Focal adhesion kinase is redistributed to focal complexes and mediates cell spreading in macrophages in response to M-CSF

The macrophage colony-stimulating factor (M-CSF, CSF-1) regulates survival, proliferation and differentiation of mononuclear phagocytes, as well as macrophage motility and morphology. The latter features are usually regulated by ECM-mediated activation of integrins and subsequent tyrosine phosphorylation of cellular proteins, including focal adhesion kinase (FAK). FAK is phosphorylated by downstream receptor tyrosine kinases as well. We addressed the question whether M-CSF regulates FAK tyrosine phosphorylation in macrophages, and found that M-CSF induces FAK phosphorylation at all known tyrosine residues. This phosphorylation was dependent on Src. Extracellularly-regulated kinase (ERK), Jun N-terminal kinase (JNK) and phosphatidylinositol-3-kinase (PI3K) were found to be negatively involved in M-CSF-induced FAK phosphorylation, as their inhibition resulted in FAK hyper-phosphorylation. Following M-CSF treatment, FAK and the active forms of M-CSFR and Src were redistributed to the cytoskeleton, where active ERK, JNK and PI3K were detectable. Immunofluorescence showed the presence of FAK and its active form in focal complexes following M-CSF treatment. Moreover, cell spreading and adhesion were impaired when FAK tyrosine phosphorylation was abrogated by either transfection with FRNK, a dominant negative form of FAK, or treatment with a number of inhibitors of upstream FAK-activating signals. These results point to a relevant role for FAK in the regulation of cell spreading and adhesion in macrophages.     

Hck enhances the adherence of lipopolysaccharide-stimulated macrophages via Cbl and phosphatidylinositol 3-kinase

Src family tyrosine kinases have previously been proposed to mediate some of the biological effects of lipopolysaccharide on macrophages. Accordingly, we have sought to identify substrates of Src family kinases in lipopolysaccharide-stimulated macrophages. Stimulation of Bac1.2F5 macrophage cells with lipopolysaccharide was found to induce gradual and persistent tyrosine phosphorylation of Cbl in an Src family kinase-dependent manner. Immunoprecipitation experiments revealed that Cbl associates with Hck in Bac1.2F5 cells, while expression of an activated form of Hck in Bac1.2F5 cells induces tyrosine phosphorylation of Cbl in the absence of lipopolysaccharide stimulation. The Src homology 3 domain of Hck can directly bind Cbl, and this interaction is important for phosphorylation of Cbl. Association of the p85 subunit of phosphatidylinositol (PI) 3-kinase with Cbl is enhanced following lipopolysaccharide stimulation of Bac1.2F5 cells, and transient expression experiments indicate that phosphorylation of Cbl by Hck can facilitate the association of p85 with Cbl. Lipopolysaccharide treatment also stimulates the partial translocation of Hck to the cytoskeleton of Bac1.2F5 cells. Notably, lipopolysaccharide enhances the adherence of Bac1.2F5 cells, an effect that is dependent on the activity of Src family kinases and PI 3-kinase. Thus, we postulate that Hck enhances the adherence of lipopolysaccharide-stimulated macrophages, at least in part, via Cbl and PI 3-kinase.     

Integrin-mediated Signals Regulated by Members of the Rho Family of GTPases

The organization of the actin cytoskeleton can be regulated by soluble factors that trigger signal transduction events involving the Rho family of GTPases. Since adhesive interactions are also capable of organizing the actin-based cytoskeleton, we examined the role of Cdc42-, Rac-, and Rho-dependent signaling pathways in regulating the cytoskeleton during integrin-mediated adhesion and cell spreading using dominant-inhibitory mutants of these GTPases. When Rat1 cells initially adhere to the extracellular matrix protein fibronectin, punctate focal complexes form at the cell periphery. Concomitant with focal complex formation, we observed some phosphorylation of the focal adhesion kinase (FAK) and Src, which occurred independently of Rho family GTPases. However, subsequent phosphorylation of FAK and paxillin occurs in a Rho-dependent manner. Moreover, we found Rho dependence of the assembly of large focal adhesions from which actin stress fibers radiate. Initial adhesion to fibronectin also stimulates membrane ruffling; we show that this ruffling is independent of Rho but is dependent on both Cdc42 and Rac. Furthermore, we observed that Cdc42 controls the integrin-dependent activation of extracellular signal–regulated kinase 2 and of Akt, a kinase whose activity has been demonstrated to be dependent on phosphatidylinositol (PI) 3-kinase. Since Rac-dependent membrane ruffling can be stimulated by PI 3-kinase, it appears that Cdc42, PI 3-kinase, and Rac lie on a distinct pathway that regulates adhesion-induced membrane ruffling. In contrast to the differential regulation of integrin-mediated signaling by Cdc42, Rac, and Rho, we observed that all three GTPases regulate cell spreading, an event that may indirectly control cellular architecture. Therefore, several separable signaling pathways regulated by different members of the Rho family of GTPases converge to control adhesion-dependent changes in the organization of the cytoskeleton, changes that regulate cell morphology and behavior.     

M-CSF stimulated differentiation requires persistent MEK activity and MAPK phosphorylation independent of Grb2-Sos association and phosphatidylinositol 3-kinase activity

Macrophage colony-stimulating factor (M-CSF) is a physiological regulator of monocyte-macrophage lineage. Ectopic expression of the M-CSF receptor (M-CSFR, or Fms) in murine myeloid cell line FDC-P1 (FD/Fms cells) results in M-CSF-dependent macrophage differentiation. Previously, we observed that M-CSF induces two temporally distinct phases of mitogen-activated protein kinase (MAPK) phosphorylation. Here we show that levels of phosphorylated MAPK kinase MEK1 follow the same kinetics as MAPK phosphorylation, characterized by an early and transient phase (the first 30 min of M-CSF stimulation) and a late and persistent phase from 4 h of stimulation. The MEK inhibitor U0126 strongly inhibited both phases of MAPK phosphorylation as well as FD/Fms cell differentiation, indicating that MAPK may relay M-CSF differentiation signaling downstream of M-CSFR. Treatment of FD/Fms cells with U0126 during the first hour of M-CSF stimulation reversibly blocked the early phase of MAPK phosphorylation but did not affect differentiation. In contrast, U0126 still inhibited FD/Fms cell differentiation when its addition was delayed by 24 h. This demonstrated that late and persistent MEK activity is specifically required for macrophage differentiation to occur. Furthermore, disrupting Grb2-Sos complexes with a specific blocking peptide did not prevent FD/Fms cells differentiation in response to M-CSF, nor did it abolish MAPK phosphorylation. The role of phosphatidylinositol 3-kinase (PI 3-kinase), another potential regulator of the MAPK pathway, was examined using the specific inhibitor LY294002. This compound could not impede FD/Fms cell commitment to macrophage differentiation and did not significantly affect MAPK phosphorylation in response to M-CSF. Therefore, M-CSF differentiation signaling in myeloid progenitor cells is mediated through persistent MEK activity but it is not strictly dependent upon Grb2-Sos interaction or PI 3-kinase activity.     

Involvement of c-Src Tyrosine Kinase Upstream of Class I Phosphatidylinositol (PI) 3-Kinases in Salmonella Enteritidis Rck Protein-mediated Invasion

The Salmonella outer membrane protein Rck mediates a Zipper entry mechanism controlled by tyrosine phosphorylation and class I phosphatidylinositol 3-kinase (PI 3-kinase). However, the underlying mechanism leading to this signaling cascade remains unclear. The present study showed that using Rck-coated beads or Rck-overexpressing Escherichia coli, Rck-mediated actin polymerization and invasion were blocked by PP2, a Src family tyrosine kinase inhibitor. In addition, phosphorylation of Src family kinases significantly increased after stimulation with Rck. The specific contribution of c-Src, one member of the Src family kinases, was demonstrated using c-Src-deficient fibroblasts or c-Src siRNA transfected epithelial cells. We also observed that Rck-mediated internalization led to the formation of a complex between c-Src and at least one tyrosine-phosphorylated protein. Furthermore, our results revealed that the c-Src signal molecule was upstream of PI 3-kinase during the Rck-mediated signaling pathway as Rck-mediated PI 3-kinase activation was blocked by PP2, and PI 3-kinase inhibitor had no effect on the Src phosphorylation. These results demonstrate the involvement of c-Src upstream of the PI 3-kinase in the Zipper entry process mediated by Rck.     

Amyloid-beta peptides induce cell proliferation and macrophage colony-stimulating factor expression via the PI3-kinase/Akt pathway in cultured Ra2 microglial cells

Alzheimer's disease is characterized by numerous amyloid-beta peptide (Abeta) plaques surrounded by microglia. Here we report that Abeta induces the proliferation of the mouse microglial cell line Ra2 by increasing the expression of macrophage colony-stimulating factor (M-CSF). We examined signal cascades for Abeta-induced M-CSF mRNA expression. The induction of M-CSF was blocked by a phosphatidylinositol 3 kinase (PI3-kinase) inhibitor (LY294002), a Src family tyrosine kinase inhibitor (PP1) and an Akt inhibitor. Electrophoretic mobility shift assays showed that Abeta enhanced NF-kappaB binding activity to the NF-kappaB site of the mouse M-CSF promoter, which was blocked by LY294002. These results indicate that Abeta induces M-CSF mRNA expression via the PI3-kinase/Akt/NF-kappaB pathway.     

The SH3 domain directs acto-myosin-dependent targeting of v-Src to focal adhesions via phosphatidylinositol 3-kinase

The v-Src oncoprotein is translocated to integrin-linked focal adhesions, where its tyrosine kinase activity induces adhesion disruption and cell transformation. We previously demonstrated that the intracellular targeting of Src is dependent on the actin cytoskeleton, under the control of the Rho family of small G proteins. However, the assembly of v-Src into focal adhesions does not require its catalytic activity or myristylation-dependent membrane association. Here, we report that the SH3 domain is essential for the assembly of focal adhesions containing the oncoprotein by mediating a switch from a microtubule-dependent, perinuclear localization to actin-associated focal adhesions; furthermore, v-Src translocation to focal adhesions requires myosin activity, at least under normal conditions when the actin cytoskeleton is being dynamically regulated. Although the SH3 domain of v-Src is also necessary for its association with focal adhesion kinase (FAK), which is often considered a likely candidate mediator of focal adhesion targeting via its carboxy-terminal targeting sequence, we show here that binding to FAK is not essential for the targeting of v-Src to focal adhesions. The p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase also associates with v-Src in an SH3-dependent manner, but in this case inhibition of PI 3-kinase activity suppressed assembly of focal adhesions containing the oncoprotein. Thus, the Src SH3 domain, which binds PI 3-kinase and which is necessary for activation of Akt downstream, is required for the actin-dependent targeting of v-Src to focal adhesions.     

Intracellular movement of green fluorescent protein-tagged phosphatidylinositol 3-kinase in response to growth factor receptor signaling.

Phosphatidylinositol 3-kinase (PI 3-kinase) is a lipid kinase which has been implicated in mitogenesis, protein trafficking, inhibition of apoptosis, and integrin and actin functions. Here we show using a green fluorescent protein-tagged p85 subunit that phosphatidylinositol 3-kinase is distributed throughout the cytoplasm and is localized to focal adhesion complexes in resting NIH-3T3, A431, and MCF-7 cells. Ligand stimulation of an epidermal growth factor receptor/c-erbB-3 chimera expressed in these cells results in a redistribution of p85 to the cell membrane which is independent of the catalytic activity of the enzyme and the integrity of the actin cytoskeleton. The movement is, however, dependent on the phosphorylation status of the erbB-3 chimera. Using rhodamine-labeled epidermal growth factor we show that the phosphatidylinositol 3-kinase and the receptors colocalize in discrete patches on the cell surface. Low concentrations of ligand cause patching only at the periphery of the cells, whereas at high concentrations patches were seen over the whole cell surface. Using green fluorescent protein-tagged fragments of p85 we show that binding to the receptor requires the NH(2)-terminal part of the protein as well as its SH2 domains.     

Inhibiting Src family tyrosine kinase activity blocks glutamate signalling to ERK1/2 and Akt/PKB but not JNK in cultured striatal neurones

Glutamate receptor activation of mitogen-activated protein (MAP) kinase signalling cascades has been implicated in diverse neuronal functions such as synaptic plasticity, development and excitotoxicity. We have previously shown that Ca2+-influx through NMDA receptors in cultured striatal neurones mediates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt/protein kinase B (PKB) through a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. Exposing neurones to the Src family tyrosine kinase inhibitor PP2, but not the inactive analogue PP3, inhibited NMDA receptor-induced phosphorylation of ERK1/2 and Akt/PKB in a concentration-dependent manner, and reduced cAMP response element-binding protein (CREB) phosphorylation. To establish a link between Src family tyrosine kinase-mediated phosphorylation and PI 3-kinase signalling, affinity precipitation experiments were performed with the SH2 domains of the PI 3-kinase regulatory subunit p85. This revealed a Src-dependent phosphorylation of a focal adhesion kinase (FAK)-p85 complex on glutamate stimulation. Demonstrating that PI3-kinase is not ubiquitously involved in NMDA receptor signal transduction, the PI 3-kinase inhibitors wortmannin and LY294002 did not prevent NMDA receptor Ca2+-dependent phosphorylation of c-Jun N-terminal kinase 1/2 (JNK1/2). Further, inhibiting Src family kinases increased NMDA receptor-dependent JNK1/2 phosphorylation, suggesting that Src family kinase-dependent cascades may physiologically limit signalling to JNK. These results demonstrate that Src family tyrosine kinases and PI3-kinase are pivotal regulators of NMDA receptor signalling to ERK/Akt and JNK in striatal neurones.     

The chum salmon IGF-II gene promoter is activated by hepatocyte nuclear factor 3beta

The molecular events whereby gastrin occupancy of G/CCK(B) receptors leads to phosphatidylinositol (PI) 3-kinase activation have been examined. We report here that this peptide promotes the association between two non-receptor tyrosine kinases, p60Src and p125FAK, and elicits a parallel increase in tyrosine phosphorylation and activity of both kinases. Gastrin-induced PI 3-kinase activity was coprecipitated with p60Src and p125FAK and was inhibited by herbimycin A, the selective Src inhibitor PP-2 or cytochalasin D, which disrupts the actin cytoskeleton and prevents p125FAK activity. These results indicate, for the first time, that a p60Src/p125FAK complex acts upstream of the gastrin-stimulated PI 3-kinase pathway.     

Reactive oxygen species regulate M-CSF-induced monocyte/macrophage proliferation through SHP1 oxidation

Macrophage colony-stimulating factor (M-CSF) stimulation results in the production of reactive oxygen species (ROS) that participate in the proliferation of monocyte/macrophage. However, the molecular mechanisms whereby ROS modulate the signaling processes of M-CSF remain poorly defined. We report here that the redox-sensitive Src homology region 2 domain-containing phosphatase 1 (SHP1) is a critical regulator of M-CSF-mediated signaling in bone marrow monocyte/macrophage lineage cells (BMMs). Application of diphenylene iodonium (DPI) inhibited the responses of BMMs to M-CSF, including ROS production, cell proliferation, and phosphorylation of c-Fms as well as Akt kinase, but not of MAP kinases such as ERK, p38, and JNK. Dysregulation of SHP1 by overexpression or RNA interference in BMMs showed that SHP1 specifically regulates PI3 kinase (PI3K)/Akt signaling, but not MAP kinases in a redox-dependent manner, thereby regulating proliferation of BMMs through cyclins D1 and D2. These findings demonstrate that M-CSF-mediated ROS generation leads to SHP1 oxidation, which promotes cell proliferation through the PI3K/Akt-dependent signaling pathway.     

Translocation of the B cell antigen receptor into lipid rafts reveals a novel step in signaling

The cross-linking of the B cell Ag receptor (BCR) leads to the initiation of a signal transduction cascade in which the earliest events involve the phosphorylation of the immunoreceptor tyrosine-based activation motifs of Ig alpha and Ig beta by the Src family kinase Lyn and association of the BCR with the actin cytoskeleton. However, the mechanism by which BCR cross-linking initiates the cascade remains obscure. In this study, using various A20-transfected cell lines, biochemical and genetic evidence is provided that BCR cross-linking leads to the translocation of the BCR into cholesterol- and sphingolipid-rich lipid rafts in a process that is independent of the initiation of BCR signaling and does not require the actin cytoskeleton. Translocation of the BCR into lipid rafts did not require the Ig alpha/Ig beta signaling complex, was not dependent on engagement of the FcR, and was not blocked by the Src family kinase inhibitor PP2 or the actin-depolymerizing agents cytochalasin D or latrunculin. Thus, cross-linking or oligomerization of the BCR induces the BCR translocation into lipid rafts, defining an event in B cell activation that precedes receptor phosphorylation and association with the actin cytoskeleton.     

Verotoxin-1 stimulation of macrophage-like THP-1 cells up-regulates tissue factor expression through activation of c-Yes tyrosine kinase: Possible signal transduction in tissue factor up-regulation

Verotoxin (VT)-producing Escherichia coli (E. coli) O157:H7 infections are frequently complicated by thrombotic angiopathy, hemolytic uremic syndrome (HUS) and neurological symptoms. The present data demonstrate that VT-1 (Shiga toxin) stimulation of macrophage-like THP-1 cells up-regulates the activity, antigen and mRNA levels of tissue factor (TF), a key cofactor of the coagulation-inflammation-thrombosis circuit. This up-regulation is accompanied by phosphorylation of phosphatidylinositol 3-kinase (PI3-kinase), IkappaB kinase beta (IKKbeta) and extracellular signal-regulated kinase 2 (ERK2). Changes in TF mRNA levels were in parallel with the activation of NF-kappaB/Rel and Egr-1 activation, but not with AP-1. Inhibition of PI3-kinase attenuated VT-1-induced phosphorylation of IKKbeta and ERK2, and the up-regulation of TF mRNA levels. VT-1 stimulation rapidly activated c-Yes tyrosine kinase, a member of the Src family. Treatment of the cells with c-Yes antisense oligos attenuated the VT-1-induced phosphorylation of PI3-kinase, IKKbeta and ERK2, activations of NF-kappaB/Rel and Egr-1, and up-regulation of TF mRNA levels. These results suggest that VT-1-induced macrophage stimulation activates c-Yes, which then up-regulates TF expression through activation of the IKKbeta/proteasome/NF-kappaB/Rel and MEK/ERK2/Egr-1 pathways via activation of PI3-kinase. Induction of macrophage TF expression by VT-1 may play an important role in the acceleration of the coagulation-inflammation-thrombosis circuit during infections by VT-producing E. coli.     

beta -Arrestin-mediated recruitment of the Src family kinase Yes mediates endothelin-1-stimulated glucose transport

The insulin and the endothelin type A (ETA) receptor both can couple into the heterotrimeric G protein alpha(q/11) (Galpha(q/11)), leading to Galpha(q/11) tyrosine phosphorylation, phosphatidylinositol 3-kinase activation, and subsequent stimulation of glucose transport. In this study, we assessed the potential role of Src kinase in ET-1 signaling to glucose transport in 3T3-L1 adipocytes. Src kinase inhibitor PP2 blocked ET-1-induced Src kinase activity, Galpha(q/11) tyrosine phosphorylation, and glucose transport stimulation. To determine which Src family kinase member was involved, we microinjected anti-c-Src, -c-Fyn, or -c-Yes antibody into these cells and found that only anti-c-Yes antibody blocked GLUT4 translocation (70% decreased). Overexpression or microinjection of a dominant negative mutant (K298M) of Src kinase also inhibited ET-1-induced Galpha(q/11) tyrosine phosphorylation and GLUT4 translocation. In co-immunoprecipitation experiments, we found that beta-arrestin 1 associated with the ETA receptor in an agonist-dependent manner and that beta-arrestin 1 recruited Src kinase to a molecular complex that included the ETA receptor. Microinjection of beta-arrestin 1 antibody inhibited ET-1- but not insulin-stimulated GLUT4 translocation. In conclusion, 1) the Src kinase Yes can induce tyrosine phosphorylation of Galpha(q/11) in response to ET-1 stimulation, and 2) beta-arrestin 1 and Src kinase form a molecular complex with the ETA receptor to mediate ET-1 signaling to Galpha(q/11) with subsequent glucose transport stimulation.     

Agonist-specific transactivation of phosphoinositide 3-kinase signaling pathway mediated by the dopamine D2 receptor

Bromocriptine, acting through the dopamine D2 receptor, provides robust protection against apoptosis induced by oxidative stress in PC12-D2R and immortalized nigral dopamine cells. We now report the characterization of the D2 receptor signaling pathways mediating the cytoprotection. Bromocriptine caused protein kinase B (Akt) activation in PC12-D2R cells and the inhibition of either phosphoinositide (PI) 3-kinase, epidermal growth factor receptor (EGFR), or c-Src eliminated the Akt activation and the cytoprotective effects of bromocriptine against oxidative stress. Co-immunoprecipitation studies showed that the D2 receptor forms a complex with the EGFR and c-Src that was augmented by bromocriptine, suggesting a cross-talk between these proteins in mediating the activation of Akt. EGFR repression by inhibitor or by RNA interference eliminated the activation of Akt by bromocriptine. D2 receptor stimulation by bromocriptine induced c-Src tyrosine 418 phosphorylation and EGFR phosphorylation specifically at tyrosine 845, a known substrate of Src kinase. Furthermore, Src tyrosine kinase inhibitor or dominant negative Src interfered with Akt translocation and phosphorylation. Thus, the predominant signaling cascade mediating cytoprotection by the D2 receptor involves c-Src/EGFR transactivation by D2 receptor, activating PI 3-kinase and Akt. We also found that the agonist pramipexole failed to stimulate activation of Akt in PC12-D2R cells, providing an explanation for our previous observations that, despite efficiently activating G-protein signaling, this agonist had little cytoprotective activity in this experimental system. These results support the hypothesis that specific dopamine agonists stabilize distinct conformations of the D2 receptor that differ in their coupling to G-proteins and to a cytoprotective c-Src/EGFR-mediated PI-3 kinase/Akt pathway.     

M-CSF regulates the cytoskeleton via recruitment of a multimeric signaling complex to c-Fms Tyr-559/697/721

M-CSF is known to induce cytoskeletal reorganization in macrophages and osteoclasts by activation of phosphatidylinositol 3-kinase (PI3K) and c-Src, but the detailed mechanisms remain unclear. We find, unexpectedly, that tyrosine (Tyr) to phenylalanine (Phe) mutation of Tyr-721, the PI3K binding site in the M-CSF receptor c-Fms, fails to suppress cytoskeletal remodeling or actin ring formation. In contrast, mutation of c-Fms Tyr-559 to Phe blocks M-CSF-induced cytoskeletal reorganization by inhibiting formation of a Src Family Kinase SFK.c-Cbl.PI3K complex and the downstream activation of Vav3 and Rac, two key mediators of actin remodeling. Using an add-back approach in which specific Tyr residues are reinserted into c-Fms inactivated by the absence of all seven functionally important Tyr residues, we find that Tyr-559 is necessary but not sufficient to transduce M-CSF-dependent cytoskeletal reorganization. Furthermore, this same add-back approach identifies important roles for Tyr-697 and Tyr-721 in collaborating with Tyr-559 to recruit a multimeric signaling complex that can transduce signals from c-Fms to the actin cytoskeleton.     

Involvement of the Src kinase Lyn in phospholipase C-gamma 2 phosphorylation and phosphatidylinositol 3-kinase activation in Epo signalling

We examined the role of the Src kinase Lyn in phospholipase C-gamma 2 (PLC-gamma 2) and phosphatidylinositol (PI) 3-kinase activation in erythropoietin (Epo)-stimulated FDC-P1 cells transfected with a wild type (WT) Epo-receptor (Epo-R). We showed that two inhibitors of Src kinases, PP1 and PP2, abolish both PLC-gamma 2 tyrosine phosphorylation and PI 3-kinase activity in WT Epo-R FDC-P1 cells. We also demonstrated that Epo-phosphorylated Lyn is associated with tyrosine phosphorylated PLC-gamma 2 and PI 3-kinase in WT Epo-R FDC-P1-stimulated cells. Moreover Epo-activated Lyn phosphorylates in vitro PLC-gamma 2 immunoprecipitated from unstimulated cells. Our results suggest that the Src kinase Lyn is involved in PLC-gamma 2 phosphorylation and PI 3-kinase activation induced by Epo.     

Differential Regulation of Focal Adhesion Kinase and Mitogen-Activated Protein Kinase Tyrosine Phosphorylation During Insulin-Like Growth Factor-I-Mediated Cytoskeletal Reorganization

Abstract: In SH-SY5Y human neuroblastoma cells, insulin-like growth factor (IGF)-I mediates membrane ruffling and growth cone extension. We have previously shown that IGF-I activates the tyrosine phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated protein kinase (ERK) 2. In the current study, we examined which signaling pathway underlies IGF-I-mediated FAK phosphorylation and cytoskeletal changes and determined if an intact cytoskeleton was required for IGF-I signaling. Treatment of SH-SY5Y cells with cytochalasin D disrupted the actin cytoskeleton and prevented any morphological changes induced by IGF-I. Inhibitors of phosphatidylinositol 3-kinase (PI 3-K) blocked IGF-I-mediated changes in the actin cytoskeleton as measured by membrane ruffling. In contrast, PD98059, a selective inhibitor of ERK kinase, had no effect on IGF-I-induced membrane ruffling. In parallel with effects on the actin cytoskeleton, cytochalasin D and PI 3-K inhibitors blocked IGF-I-induced FAK tyrosine phosphorylation, whereas PD98059 had no effect. It is interesting that cytochalasin D did not block IGF-I-induced ERK2 tyrosine phosphorylation. Therefore, it is likely that FAK and ERK2 tyrosine phosphorylations are regulated by separate pathways during IGF-I signaling. Our study suggests that integrity as well as dynamic motility of the actin cytoskeleton mediated by PI 3-K is required for IGF-I-induced FAK tyrosine phosphorylation, but not for ERK2 activation.     

Src kinase mediates phosphatidylinositol 3-kinase/Akt-dependent rapid endothelial nitric-oxide synthase activation by estrogen

17beta-Estradiol activates endothelial nitric oxide synthase (eNOS), enhancing nitric oxide (NO) release from endothelial cells via the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway. The upstream regulators of this pathway are unknown. We now demonstrate that 17beta-estradiol rapidly activates eNOS through Src kinase in human endothelial cells. The Src family kinase specific-inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) abrogates 17beta-estradiol- but not ionomycin-stimulated NO release. Consistent with these results, PP2 blocked 17beta-estradiol-induced Akt phosphorylation but did not inhibit NO release from cells transduced with a constitutively active Akt. PP2 abrogated 17beta-estradiol-induced activation of PI3-kinase, indicating that the PP2-inhibitable kinase is upstream of PI3-kinase and Akt. A 17beta-estradiol-induced estrogen receptor/c-Src association correlated with rapid c-Src phosphorylation. Moreover, transfection of kinase-dead c-Src inhibited 17beta-estradiol-induced Akt phosphorylation, whereas constitutively active c-Src increased basal Akt phosphorylation. Estrogen stimulation of murine embryonic fibroblasts with homozygous deletions of the c-src, fyn, and yes genes failed to induce Akt phosphorylation, whereas cells maintaining c-Src expression demonstrated estrogen-induced Akt activation. Estrogen rapidly activated c-Src inducing an estrogen receptor, c-Src, and P85 (regulatory subunit of PI3-kinase) complex formation. This complex formation results in the successive activation of PI3-kinase, Akt, and eNOS with consequent enhanced NO release, implicating c-Src as a critical upstream regulator of the estrogen-stimulated PI3-kinase/Akt/eNOS pathway.     

Properties of BK<Subscript>Ca</Subscript> Channels Formed by Bicistronic Expression of <Emphasis Type="Italic">hSlo</Emphasis>α and β1–4 Subunits in HEK293 Cells

The basolateral Na+/HCO3- cotransporter (NBC) is the major pathway for bicarbonate reabsorption in the renal proximal tubule cells. The cotransporter activity is enhanced by 10% CO2. Phosphatidylinositol 3-kinase (PI3K) has been shown to regulate the function and trafficking of cellular proteins by promoting their translocation to the plasma membrane. Therefore, we sought to examine the role of PI3K in CO2-mediated stimulation of NBC activity in OK cells. Our studies showed that wortmannin, a well-characterized PI3K inhibitor, had no effect on baseline NBC activity but prevented the stimulatory effect of 10% CO2. This effect was concentration-dependent and time-dependent. Another inhibitor of PI3K, LY294002, also prevented the CO2-mediated increase in NBC activity. CO2 stimulation of the cotransporter was paralleled by an increase in PI3K enzyme activity and this effect was blocked by wortmannin. Biotinylation studies also showed that 10% CO2 increased the immunoreactive NBC in the basolateral membranes and this was prevented by wortmannin. We previously showed that 10% CO2 stimulation of NBC activity involves the Src family kinase pathway. In the current studies, CO2 stimulation significantly increased Src phosphorylation and this effect was abrogated by wortmannin. In summary, CO2 stimulation of NBC is mediated at least in part by increased immunoreactive NBC protein in the basolateral membrane, a process which requires the interaction of PI3K with Src family kinase.