Epidermal growth factor stimulates serine/threonine phosphorylation of the focal adhesion protein paxillin in a MEK-dependent manner in normal rat kidney cells

Epidermal growth factor (EGF)-stimulated proliferation of renal epithelial cells plays an important role in the recovery of kidney tubule epithelia following exposure to insult. Numerous studies have demonstrated that tyrosine phosphorylation of the focal adhesion protein paxillin mediates in part the effects of growth factors on cell growth, migration, and organization of the actin-based cytoskeleton. The experiments in this report were designed to determine the effect of EGF on paxillin phosphorylation in normal rat kidney (NRK) epithelial cells. Interestingly, treatment of NRK cells with EGF stimulated paxillin serine/threonine phosphorylation, which caused a reduction in the mobility of paxillin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The EGF-stimulated mobility shift of paxillin was independent of an intact cytoskeleton, phosphatidylinositol 3-kinase (PI 3-kinase) activation, protein kinase C (PKC) activation, and cellular adhesion. However, inhibitors of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase abrogated the EGF-stimulated change in paxillin mobility. In addition, the EGF-stimulated change in paxillin serine/threonine phosphorylation was not accompanied by a profound reorganization of the actin cytoskeleton. These results identify paxillin as a component EGF signaling in renal epithelial cells and implicate members of the MAP kinase pathway as critical regulators of paxillin serine/threonine phosphorylation.     

Epidermal growth factor stimulates Rac activation through Src and phosphatidylinositol 3-kinase to promote colonic epithelial cell migration

Regulated intestinal epithelial cell migration plays a key role in wound healing and maintenance of a healthy gastrointestinal tract. Epidermal growth factor (EGF) stimulates cell migration and wound closure in intestinal epithelial cells through incompletely understood mechanisms. In this study we investigated the role of the small GTPase Rac in EGF-induced cell migration using an in vitro wound-healing assay. In mouse colonic epithelial (MCE) cell lines, EGF-stimulated wound closure was accompanied by a doubling of the number of cells containing lamellipodial extensions at the wound margin, increased Rac membrane translocation in cells at the wound margin, and rapid Rac activation. Either Rac1 small interfering (si)RNA or a Rac1 inhibitor completely blocked EGF-stimulated wound closure. Whereas EGF failed to activate Rac in colon cells from EGF receptor (EGFR) knockout mice, stable expression of wild-type EGFR restored EGF-stimulated Rac activation and migration. Pharmacological inhibition of either phosphatidylinositol 3-kinase (PI3K) or Src family kinases reduced EGF-stimulated Rac activation. Cotreatment of cells with both inhibitors completely blocked EGF-stimulated Rac activation and localization to the leading edge of cells and lamellipodial extension. Our results present a novel mechanism by which the PI3K and Src signaling cascades cooperate to activate Rac and promote intestinal epithelial cell migration downstream of EGFR.     

Specific requirement for the p85-p110alpha phosphatidylinositol 3-kinase during epidermal growth factor-stimulated actin nucleation in breast cancer cells

We have studied the role of phosphatidylinositol 3-kinases (PI 3-kinases) in the regulation of the actin cytoskeleton in MTLn3 rat adenocarcinoma cells. Stimulation of MTLn3 cells with epidermal growth factor (EGF) induced a rapid increase in actin polymerization, with production of lamellipodia within 3 min. EGF-stimulated lamellipodia were blocked by 100 nM wortmannin, suggesting the involvement of a class Ia PI 3-kinase. MTLn3 cells contain equal amounts of p110alpha and p110beta, and do not contain p110delta. Injection of specific inhibitory antibodies to p110alpha induced cell rounding and blocked EGF-stimulated lamellipod extension, whereas control or anti-p110beta antibodies had no effect. In contrast, both antibodies inhibited EGF-stimulated DNA synthesis. An in situ assay for actin nucleation showed that EGF-stimulated formation of new barbed ends was blocked by injection of anti-p110alpha antibodies. In summary, the p110alpha isoform of PI 3-kinase is specifically required for EGF-stimulated actin nucleation during lamellipod extension in breast cancer cells.     

Novel role of Giα2 in cell migration: Downstream of PI3-kinase–AKT and Rac1 in prostate cancer cells

Tumor cell motility is the essential step in cancer metastasis. Previously, we showed that oxytocin and epidermal growth factor (EGF) effects on cell migration in prostate cancer cells require Giα2 protein. In the current study, we investigated the interactions among G-protein coupled receptor (GPCR), Giα2, PI3-kinase, and Rac1 activation in the induction of migratory and invasive behavior by diverse stimuli. Knockdown and knockout of endogenous Giα2 in PC3 cells resulted in attenuation of transforming growth factor β1 (TGFβ1), oxytocin, SDF-1α, and EGF effects on cell migration and invasion. In addition, knockdown of Giα2 in E006AA cells attenuated cell migration and overexpression of Giα2 in LNCaP cells caused significant increase in basal and EGF-stimulated cell migration. Pretreatment of PC3 cells with Pertussis toxin resulted in attenuation of TGFβ1- and oxytocin-induced migratory behavior and PI3-kinase activation without affecting EGF-induced PI3-kinase activation and cell migration. Basal- and EGF-induced activation of Rac1 in PC3 and DU145 cells were not affected in cells after Giα2 knockdown. On the other hand, Giα2 knockdown abolished the migratory capability of PC3 cells overexpressing constitutively active Rac1. The knockdown or knockout of Giα2 resulted in impaired formation of lamellipodia at the leading edge of the migrating cells. We conclude that Giα2 protein acts at two different levels which are both dependent and independent of GPCR signaling to induce cell migration and invasion in prostate cancer cells and its action is downstream of PI3-kinase–AKT–Rac1 axis.     

Over-expression of the p110beta but not p110alpha isoform of PI 3-kinase inhibits motility in breast cancer cells

Phosphoinositide 3-kinase (PI 3-kinase) activity is required for growth factor-induced cytoskeletal regulation and cell migration. We previously found that in MTLn3 rat adenocarcinoma cells, EGF-stimulated induction of actin barbed ends and lamellipod extension specifically requires the p85/p110alpha isoform of PI 3-kinase. To further characterize signaling by distinct PI 3-kinase isoforms, we have developed MTLn3 cells that transiently or stably overexpress either p110alpha or p110beta. Transient overexpression of p110beta inhibited EGF-stimulated lamellipod extension, whereas p110alpha-transfected cells showed normal EGF-stimulated lamellipod extension. Similar results were obtained by overexpression of kinase-dead p110beta, suggesting that effects on cytoskeletal signaling were due to competition with p85/p110alpha complexes. Stable overexpression of p110alpha appeared to be toxic, based on the difficulty in obtaining stable overexpressing clones. In contrast, cells expressing a 2-fold increase in p110beta were readily obtainable. Interestingly, cells stably expressing p110beta showed a marked inhibition of EGF-stimulated lamellipod extension. Using computer-assisted analysis of time-lapse images, we found that overexpression of p110beta caused a nearly complete inhibition of motility. Cells overexpressing p110beta showed normal activation of Akt and Erk, suggesting that overall PI 3-kinase signaling was intact. A chimeric p110 molecule containing the p85-binding and Ras-binding domains of p110alpha and the C2, helical, and kinase domains of p110beta, was catalytically active yet also inhibited EGF-stimulated lamellipod extension. These data highlight the differential signaling by distinct p110 isoforms. Identification of effectors that are differently regulated by p110alpha versus p110beta will be important for understanding cell migration and its role in metastasis.     

Osteopontin stimulates gelsolin-associated phosphoinositide levels and phosphatidylinositol triphosphate-hydroxyl kinase.

Based on previous studies demonstrating activation of phosphatidylinositol 3-hydroxyl kinase (PI3-kinase) and stimulation of a change in cell shape, we examined the effect of osteopontin on the association of phospholipids with gelsolin, an actin-capping/severing protein. Osteopontin stimulated a rapid increase in phosphatidylinositol bisphosphate and phosphatidylinositol triphosphate levels associated with gelsolin in Triton-soluble fractions of cell lysates. The increased levels of phosphatidylinositol triphosphate associated with gelsolin were due to stimulation of PI3-kinase activity associated with gelsolin in the Triton-soluble fractions, and they were blocked by the PI3-kinase inhibitor wortmannin. Osteopontin stimulated translocation of PI3-kinase from the Triton-insoluble to Triton-soluble gelsolin. Osteopontin also decreased Triton-soluble gelsolin/actin complexes consistent with actin uncapping, and increased F-actin levels, which were also blocked by wortmannin. The osteopontin effects were mediated through binding to the alpha(v)beta 3 integrin. Taken together, our studies indicate that osteopontin/alpha(v)beta 3-mediated changes in gelsolin-associated phosphoinositide levels and PI3-kinase activity are related to stimulation of F-actin formation in osteoclasts.     

Epidermal growth factor-stimulated intestinal epithelial cell migration requires Src family kinase-dependent p38 MAPK signaling

Members of the epidermal growth factor (EGF) family of ligands and their receptors regulate migration and growth of intestinal epithelial cells. However, our understanding of the signal transduction pathways determining these responses is incomplete. In this study we tested the hypothesis that p38 is required for EGF-stimulated intestinal epithelial monolayer restitution. EGF-stimulated migration in a wound closure model required continuous presence of ligand for several hours for maximal response, suggesting a requirement for sustained signal transduction pathway activation. In this regard, prolonged exposure of cells to EGF activated p38 for up to 5 h. Furthermore genetic or pharmacological blockade of p38 signaling inhibited the ability of EGF to accelerate wound closure. Interestingly p38 inhibition was associated with increased EGF-stimulated ERK1/ERK2 phosphorylation and cell proliferation, suggesting that p38 regulates the balance of proliferation/migration signaling in response to EGF receptor activity. Activation of p38 in intestinal epithelial cells through EGF receptor was abolished by blockade of Src family tyrosine kinase signaling but not inhibition of phosphatidylinositol 3-kinase or protein kinase C. Taken together, these data suggest that Src family kinase-dependent p38 activation is a key component of a signaling switch routing EGF-stimulated responses to epithelial cell migration/restitution rather than proliferation during wound closure.     

The class II phosphoinositide 3-kinase PI3K-C2beta regulates cell migration by a PtdIns3P dependent mechanism

The biological and pathophysiological significance of class II phosphoinositide 3-kinase enzyme expression currently remains unclear. Using an in vitro scrape wound assay and time-lapse video microscopy, we demonstrate that cell motility is increased in cultures expressing recombinant PI3K-C2beta enzyme. In addition, overexpression of PI3K-C2beta transiently decreased cell adhesion, stimulated the formation of cytoplasmic processes, and decreased the rate of cell proliferation. Consistent with these observations, expression of PI3K-C2beta also decreased expression of alpha4 beta1 integrin subunits. Using asynchronous cultures, we show that endogenous PI3K-C2beta is present in lamellipodia of motile cells. When cells expressing recombinant PI3K-C2beta were plated onto fibronectin, cortical actin staining increased markedly and actin rich lamellipodia and filopodia became evident. Overexpression of a 2xFYVE(Hrs) domain fusion protein abolished this response demonstrating that the effect of PI3K-C2beta on the reorganization of actin filaments is dependent upon PtdIns3P. Finally, overexpression of PI3K-C2beta increased GTP loading of Cdc42. Our data demonstrates for the first time, that PI3K-C2beta plays a regulatory role in cell motility and that the mechanism by which it reorganizes the actin cytoskeleton is dependent upon PtdIns3P production.     

Galectin binding to Mgat5-modified N-glycans regulates fibronectin matrix remodeling in tumor cells

Oncogenic signaling stimulates the dynamic remodeling of actin microfilaments and substrate adhesions, essential for cell spreading and motility. Transformation is associated with increased expression of beta1,6GlcNAc-branched N-glycans, products of Golgi beta1,6-acetylglucosaminyltransferase V (Mgat5) and the favored ligand for galectins. Herein we report that fibronectin fibrillogenesis and fibronectin-dependent cell spreading are deficient in Mgat5(-/-) mammary epithelial tumor cells and inhibited in Mgat5(+/+) cells by blocking Golgi N-glycan processing with swainsonine or by competitive inhibition of galectin binding. At an optimum dosage, exogenous galectin-3 added to Mgat5(+/+) cells activates focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K), recruits conformationally active alpha5beta1-integrin to fibrillar adhesions, and increases F-actin turnover. RGD peptide inhibits PI3K-dependent fibronectin matrix remodeling and fibronectin-dependent cell motility, while galectin-3 stimulates and overrides the inhibitory effects of RGD. Antibodies to the galectin-3 N-terminal oligomerization domain stimulate alpha5beta1 activation and recruitment to fibrillar adhesions in Mgat5(+/+) cells, an effect that is blocked by disrupting galectin-glycan binding. Our results demonstrate that fibronectin polymerization and tumor cell motility are regulated by galectin-3 binding to branched N-glycan ligands that stimulate focal adhesion remodeling, FAK and PI3K activation, local F-actin instability, and alpha5beta1 translocation to fibrillar adhesions.     

Novel role for Na,K-ATPase in phosphatidylinositol 3-kinase signaling and suppression of cell motility

The Na,K-ATPase, consisting of alpha- and beta-subunits, regulates intracellular ion homeostasis. Recent studies have demonstrated that Na,K-ATPase also regulates epithelial cell tight junction structure and functions. Consistent with an important role in the regulation of epithelial cell structure, both Na,K-ATPase enzyme activity and subunit levels are altered in carcinoma. Previously, we have shown that repletion of Na,K-ATPase beta1-subunit (Na,K-beta) in highly motile Moloney sarcoma virus-transformed Madin-Darby canine kidney (MSV-MDCK) cells suppressed their motility. However, until now, the mechanism by which Na,K-beta reduces cell motility remained elusive. Here, we demonstrate that Na,K-beta localizes to lamellipodia and suppresses cell motility by a novel signaling mechanism involving a cross-talk between Na,K-ATPase alpha1-subunit (Na,K-alpha) and Na,K-beta with proteins involved in phosphatidylinositol 3-kinase (PI3-kinase) signaling pathway. We show that Na,K-alpha associates with the regulatory subunit of PI3-kinase and Na,K-beta binds to annexin II. These molecular interactions locally activate PI3-kinase at the lamellipodia and suppress cell motility in MSV-MDCK cells, independent of Na,K-ATPase ion transport activity. Thus, these results demonstrate a new role for Na,K-ATPase in regulating carcinoma cell motility.     

Indomethacin delays gastric restitution: association with the inhibition of focal adhesion kinase and tensin phosphorylation and reduced actin stress fibers

Repair of superficial gastric mucosal injury is accomplished by the process of restitution-migration of epithelial cells to restore continuity of the mucosal surface. Actin filaments, focal adhesions, and focal adhesion kinase (FAK) play crucial roles in cell motility essential for restitution. We studied whether epidermal growth factor (EGF) and/or indomethacin (IND) affect cell migration, actin stress fiber formation, and/or phosphorylation of FAK and tensin in wounded gastric monolayers. Human gastric epithelial monolayers (MKN 28 cells) were wounded and treated with either vehicle or 0.5 mM IND for 16 hr followed by EGF. EGF treatment significantly stimulated cell migration and actin stress fiber formation, and increased FAK localization to focal adhesions, and phosphorylation of FAK and tensin, whereas IND inhibited all these at the baseline and EGF-stimulated conditions. IND-induced inhibition of FAK phosphorylation preceded changes in actin polymerization, indicating that actin depolymerization might be the consequence of decreased FAK activity. In in vivo experiments, rats received either vehicle or IND (5 mg/kg i.g.), and 3 min later, they received water or 5% hypertonic NaCl; gastric mucosa was obtained at 1, 4, and 8 hr after injury. Four and 8 hr after hypertonic injury, FAK phosphorylation was induced in gastric mucosa compared with controls. IND pretreatment significantly delayed epithelial restitution in vivo, and reduced FAK phosphorylation and recruitment to adhesion points, as well as actin stress fiber formation in migrating surface epithelial cells. Our study indicates that FAK, tensin, and actin stress fibers are likely mediators of EGF-stimulated cell migration in wounded human gastric monolayers and potential targets for IND-induced inhibition of restitution.     

Involvement of Phosphatidylinositide 3′-Kinase and Rac in Platelet-Derived Growth Factor-Induced Actin Reorganization and Chemotaxis

Previous work has suggested a role for phosphatidylinositide 3′-kinase (PI3-kinase) in platelet-derived growth factor (PDGF)-induced actin reorganization and chemotaxis. In support of this notion, we show in this report that the PI3-kinase inhibitor wortmannin inhibits chemotaxis of PDGF β-receptor expressing porcine aortic endothelial (PAE/PDGFR-β) cells. Treatment with wortmannin resulted in a dose-dependent decrease in chemotaxis with an IC₅₀value of about 15–20 nM.Higher concentrations of wortmannin also reduced basal random migration of transfected cells in the absence of PDGF. We also investigated the role of Rac in PDGF-induced actin reorganization and cell motility. Overexpression of wt Rac in PAE/PDGFR-β cells led to an increased cell motility and edge ruffling in response to PDGF-BB, compared to control cells. In PAE/PDGFR-β cells transfected with inducible V12Rac (a constitutively active Rac mutant), membrane ruffling occurred in the absence of PDGF stimulation and was independent of PI3-kinase activity. On the other hand, PAE/PDGFR-β cells transfected with inducible N17Rac (a dominant negative Rac mutant) failed to show membrane ruffling in response to PDGF stimulation. Together with previous observations, these data indicate that activation of PI3-kinase is crucial for initiation of PDGF-induced cell motility responses and that Rac has a major role downstream of PI3-kinase, in this pathway.     

Cdc42 and phosphoinositide 3-kinase drive Rac-mediated actin polymerization downstream of c-Met in distinct and common pathways

Activation of c-Met, the hepatocyte growth factor (HGF)/scatter factor receptor induces reorganization of the actin cytoskeleton, which drives epithelial cell scattering and motility and is exploited by pathogenic Listeria monocytogenes to invade nonepithelial cells. However, the precise contributions of distinct Rho-GTPases, the phosphatidylinositol 3-kinases, and actin assembly regulators to c-Met-mediated actin reorganization are still elusive. Here we report that HGF-induced membrane ruffling and Listeria invasion mediated by the bacterial c-Met ligand internalin B (InlB) were significantly impaired but not abrogated upon genetic removal of either Cdc42 or pharmacological inhibition of phosphoinositide 3-kinase (PI3-kinase). While loss of Cdc42 or PI3-kinase function correlated with reduced HGF- and InlB-triggered Rac activation, complete abolishment of actin reorganization and Rac activation required the simultaneous inactivation of both Cdc42 and PI3-kinase signaling. Moreover, Cdc42 activation was fully independent of PI3-kinase activity, whereas the latter partly depended on Cdc42. Finally, Cdc42 function did not require its interaction with the actin nucleation-promoting factor N-WASP. Instead, actin polymerization was driven by Arp2/3 complex activation through the WAVE complex downstream of Rac. Together, our data establish an intricate signaling network comprising as key molecules Cdc42 and PI3-kinase, which converge on Rac-mediated actin reorganization essential for Listeria invasion and membrane ruffling downstream of c-Met.     

Macrophage stimulating protein-induced epithelial cell adhesion is mediated by a PI3-K-dependent, but FAK-independent mechanism

Macrophage stimulating protein (MSP) is a growth and motility factor that mediates its activity via the RON/STK receptor tyrosine kinase. MSP promotes integrin-dependent epithelial cell migration, which suggests that MSP may regulate integrin receptor functions. Integrins are cell surface receptors for extracellular matrix. Epithelial cell adhesion and motility are mediated by integrins. We studied the enhancement by MSP of cell adhesion and the molecular mechanisms mediating this effect. MSP decreased the time required for adhesion of 293 and RE7 epithelial cells to substrates coated with collagen or fibronectin. Prevention of adhesion by an RGD-containing peptide showed that the cell-substrate interaction was mediated by integrins. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), blocked MSP-dependent adhesion, which shows that PI3-K is in the MSP-induced adhesion pathway. MSP also affected focal adhesion kinase (FAK) which is important for some types of cell adhesion and motility. Although MSP caused PI3-K-independent tyrosine phosphorylation and activation of FAK, experiments with dominant-negative FAK constructs showed that FAK does not mediate the effects of MSP on cell adhesion or motility. Thus PI3-K, but not FAK, mediates MSP-induced integrin-dependent adhesion of epithelial cells. Also, we found ligand-independent association between RON and beta1 integrin, which is additional evidence for a relationship between these two receptor systems.     

Lack of correlation between changes in polyphosphoinositide levels and actin/gelsolin complexes in A431 cells treated with epidermal growth factor

The polyphosphoinositides, PIP and PIP2, have been proposed to regulate actin polymerization in vivo because they dissociate actin/gelsolin complexes in vitro. We tested this hypothesis by comparing the ability of EGF and bradykinin to affect PI metabolism and the actin cytoskeleton in A431 cells. EGF, but not bradykinin, was found to induce ruffling and dissociation of actin/gelsolin complexes in these cells. However, both EGF and bradykinin stimulated the accumulation of inositol phosphates in [3H]inositol-labeled cells indicating that stimulation of PI turnover is not sufficient for the induction of changes in actin/gelsolin complex levels. EGF stimulated a twofold increase in the level of PIP in A431 cells. Other phosphoinositide levels were not markedly altered. Treatment of the cells with cholera toxin abrogated the EGF-induced rise in PIP levels without altering the dissociation of actin from gelsolin. These data indicate that increases in PIP and/or PIP2 are not necessary for dissociation of actin/gelsolin complexes. Overall, these experiments suggest that in A431 cells, the effects of EGF on the actin cytoskeleton are unlikely to be mediated through changes in PIP or PIP2 levels.     

Regulation of podosomes by integrin alphavbeta3 and Rho GTPase-facilitated phosphoinositide signaling

In osteoclasts, polyphosphoinositides such as phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5 trisphosphate (PI(3,4,5)P3) are produced in response to integrin alphavbeta3 signaling and they have a critical role in actin cytoskeleton remodeling. The levels of PI(4,5)P2 and PI(3,4,5)P3 are regulated by Rho GTPase through the activation of phosphatidylinositol 4-phosphate 5-kinase (PI4P-5 kinase) and phospatidylinositol 3-kinase (PI3 kinase), respectively. Interaction of PI(4,5)P2 with gelsolin and Wiscott-Aldrich syndrome protein (WASP) is critical for podosome assembly/disassembly and actin ring formation in osteoclasts. Interaction of PI(3,4,5)P3 with gelsolin functions in orchestrating the podosome signaling complex consisting of several key signaling molecules. Gelsolin deficiency has been shown to block podosome assembly and motility in mouse osteoclasts. However, these osteoclasts are able to form a WASP-containing actin ring and retain their resorptive function. The TAT-mediated delivery of gelsolin phosphoinositide-binding domains into osteoclasts resulted in production of podosome clusters and disruption of actin ring formation. Hence, these osteoclasts were hypomotile and less resorptive. Our observations suggest that both PI(4,5)P2 and PI(3,4,5)P3 are involved in regulating osteoclast functions through modulation of severing, capping, and nucleating functions of actin-binding proteins.     

c-kit receptor signaling through its phosphatidylinositide-3'-kinase-binding site and protein kinase C: role in mast cell enhancement of degranulation, adhesion, and membrane ruffling.

In bone marrow-derived mast cells (BMMCs), the Kit receptor tyrosine kinase mediates diverse responses including proliferation, survival, chemotaxis, migration, differentiation, and adhesion to extracellular matrix. In connective tissue mast cells, a role for Kit in the secretion of inflammatory mediators has been demonstrated as well. We recently demonstrated a role for phosphatidylinositide-3' (PI 3)-kinase in Kit-ligand (KL)-induced adhesion of BMMCs to fibronectin. Herein, we investigated the mechanism by which Kit mediates enhancement of Fc epsilon RI-mediated degranulation, cytoskeletal rearrangements, and adhesion in BMMCs. Wsh/Wsh BMMCs lacking endogenous Kit expression, were transduced to express normal and mutant Kit receptors containing Tyr-->Phe substitution at residues 719 and 821. Although the normal Kit receptor fully restored KL-induced responses in Wsh/Wsh BMMCs, Kit gamma 719F, which fails to bind and activate PI 3-kinase, failed to potentiate degranulation and is impaired in mediating membrane ruffling and actin assembly. Inhibition of PI 3-kinase with wortmannin or LY294002 also inhibited secretory enhancement and cytoskeletal rearrangements mediated by Kit. In contrast, secretory enhancement and adhesion stimulated directly through protein kinase C (PKC) do not require PI 3-kinase. Calphostin C, an inhibitor of PKC, blocked Kit-mediated adhesion to fibronectin, secretory enhancement, membrane ruffling, and filamentous actin assembly. Although cytochalasin D inhibited Kit-mediated filamentous actin assembly and membrane ruffling, secretory enhancement and adhesion to fibronectin were not affected by this drug. Therefore, Kit-mediated cytoskeletal rearrangements that are dependent on actin polymerization can be uncoupled from the Kit-mediated secretory and adhesive responses. Our results implicate receptor-proximal PI 3-kinase activation and activation of a PKC isoform in Kit-mediated secretory enhancement, adhesion, and cytoskeletal reorganization.     

N-terminal domains of the class ia phosphoinositide 3-kinase regulatory subunit play a role in cytoskeletal but not mitogenic signaling

Phosphoinositide (PI) 3-kinases are required for the acute regulation of the cytoskeleton by growth factors. We have shown previously that in the MTLn3 rat adenocarcinoma cells line, the p85/p110alpha PI 3-kinase is required for epidermal growth factor (EGF)-stimulated lamellipod extension and formation of new actin barbed ends at the leading edge of the cell. We have now examined the role of the p85alpha regulatory subunit in greater detail. Microinjection of recombinant p85alpha into MTLn3 cells blocked both EGF-stimulated mitogenic signaling and lamellipod extension. In contrast, a truncated p85(1-333), which lacks the SH2 and iSH2 domains and does not bind p110, had no effect on EGF-stimulated mitogenesis but still blocked EGF-stimulated lamellipod extension. Additional deletional analysis showed that the SH3 domain was not required for inhibition of lamellipod extension, as a construct containing only the proline-rich and breakpoint cluster region (BCR) homology domains was sufficient for inhibition. Although the BCR domain of p85 binds Rac, the effects of the p85 constructs were not because of a general inhibition of Rac signaling, because sorbitol-induced JNK activation in MTLn3 cells was not inhibited. These data show that the proline-rich and BCR homology domains of p85 are involved in the coupling of p85/p110 PI 3-kinases to regulation of the actin cytoskeleton. These data provide evidence of a distinct cellular function for the N-terminal domains of p85.     

FAK integrates growth-factor and integrin signals to promote cell migration

Here we show that cells lacking focal adhesion kinase (FAK) are refractory to motility signals from platelet-derived and epidermal growth factors (PDGF and EGF respectively), and that stable re-expression of FAK rescues these defects. FAK associates with activated PDGF- and EGF-receptor (PDGFR and EGFR) signalling complexes, and expression of the band-4.1-like domain at the FAK amino terminus is sufficient to mediate an interaction with activated EGFR. However, efficient EGF-stimulated cell migration also requires FAK to be targeted, by its carboxy-terminal domain, to sites of integrin-receptor clustering. Although the kinase activity of FAK is not needed to promote PDGF- or EGF-stimulated cell motility, kinase-inactive FAK is transphosphorylated at the indispensable Src-kinase-binding site, FAK Y397, after EGF stimulation of cells. Our results establish that FAK is an important receptor-proximal link between growth-factor-receptor and integrin signalling pathways.