect HUVEC proliferation. The effect of Ang II on FAK and paxillin phosphorylation was markedly attenuated in cells pretreated with wortmannin and LY294002, indicating that phosphoinositide 3-kinase (PI3K) plays an important role in regulating FAK activation. Similar results were observed when HUVEC were pretreated with genistein, a non-selective tyrosine kinases inhibitor, or with the specific inhibitor PP2 for Src family kinases, demonstrating the involvement of protein tyrosine kinases, and particularly Src family of tyrosine kinases, in the downstream signalling pathway of Ang II receptors. Furthermore, FAK and paxillin phosphorylation was markedly blocked after treatment of HUVEC with AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) phosphorylation. Pretreatment of cells with inhibitors of PI3K, Src family tyrosine kinases, and EGFR also decreased HUVEC migration. In conclusion, these results suggest that Ang II mediates an increase in FAK and paxillin phosphorylation and induces HUVEC migration through signal transduction pathways dependent on PI3K and Src tyrosine kinase activation and EGFR transactivation.     

Knockdown of sphingosine kinase 1 inhibits the migration and invasion of human rheumatoid arthritis fibroblast-like synoviocytes by down-regulating the PI3K/AKT activation and MMP-2/9 production in vitro

To investigate the potential regulation of sphingosine kinase 1 (SPHK1) on the migration, invasion, and matrix metalloproteinase (MMP) expression in human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). RA-FLS were transfected control siRNA or SPHK1 siRNA. The migration and invasion of unmanipulated control, control siRNA or SPHK1 siRNA- transfected RA-FLS in vitro were measured by the transwell system. The relative levels of SPHK1, PI3K, and AKT as well as AKT phosphorylation in RA-FLS were determined by Western blot. The levels of MMP-2/9 secreted by RA-FLS were detected by ELISA. Knockdown of SPHK1 significantly inhibited the spontaneous migration and invasion of RA-FLS, accompanied by significantly reduced levels of PI3K expression and AKT phosphorylation. Similarly, treatment with LY294002, an inhibitor of the PI3K/AKT pathway, inhibited the migration and invasion of RA-FLS. Knockdown of SPHK1 and treatment with the inhibitor synergistically inhibited the migration and invasion of RA-FLS, by further reducing the levels of PI3K expression and AKT phosphorylation. In addition, knockdown of SPHK1 or treatment with LY294002 inhibited the secretion of MMP-2 and MMP-9, and both synergistically reduced the production of MMP-2 and MMP-9 in RA-FLS in vitro. Knockdown of SPHK1 expression inhibits the PI3K/AKT activation, MMP-2 and MMP-9 expression, and human RA-FLS migration and invasion in vitro. Potentially, SPHK1 may be a novel therapeutic target for RA.     

Regulation of PTEN stability and activity by Plk3

By studying primary isogenic murine embryonic fibroblasts (MEFs), we have shown that PLK3 null MEFs contain a reduced level of phosphatase and tensin homolog (PTEN) and increased Akt1 activation coupled with decreased GSK3β activation under normoxia and hypoxia. Purified recombinant Plk3, but not a kinase-defective mutant, efficiently phosphorylates PTEN in vitro. Mass spectrometry identifies threonine 366 and serine 370 as two putative residues that are phosphorylated by Plk3. Immunoblotting using a phosphospecific antibody confirms these sites as Plk3 phosphorylation sites. Moreover, treatment of MEFs with LiCl, an inhibitor of GSK3β and CK2, only partially suppresses the phosphorylation, suggesting Plk3 as an additional kinase that phosphorylates these sites in vivo. Plk3-targeting mutants of PTEN are expressed at a reduced level in comparison with the wild-type counterpart, which is associated with an enhanced activity of PDK1, an upstream activator of Akt1. Furthermore, the reduced level of PTEN in PLK3 null MEFs is stabilized by treatment with MG132, a proteosome inhibitor. Combined, our study identifies Plk3 as a new player in the regulation of the PI3K/PDK1/Akt signaling axis by phosphorylation and stabilization of PTEN.     

cAMP-guanine exchange factor protection from bile acid-induced hepatocyte apoptosis involves glycogen synthase kinase regulation of c-Jun NH2-terminal kinase

Cholestatic liver disorders are accompanied by the hepatic accumulation of cytotoxic bile acids that induce cell death. Increases in cAMP protect hepatocytes from bile acid-induced apoptosis by a cAMP-guanine exchange factor (cAMP-GEF)/phosphoinositide-3-kinase (PI3K)/Akt pathway. The aim of these studies was to identify the downstream substrate in this pathway and to determine at what level in the apoptotic cascade cytoprotection occurs. Since inhibitory phosphorylation of glycogen synthase kinase-3 (GSK) occurs downstream of PI3K/Akt and this phosphorylation has been implicated in cell survival, we conducted studies to determine whether GSK was downstream in cAMP-GEF/PI3K/Akt-mediated cytoprotection. Our results show that treatment of hepatocytes with the cAMP-GEF-specific analog, 4-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cAMP, results in PI3K-dependent phosphorylation of GSK. Direct chemical inhibition of GSK in rat hepatocytes or human HUH7-NTCP cells with several structurally and functionally distinct inhibitors including bromoindirubin-3'-oxime (BIO), maleimides (SB216763, SB415286), thiadiazolidine derivatives, and LiCl attenuates apoptosis induced by glycochenodeoxycholate (GCDC). In addition, genetic silencing of the GSK β isoform with small interfering RNA attenuates GCDC apoptosis in HUH7-NTCP cells. Adenoviral inhibition of the Rap1 blocks both cAMP-GEF-mediated cytoprotection against GCDC-induced apoptosis and Akt/GSK3β phosphorylation. GCDC-induced phosphorylation of the proapoptotic kinase, c-Jun NH(2)-terminal kinase (JNK) is inhibited by GSK inhibition or cAMP-GEF activation. GCDC-induced apoptosis is accompanied by phosphorylation of the endoplasmic reticulum stress markers pIEF2α and IRE-1, and pretreatment with the cAMP-GEF analog or GSK inhibitors prevents this phosphorylation. Collectively, our results support the presence of a cAMP/cAMP-GEF/Rap1/PI3K/Akt/GSKβ survival pathway in hepatocytes that inhibits bile acid-induced JNK phosphorylation.     

TRPC1 protein channel is major regulator of epidermal growth factor receptor signaling

TRP channels have been associated with cell proliferation and aggressiveness in several cancers. In particular, TRPC1 regulates cell proliferation and motility, two processes underlying cancer progression. We and others have described the mechanisms of TRPC1-dependent cell migration. However, the involvement of TRPC1 in cell proliferation remains unexplained. In this study, we show that siRNA-mediated TRPC1 depletion in non small cell lung carcinoma cell lines induced G(0)/G(1) cell cycle arrest resulting in dramatic decrease in cell growth. The expression of cyclins D1 and D3 was reduced after TRPC1 knockdown, pointing out the role of TRPC1 in G(1)/S transition. This was associated with a decreased phosphorylation and activation of EGFR and with a subsequent disruption of PI3K/Akt and MAPK downstream pathways. Stimulation of EGFR by its natural ligand, EGF, induced Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry through TRPC1. Ca(2+) entry through TRPC1 conversely activated EGFR, suggesting that TRPC1 is a component of a Ca(2+)-dependent amplification of EGF-dependent cell proliferation.     

Rho-associated Coiled-coil Kinase (ROCK) Protein Controls Microtubule Dynamics in a Novel Signaling Pathway That Regulates Cell Migration

The two members of the Rho-associated coiled-coil kinase (ROCK1 and 2) family are established regulators of actin dynamics that are involved in the regulation of the cell cycle as well as cell motility and invasion. Here, we discovered a novel signaling pathway whereby ROCK regulates microtubule (MT) acetylation via phosphorylation of the tubulin polymerization promoting protein 1 (TPPP1/p25). We show that ROCK phosphorylation of TPPP1 inhibits the interaction between TPPP1 and histone deacetylase 6 (HDAC6), which in turn results in increased HDAC6 activity followed by a decrease in MT acetylation. As a consequence, we show that TPPP1 phosphorylation by ROCK increases cell migration and invasion via modulation of cellular acetyl MT levels. We establish here that the ROCK-TPPP1-HDAC6 signaling pathway is important for the regulation of cell migration and invasion.     

ERK1/2 antagonizes glycogen synthase kinase-3beta-induced apoptosis in cortical neurons

Inhibition of glycogen synthase kinase-3beta (GSK3beta) is one of the mechanisms by which phosphatidylinositol 3-kinase (PI3K) activation protects neurons from apoptosis. Here, we report that inhibition of ERK1/2 increased the basal activity of GSK3beta in cortical neurons and that both ERK1/2 and PI3K were required for brain-derived neurotrophic factor (BDNF) suppression of GSK3beta activity. Moreover, cortical neuron apoptosis induced by expression of recombinant GSK3beta was inhibited by coexpression of constitutively active MKK1 or PI3K. Activation of both endogenous ERK1/2 and PI3K signaling pathways was required for BDNF to block apoptosis induced by expression of recombinant GSK3beta. Furthermore, cortical neuron apoptosis induced by LY294002-mediated activation of endogenous GSK3beta was blocked by expression of constitutively active MKK1 or by BDNF via stimulation of the endogenous ERK1/2 pathway. Although both PI3K and ERK1/2 inhibited GSK3beta activity, neither had an effect on GSK3beta phosphorylation at Tyr-216. Interestingly, PI3K (but not ERK1/2) induced the inhibitory phosphorylation of GSK3beta at Ser-9. Significantly, coexpression of constitutively active MKK1 (but not PI3K) still suppressed neuronal apoptosis induced by expression of the GSK3beta(S9A) mutant. These data suggest that activation of the ERK1/2 signaling pathway protects neurons from GSK3beta-induced apoptosis and that inhibition of GSK3beta may be a common target by which ERK1/2 and PI3K protect neurons from apoptosis. Furthermore, ERK1/2 inhibits GSK3beta activity via a novel mechanism that is independent of Ser-9 phosphorylation and likely does not involve Tyr-216 phosphorylation.