Identification of signaling molecules mediating corticotropin-releasing hormone-R1alpha-mitogen-activated protein kinase (MAPK) interactions: the critical role of phosphatidylinositol 3-kinase in regulating ERK1/2 but not p38 MAPK activation

In most target cells, activation of the type 1 CRH receptor (CRH-R1) by CRH or urocortin (UCN I) leads to stimulation of the Gs-protein/adenylyl cyclase/protein kinase A cascade. Signal transduction of CRH-R1 also involves alternative pathways such as phosphorylation of ERK1/2 and p38 MAPK, two members of the MAPK family that mediate important pathophysiological responses. The intracellular pathways by which CRH-R1 activates these MAPK are only partially understood; here we characterized further signaling mechanisms and molecules involved in CRH-R1-mediated ERK1/2 and p38 MAPK activation. In human embryonic kidney 293 cells overexpressing recombinant CRH-R1alpha, UCN I induced ERK1/2 and p38 MAPK activation was dependent on signaling molecules involved in agonist-induced CRH-R1alpha trafficking and endocytosis. Furthermore, time course studies and use of selective inhibitors demonstrated that ERK1/2 activation occured within 5 min, was sustained for at least 60 min, and was dependent on both phosphatidylinositol 3-kinase (PI3-K)/Akt activation and epidermoid growth factor receptor transactivation involving matrix metelloproteinases. UCN I effect on p38 MAPK phosphorylation was more transient, returned to basal within 40 min and was dependent on epidermoid growth factor receptor transactivation, but not PI3-K/Akt activation. Overexpression of G(alpha-)transducin, showed that G(betagamma)-subunit activation is only partially required for ERK1/2 phosphorylation and does not play a role in p38 MAPK phosphorylation, whereas overexpression of a dominant-negative Ras (Ras N17) attenuated both ERK and p38 MAPK activation. In conclusion, a complex signaling network appears to mediate CRH-R1alpha-MAPK interactions; PI3-K might play a critical role in the regulation of CRH-R1alpha signaling selectivity and cellular responses.     

Bradykinin-induced p42/p44 MAPK phosphorylation and cell proliferation via Src, EGF receptors, and PI3-K/Akt in vascular smooth muscle cells

In our previous study, bradykinin (BK) exerts its mitogenic effect through Ras/Raf/MEK/MAPK pathway in vascular smooth muscle cells (VSMCs). In addition to this pathway, the non-receptor tyrosine kinases (Src), EGF receptor (EGFR), and phosphatidylinositol 3-kinase (PI3-K) have been implicated in linking a variety of G-protein coupled receptors to MAPK cascades. Here, we investigated whether these different mechanisms participating in BK-induced activation of p42/p44 MAPK and cell proliferation in VSMCs. We initially observed that BK- and EGF-dependent activation of Src, EGFR, Akt, and p42/p44 MAPK and [3H]thymidine incorporation were mediated by Src and EGFR, because the Src inhibitor PP1 and EGFR kinase inhibitor AG1478 abrogated BK- and EGF-dependent effects. Inhibition of PI3-K by LY294002 attenuated BK-induced Akt and p42/p44 MAPK phosphorylation and [3H]thymidine incorporation, but had no effect on EGFR phosphorylation, suggesting that EGFR may be an upstream component of PI3-K/Akt and MAPK in these responses. This hypothesis was supported by the tranfection with dominant negative plasmids of p85 and Akt which significantly attenuated BK-induced Akt and p42/p44 MAPK phosphorylation. Pretreatment with U0126 (a MEK1/2 inhibitor) attenuated the p42/p44 MAPK phosphorylation and [3H]thymidine incorporation stimulated by BK, but had no effect on Akt activation. Moreover, BK-induced transactivation of EGFR and cell proliferation was blocked by matrix metalloproteinase inhibitor GM6001. These results suggest that, in VSMCs, the mechanism of BK-stimulated activation of p42/p44 MAPK and cell proliferation was mediated, at least in part, through activation of Src family kinases, EGFR transactivation, and PI3-K/Akt.     

PGE2 inhibits apoptosis in human adenocarcinoma Caco-2 cell line through Ras-PI3K association and cAMP-dependent kinase A activation

PGE2 plays a critical role in colorectal carcinogenesis. We have previously shown that COX-2 expression and PGE2 synthesis are mediated by IGF-II/IGF-I receptor signaling in the Caco-2 cell line and that the pathway of phosphatidylinositol 3-kinase (PI3K)/Akt protects the cell from apoptosis. In the present study, we demonstrate that PGE2 has the ability to increase Ras and PI3K association and decrease the level of apoptosis in the same experimental system. The effect of PGE2 on PI3K/Ras association is dependent on the activation of EP4 receptor, the increase of cAMP levels, and the activation of PKA. In fact, treatment of cells with the PKA inhibitor H89 decreases the association of Ras and PI3K and Ras-associated PI3K activity. PKA inhibitor H89 is able to decrease threonine phosphorylation of Akt and to increase serine phosphorylation of Akt by p38 MAPK and counteracts the cytoprotective effect induced by PGE2. In addition, PGE2 is able to activate p38 MAPK and the inhibition of p38 MAPK, with SB203580 specific inhibitor or with dominant negative MKK6 kinase, is able to revert the apoptotic effect of H89 and serine phosphorylation of Akt. The effect of PGE2 on Caco-2 cell survival through PKA activation is mediated and regulated by the balance of threonine/serine phosphorylation of Akt by p38 kinase and PI3K. In conclusion, our data elucidate a novel mechanism for regulation of colon cancer cell survival and provide evidences for new combinatory treatments of colon cancer.     

Flt3 ligand induces tyrosine phosphorylation of gab1 and gab2 and their association with shp-2, grb2, and PI3 kinase

The receptor tyrosine kinase Flt3 has been shown to play an important role in proliferation, differentiation, and survival of hematopoietic stem and progenitor cells. Although some postreceptor signaling events of Flt3 have been characterized, the involvement of Gab family proteins in Flt3 signaling is not known. In this study, we show that both Gab1 and Gab2 are rapidly tyrosine phosphorylated after Flt3 ligand stimulation of Flt3 ligand-responsive cells. They interact with tyrosine-phosphorylated Shp-2, p85, Grb2, and Shc. The results suggest that Gab proteins are engaged in Flt3 signaling to mediate downstream activation of Shp-2 and PI3 kinase pathways and possibly the Ras/Raf/MAPK pathway.     

接头蛋白Gab1的结构及功能研究进展

Gab1蛋白属于接头蛋白Gab家族,该家族蛋白因能与生长因子受体结合蛋白2（Grb2）相结合而得名。作为接头蛋白,Gab1蛋白能被多种受体酪氨酸激酶或非受体酪氨酸激酶激活,接受胞外多种生长因子、细胞因子和一些T/B细胞抗原受体的刺激,介导PI3K/Akt和Ras/MAPK等多条信号转导途径,具有促进细胞生长、迁移、调节免疫等多种生物学功能,与糖尿病、肿瘤、心血管疾病等的发生发展密切相关。     

The docking protein Gab1 is an essential component of an indirect mechanism for fibroblast growth factor stimulation of the phosphatidylinositol 3-kinase/Akt antiapoptotic pathway

The docking protein Gab1 has been implicated as a mediator of multiple signaling pathways that are activated by a variety of receptor tyrosine kinases and cytokines. We have previously proposed that fibroblast growth factor 1 (FGF1) stimulation of tyrosine phosphorylation of Gab1 and recruitment of phosphatidylinositol (PI) 3-kinase are mediated by an indirect mechanism in which the docking protein fibroblast receptor substrate 2alpha (FRS2alpha) plays a critical role. In this report, we explore the role of Gab1 in FGF1 signaling by using mouse embryo fibroblasts (MEFs) derived from Gab1(-/-) or FRS2alpha(-/-) mice. We demonstrate that Gab1 is essential for FGF1 stimulation of both PI 3-kinase and the antiapoptotic protein kinase Akt, while FGF1-induced mitogen-activated protein kinase (MAPK) stimulation is not affected by Gab1 deficiency. To test the indirect mechanism for FGF1 stimulation of PI 3-kinase and Akt, we use a chimeric docking protein composed of the membrane targeting signal and the phosphotyrosine-binding domain of FRS2alpha fused to the C-terminal portion of Gab1, the region including the binding sites for the complement of signaling proteins that are recruited by Gab1. We demonstrate that expression of the chimeric docking protein in Gab1(-/-) MEFs rescues PI 3-kinase and the Akt responses, while expression of the chimeric docking protein in FRS2alpha(-/-) MEFs rescues stimulation of both Akt and MAPK. These experiments underscore the essential role of Gab1 in FGF1 stimulation of the PI 3-kinase/Akt signaling pathway and provide further support for the indirect mechanism for FGF1 stimulation of PI 3-kinase involving regulated assembly of a multiprotein complex.     

Epidermal growth factor-induced DNA synthesis. Key role for Src phosphorylation of the docking protein Gab2

We have previously demonstrated that phosphatidylinositol 3-kinase (PI3-kinase) is necessary and sufficient to account for epidermal growth factor (EGF)-induced mitogenesis in rat primary hepatocytes. A cytosolic Gab2-containing complex accounts for >80% of the total EGF-induced PI3-kinase activity (Kong, M., Mounier, C., Wu, J., and Posner, B. I. (2000) J. Biol. Chem. 275, 36035-36042), suggesting a key role for Gab2 in EGF-induced mitogenesis. Here, we demonstrate that PP1, a selective inhibitor of Src family kinases, blocks the EGF-induced Gab2 tyrosine phosphorylation without inhibiting EGF-induced phosphorylation of the EGF receptor, ErbB3, or Shc. We also show that Gab2 phosphorylation is increased in Csk knockout cells in which Src family kinases are constitutively activated. Furthermore, PP1 blocks Gab2-associated downstream events including EGF-induced PI3-kinase activation, Akt phosphorylation, and DNA synthesis. We demonstrate that Gab2 and Src are constitutively associated. Since this association involves the proline-rich sequences of Gab2, it probably involves the Src homology 3 domain of Src kinase. Mutation of the proline-rich sequences in Gab2 prevented EGF-induced Gab2 phosphorylation, PI3-kinase/Akt activation, and DNA synthesis, demonstrating that Gab2 phosphorylation is critical for EGF-induced mitogenesis and is not complemented by ErbB3 or Shc phosphorylation. We also found that overexpression of a Gab2 mutant lacking SHP2 binding sites increased EGF-induced Gab2 phosphorylation and the activation of PI3-kinase but blocked activation of MAPK. In addition, we demonstrated that the Src-induced response was down-regulated by Gab2-associated SHP2. In summary, our results have defined the role for Src activation in EGF-induced hepatic mitogenesis through the phosphorylation of Gab2 and the activation of the PI3-kinase cascade.     

In vivo functional analysis of the daughter of sevenless protein in receptor tyrosine kinase signaling

One mechanism used by receptor tyrosine kinases to relay a signal to different downstream effector molecules is to use adaptor proteins that provide docking sites for a variety of proteins. The daughter of sevenless (dos) gene was isolated in a genetic screen for components acting downstream of the Sevenless (Sev) receptor tyrosine kinase. Dos contains a N-terminally located PH domain and several tyrosine residues within consensus binding sites for a number of SH2 domain containing proteins. The structural features of Dos and experiments demonstrating tyrosine phosphorylation of Dos upon Sev activation suggested that Dos belongs to the family of multisite adaptor proteins that include the Insulin Receptor Substrate (IRS) proteins, Gab1, and Gab2. Here, we studied the structural requirements for Dos function in receptor tyrosine kinase mediated signaling processes by expressing mutated dos transgenes in the fly. We show that mutant Dos proteins lacking the putative binding sites for the SH2 domains of Shc, PhospholipaseC-γ (PLC-γ) and the regulatory subunit of Phosphoinositide 3-kinase (PI3-K) can substitute the loss of endogenous Dos function during development. In contrast, tyrosine 801, corresponding to a predicted Corkscrew (Csw) tyrosine phosphatase SH2 domain binding site, is essential for Dos function. Furthermore, we assayed whether the Pleckstrin homology (PH) domain is required for Dos function and localization. Evidence is provided that deletion or mutation of the PH domain interferes with the function but not with localization of the Dos protein. The Dos PH domain can be replaced by the Gab1 PH domain but not by a heterologous membrane anchor, suggesting a specific function of the PH domain in regulating signal transduction.     

Computational Model of Gab1/2-Dependent VEGFR2 Pathway to Akt Activation

Vascular endothelial growth factor (VEGF) signal transduction is central to angiogenesis in development and in pathological conditions such as cancer, retinopathy and ischemic diseases. However, no detailed mass-action models of VEGF receptor signaling have been developed. We constructed and validated the first computational model of VEGFR2 trafficking and signaling, to study the opposing roles of Gab1 and Gab2 in regulation of Akt phosphorylation in VEGF-stimulated endothelial cells. Trafficking parameters were optimized against 5 previously published in vitro experiments, and the model was validated against six independent published datasets. The model showed agreement at several key nodes, involving scaffolding proteins Gab1, Gab2 and their complexes with Shp2. VEGFR2 recruitment of Gab1 is greater in magnitude, slower, and more sustained than that of Gab2. As Gab2 binds VEGFR2 complexes more transiently than Gab1, VEGFR2 complexes can recycle and continue to participate in other signaling pathways. Correspondingly, the simulation results show a log-linear relationship between a decrease in Akt phosphorylation and Gab1 knockdown while a linear relationship was observed between an increase in Akt phosphorylation and Gab2 knockdown. Global sensitivity analysis demonstrated the importance of initial-concentration ratios of antagonistic molecular species (Gab1/Gab2 and PI3K/Shp2) in determining Akt phosphorylation profiles. It also showed that kinetic parameters responsible for transient Gab2 binding affect the system at specific nodes. This model can be expanded to study multiple signaling contexts and receptor crosstalk and can form a basis for investigation of therapeutic approaches, such as tyrosine kinase inhibitors (TKIs), overexpression of key signaling proteins or knockdown experiments.     

Both src-dependent and -independent mechanisms mediate phosphatidylinositol 3-kinase regulation of colony-stimulating factor 1-activated mitogen-activated protein kinases in myeloid progenitors

Colony-stimulating factor 1 (CSF-1) supports the proliferation, survival, and differentiation of bone marrow-derived cells of the monocytic lineage. In the myeloid progenitor 32D cell line expressing CSF-1 receptor (CSF-1R), CSF-1 activation of the extracellular signal-regulated kinase (ERK) pathway is both Ras and phosphatidylinositol 3-kinase (PI3-kinase) dependent. PI3-kinase inhibition did not influence events leading to Ras activation. Using the activity of the PI3-kinase effector, Akt, as readout, studies with dominant-negative and oncogenic Ras failed to place PI3-kinase downstream of Ras. Thus, PI3-kinase appears to act in parallel to Ras. PI3-kinase inhibitors enhanced CSF-1-stimulated A-Raf and c-Raf-1 activities, and dominant-negative A-Raf but not dominant-negative c-Raf-1 reduced CSF-1-provoked ERK activation, suggesting that A-Raf mediates a part of the stimulatory signal from Ras to MEK/ERK, acting in parallel to PI3-kinase. Unexpectedly, a CSF-1R lacking the PI3-kinase binding site (DeltaKI) remained capable of activating MEK/ERK in a PI3-kinase-dependent manner. To determine if Src family kinases (SFKs) are involved, we demonstrated that CSF-1 activated Fyn and Lyn in cells expressing wild-type (WT) or DeltaKI receptors. Moreover, CSF-1-induced Akt activity in cells expressing DeltaKI is SFK dependent since Akt activation was prevented by pharmacological or genetic inhibition of SFK activity. The docking protein Gab2 may link SFK to PI3-kinase. CSF-1 induced Gab2 tyrosyl phosphorylation and association with PI3-kinase in cells expressing WT or DeltaKI receptors. However, only in DeltaKI cells are these events prevented by PP1. Thus in myeloid progenitors, CSF-1 can activate the PI3-kinase/Akt pathway by at least two mechanisms, one involving direct receptor binding and one involving SFKs.     

Prolactin-stimulated activation of ERK1/2 mitogen-activated protein kinases is controlled by PI3-kinase/Rac/PAK signaling pathway in breast cancer cells

There is strong evidence that deregulation of prolactin (PRL) signaling contributes to pathogenesis and chemoresistance of breast cancer. Therefore, understanding cross-talk between distinct signal transduction pathways triggered by activation of the prolactin receptor (PRL-R), is essential for elucidating the pathogenesis of metastatic breast cancer.In this study, we applied a sequential inhibitory analysis of various signaling intermediates to examine the hierarchy of protein interactions within the PRL signaling network and to evaluate the relative contributions of multiple signaling branches downstream of PRL-R to the activation of the extracellular signal-regulated kinases ERK1 and ERK2 in T47D and MCF-7 human breast cancer cells.Quantitative measurements of the phosphorylation/activation patterns of proteins showed that PRL simultaneously activated Src family kinases (SFKs) and the JAK/STAT, phosphoinositide-3 (PI3)-kinase/Akt and MAPK signaling pathways. The specific blockade or siRNA-mediated suppression of SFK/FAK, JAK2/STAT5, PI3-kinase/PDK1/Akt, Rac/PAK or Ras regulatory circuits revealed that (1) the PI3-kinase/Akt pathway is required for activation of the MAPK/ERK signaling cascade upon PRL stimulation; (2) PI3-kinase-mediated activation of the c-Raf-MEK1/2-ERK1/2 cascade occurs independent of signaling dowstream of STATs, Akt and PKC, but requires JAK2, SFKs and FAK activities; (3) activated PRL-R mainly utilizes the PI3-kinase-dependent Rac/PAK pathway rather than the canonical Shc/Grb2/SOS/Ras route to initiate and sustain ERK1/2 signaling. By interconnecting diverse signaling pathways PLR may enhance proliferation, survival, migration and invasiveness of breast cancer cells.     

Epstein-Barr virus latent membrane protein 2A mediates transformation through constitutive activation of the Ras/PI3-K/Akt Pathway

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is widely expressed in EBV-infected cells within the infected human host and EBV-associated malignancies, suggesting that LMP2A is important for EBV latency, persistence, and EBV-associated tumorigenesis. Previously, we demonstrated that LMP2A provides an antiapoptotic signal through the activation of phosphatidylinositol 3-kinase (PI3-K)/Akt pathway in vitro. However, the exact function of LMP2A in tumor progression is not well understood. In this study, we found that LMP2A did not induce anchorage-independent cell growth in a human keratinocyte cell line, HaCaT, but did in a human gastric carcinoma cell line, HSC-39. In addition, LMP2A activated the PI3-K/Akt pathway in both HaCaT and HSC-39 cells; however, LMP2A did not activate Ras in HaCaT cells but did in HSC-39 cells. Furthermore, the Ras inhibitors manumycin A and a dominant-negative form of Ras (RasN17) and the PI3-K inhibitor LY294002 blocked LMP2A-mediated Akt phosphorylation and anchorage-independent cell growth in HSC-39 cells. These results suggest that constitutive activation of the Ras/PI3-K/Akt pathway by LMP2A is a key factor for LMP2A-mediated transformation.     

Cyclophosphamide-induced apoptosis in A431 cells is inhibited by fucosyltransferase IV

Fucosyltransferase IV (FUT4) is an essential enzyme that catalyzes the synthesis of difucosylated oligosaccharide LeY which is overexpressed in the cancers derived from the epithelial tissues. Our previous studies have shown that FUT4 overexpression promotes A431 cell proliferation through the MAPK and PI3K/Akt signaling pathways, but the relationship between FUT4 and apoptosis remained unclear. Here, we investigated the effect of FUT4 overexpression on cyclophosphamide (CPA)-induced apoptosis in A431 cells. Western blot analysis showed that FUT4 overexpression decreased expression of Bax, Caspase 3, and PARP proteins, and increased anti-apoptotic Bcl-2 protein in A431 cells. The anti-apoptosis effect of FUT4 was confirmed both by Annexin-V/PI and JC-1 assays. The results showed that FUT4 overexpression up-regulated phosphorylation of ERK1/2 and Akt which was inhibited by CPA in dose-dependent manner. By blocking the ERK/MAPK and PI3K/Akt pathways with specific inhibitors, we demonstrated that these two pathways were required in mediating the anti-apoptosis effect of FUT4. We concluded that FUT4 inhibited cell apoptosis induced by CPA through decreasing the expression of apoptotic proteins Bax, Caspase 3, and PARP and increasing the expression of anti-apoptotic protein Bcl-2 via the ERK/MAPK and PI3K/Akt signaling pathways in A431 cells.     

Latent membrane protein 2A inhibits transforming growth factor-beta 1-induced apoptosis through the phosphatidylinositol 3-kinase/Akt pathway

Latent membrane protein 2A (LMP2A) blocks B-cell receptor signal transduction in vitro by binding the Syk and Lyn protein tyrosine kinases. As well as blocking B-cell signal transduction, LMP2A has been shown to activate the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway, which acts as a survival signal in both B cells and epithelial cells. Transforming growth factor beta 1 (TGF-beta 1) is a multifunctional cytokine that plays important roles in regulating cell growth and differentiation in many biological systems. The loss of the growth-inhibitory response to the TGF-beta 1 signal is found in many cancers and is widely thought to promote tumor development. In this study, we found that LMP2A induced the phosphorylation of Akt (serine 473) in Burkitt's lymphoma cell line Ramos and in gastric carcinoma cell line HSC-39 and partially enhanced cell viability following TGF-beta 1 treatment. In addition, LMP2A partially inhibited TGF-beta 1-induced DNA fragmentation and cleavage of poly(ADP-ribose) polymerase (PARP). In the presence of LY294002, an inhibitor of PI3-K, the LMP2A-mediated inhibitory effects on TGF-beta 1-induced DNA fragmentation and cleavage of PARP were alleviated. Furthermore, LMP2A did not alter the levels of expression of type I and type II TGF-beta 1 receptors. Taken together, these results suggest that LMP2A may inhibit TGF-beta 1-mediated apoptosis through activation of the PI3-K/Akt pathway.     

Flow shear stress stimulates Gab1 tyrosine phosphorylation to mediate protein kinase B and endothelial nitric-oxide synthase activation in endothelial cells

Fluid shear stress generated by blood flow modulates endothelial cell function via specific intracellular signaling events. We showed previously that flow activated the phosphatidylinositol 3-kinase (PI3K), Akt, and endothelial nitric-oxide synthase (eNOS) via Src kinase-dependent transactivation of vascular endothelial growth factor receptor 2 (VEGFR2). The scaffold protein Gab1 plays an important role in receptor tyrosine kinase-mediated signal transduction. We found here that laminar flow (shear stress = 12 dynes/cm2) rapidly stimulated Gab1 tyrosine phosphorylation in both bovine aortic endothelial cells and human umbilical vein endothelial cells, which correlated with activation of Akt and eNOS. Gab1 phosphorylation as well as activation of Akt and eNOS by flow was inhibited by the Src kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and VEGFR2 kinase inhibitors SU1498 and VTI, suggesting that flow-mediated Gab1 phosphorylation is Src kinase-dependent and VEGFR2-dependent. Tyrosine phosphorylation of Gab1 by flow was functionally important, because flow stimulated the association of Gab1 with the PI3K subunit p85 in a time-dependent manner. Furthermore, transfection of a Gab1 mutant lacking p85 binding sites inhibited flow-induced activation of Akt and eNOS. Finally, knockdown of endogenous Gab1 by small interference RNA abrogated flow activation of Akt and eNOS. These data demonstrate a critical role of Gab1 in flow-stimulated PI3K/Akt/eNOS signal pathway in endothelial cells.     

PKC and Raf-1 inhibition-related apoptotic signalling in N2a cells

In this study, a neuroblastoma N2a cell line was applied to investigate mechanisms of apoptosis induced either by selective inhibition of protein kinase C (PKC) by low amounts of staurosporine (STS(10) ) or by inhibition PI3-K after wortmannin (WM) treatment. We present evidence that, in the absence of serum in the medium, decreased phosphorylation of Raf-1 and BAD112, as well as Akt and BAD136, proteins and their translocation to mitochondria coincided with STS10 - or WM-induced apoptosis, respectively. Concomitantly, release of cytochrome c into the cytosol indicated a BCL-2-dependent mode of cell death after both treatments. Furthermore, in typical 'gain of function' experiments, cells with overexpression of permanently active Raf-1 or Akt transgenes displayed a significantly higher and independent resistance to either STS10 or WM. Thus, our results indicate that PKC/Raf-1/BAD112, as well as PI3-K/Akt/BAD136 signalling pathways, are both necessary for N2a cell survival and thus are unable to functionally substitute for each other as long as the cells do not receive additional signal(s) derived from serum. However, in the presence of serum, undefined trophic signal(s) can stimulate cross-talk between these two pathways at a level upstream from Raf-1 and Akt phosphorylation. In this case, only simultaneous inhibition of PKC and PI3-K is able to induce apoptosis.     

Analysis of Ras-dependent signals that prevent caspase-3 activation and apoptosis induced by cytokine deprivation in hematopoietic cells

In hematopoietic cells, Ras has been implicated in signaling pathways that prevent apoptosis triggered by deprivation of cytokines, such as interleukin-3 (IL-3). However, the mechanism whereby Ras suppresses cell death remains incompletely understood. We have investigated the role of Ras in IL-3 signal transduction by using the cytokine-dependent BaF3 cell line. Herein, we show that the activation of the pro-apoptotic protease caspase-3 upon IL-3 removal is suppressed by expression of activated Ras, which eventually prevents cell death. For caspase-3 suppression, the Raf/extracellular signal-regulated kinase (ERK)- or phosphatidylinositol 3-kinase (PI3-K)/Akt-mediated signaling pathway downstream of Ras was required. However, inhibition of both pathways did not block activated Ras-dependent suppression of cell death-associated phenotypes, such as nuclear DNA fragmentation. Thus, a pathway that is independent of both Raf/ERK and PI3-K/Akt pathways may function downstream of Ras, preventing activated caspase-3-initiated apoptotic processes. Conditional activation of c-Raf-1 also suppressed caspase-3 activation and subsequent cell death without affecting Akt activity, providing further evidence for a PI3-K/Akt-independent mechanism.     

A critical role for phosphoinositide 3-kinase upstream of Gab1 and SHP2 in the activation of ras and mitogen-activated protein kinases by epidermal growth factor

Although the mechanisms involved in the activation of mitogen-activated protein kinases (MAPK) by receptor tyrosine kinases do not display an obvious role for phosphoinositide 3-kinases (PI3Ks), we have observed in the nontransformed cell line Vero stimulated with epidermal growth factor (EGF) that wortmannin and LY294002 nearly abolished MAPK activation. The effect was observed under strong stimulation and was independent of EGF concentration. In addition, three mutants of class Ia PI3Ks were found to inhibit MAPK activation to an extent similar to their effect on Akt/protein kinase B activation. To determine the importance of PI3K lipid kinase activity in MAPK activation, we have used the phosphatase PTEN and the pleckstrin homology domain of Tec kinase. Overexpression of these proteins, but not control mutants, was found to inhibit MAPK activation, suggesting that the lipid products of class Ia PI3K are necessary for MAPK signaling. We next investigated the location of PI3K in the MAPK cascade. Pharmacological inhibitors and dominant negative forms of PI3K were found to block the activation of Ras induced by EGF. Upstream from Ras, although association of Grb2 with its conventional effectors was independent of PI3K, we have observed that the recruitment of the tyrosine phosphatase SHP2 required PI3K. Because SHP2 was also essential for Ras activation, this suggested the existence of a PI3K/SHP2 pathway leading to the activation of Ras. In addition, we have observed that the docking protein Gab1, which is involved in PI3K activation during EGF stimulation, is also implicated in this pathway downstream of PI3K. Indeed, the association of Gab1 with SHP2 was blocked by PI3K inhibitors, and expression of Gab1 mutant deficient for binding to SHP2 was found to inhibit Ras stimulation without interfering with PI3K activation. These results show that, in addition to Shc and Grb2, a PI3K-dependent pathway involving Gab1 and SHP2 is essential for Ras activation under EGF stimulation.     

OxLDL induces mitogen-activated protein kinase activation mediated via PI3-kinase/Akt in vascular smooth muscle cells

Oxidized low-density lipoprotein (OxLDL) is a risk factor in atherosclerosis and stimulates multiple signaling pathways, including activation of phosphatidylinositol 3-kinase (PI3-K)/Akt and p42/p44 mitogen-activated protein kinase (MAPK), which are involved in mitogenesis of vascular smooth muscle cells (VSMCs). We therefore investigated the relationship between PI3-K/Akt and p42/p44 MAPK activation and cell proliferation induced by OxLDL. OxLDL stimulated Akt phosphorylation in a time- and concentration-dependent manner, as determined by Western blot analysis. Phosphorylation of Akt stimulated by OxLDL and epidermal growth factor (EGF) was attenuated by inhibitors of PI3-K (wortmannin and LY294002) and intracellular Ca2+ chelator (BAPTA/AM) plus EDTA. Pretreatment of VSMCs with pertussis toxin, cholera toxin, and forskolin for 24 h also attenuated the OxLDL-stimulated Akt phosphorylation. In addition, pretreatment of VSMCs with wortmannin or LY294002 inhibited OxLDL-stimulated p42/p44 MAPK phosphorylation and [3H]thymidine incorporation. Furthermore, treatment with U0126, an inhibitor of MAPK kinase (MEK)1/2, attenuated the p42/p44 MAPK phosphorylation, but had no effect on Akt activation in response to OxLDL and EGF. Overexpression of p85-DN or Akt-DN mutants attenuated MEK1/2 and p42/p44 MAPK phosphorylation stimulated by OxLDL and EGF. These results suggest that the mitogenic effect of OxLDL is, at least in part, mediated through activation of PI3-K/Akt/MEK/MAPK pathway in VSMCs.