Negative regulation of EGFR signalling through integrin-alpha1beta1-mediated activation of protein tyrosine phosphatase TCPTP

Integrin-mediated cell adhesion regulates a multitude of cellular responses, including proliferation, survival and cross-talk between different cellular signalling pathways. So far, integrins have been mainly shown to convey permissive signals enabling anchorage-dependent receptor tyrosine kinase signalling. Here we show that a collagen-binding integrin alpha(1)beta(1) functions as a negative regulator of epidermal growth factor receptor (EGFR) signalling through the activation of a protein tyrosine phosphatase. The cytoplasmic tail of alpha(1) integrin selectively interacts with a ubiquitously expressed protein tyrosine phosphatase TCPTP (T-cell protein tyrosine phosphatase) and activates it after cell adhesion to collagen. The activation results in reduced EGFR phosphorylation after EGF stimulation. Introduction of the alpha(1) cytoplasmic domain peptide into cells induces phosphatase activation and inhibits EGF-induced cell proliferation and anchorage-independent growth of malignant cells. These data are the first demonstration of the regulation of TCPTP activity in vivo and represent a new molecular paradigm of integrin-mediated negative regulation of receptor tyrosine kinase signalling.     

C. elegans PAT-4/ILK functions as an adaptor protein within integrin adhesion complexes

BACKGROUND: Mammalian integrin-linked kinase (ILK) was identified in a yeast two-hybrid screen for proteins binding the integrin beta(1) subunit cytoplasmic domain. ILK has been implicated in integrin-mediated signaling and is also an adaptor within integrin-associated cytoskeletal complexes. RESULTS: We identified the C. elegans pat-4 gene in previous genetic screens for mutants unable to assemble integrin-mediated muscle cell attachments. Here, we report that pat-4 encodes the sole C. elegans homolog of ILK. In pat-4 null mutants, embryonic muscle cells form integrin foci, but the subsequent recruitment of vinculin and UNC-89 as well as actin and myosin filaments to these in vivo focal adhesion analogs is blocked. Conversely, PAT-4/ILK requires the ECM component UNC-52/perlecan, the transmembrane protein integrin, and the novel cytoplasmic attachment protein UNC-112 to be properly recruited to nascent attachments. Transgenically expressed "kinase-dead" ILK fully rescues pat-4 loss-of-function mutants. We also identify UNC-112 as a new binding partner for ILK. CONCLUSIONS: Our data strengthens the emerging view that ILK functions primarily as an adaptor protein within integrin adhesion complexes and identifies UNC-112 as a new ILK binding partner.     

Binding of Paxillin to the alpha 9 Integrin Cytoplasmic Domain Inhibits Cell Spreading

alpha(9)beta(1) integrin is a member of the beta(1) integrin family, plays an important role in extravasation of neutrophils at sites of acute inflammation, and is required for the normal development of the lymphatic system. The alpha(9) and alpha(4) integrin subunits are most closely related and form a subfamily of integrin alpha subunits. Previously, we have reported that the alpha(4) cytoplasmic domain directly and tightly binds paxillin, an intracellular signaling adaptor molecule. This interaction accounts for some of the unusual functional responses to alpha(4) integrin-mediated cell adhesion, including stimulation of cell migration and inhibition of cell spreading and focal adhesion formation. In the current studies, we have examined the interaction between the alpha(9) cytoplasmic domain and paxillin. Here we report that the alpha(9) cytoplasmic domain binds paxillin directly and tightly and that the alpha(9)-paxillin association inhibits cell spreading. We have identified amino acid residues in the alpha(9) cytoplasmic domain, Trp(999) and Trp(1001), that are critical for paxillin binding, and alanine substitution of either Trp(999) or Trp(1001) blocks paxillin binding. Furthermore, these mutations also reverse the effect of the alpha(9) cytoplasmic domain on cell spreading. Thus, the alpha(9) and alpha(4) integrin subunits form a paxillin-binding subfamily of integrin alpha subunits, and direct binding of paxillin to the alpha(9) cytoplasmic domain mediates some of the biological activities of the alpha(9)beta(1) integrin.     

Alpha v beta 5 integrin-dependent programmed cell death triggered by a peptide mimic of annexin V

The diverse cytoplasmic domain sequences within the various integrin subunits are critical for integrin-mediated signaling into the cell (outside-in signaling) and for activation of ligand binding affinity (inside-out signaling). Here we introduce an approach based on phage display technology to identify molecules that specifically interact with the cytoplasmic domain of the beta 5 integrin subunit. We show that a peptide selected for binding specifically to the beta 5 cytoplasmic domain (VVISYSMPD) induces apoptosis upon internalization. The cell death process induced by VVISYSMPD is sensitive to modulation by growth factors and by protein kinase C (PKC), and it cannot be triggered in beta 5 null cells. Finally, we show that the VVISYSMPD peptide is a mimic of annexin V. Our results suggest a functional link between the alpha v beta 5 integrin, annexin V, and programmed cell death. We propose the term "endothanatos" to designate this phenomenon.     

R-Ras signals through specific integrin alpha cytoplasmic domains to promote migration and invasion of breast epithelial cells.

Specificity and modulation of integrin function have important consequences for cellular responses to the extracellular matrix, including differentiation and transformation. The Ras-related GTPase, R-Ras, modulates integrin affinity, but little is known of the signaling pathways and biological functions downstream of R-Ras. Here we show that stable expression of activated R-Ras or the closely related TC21 (R-Ras 2) induced integrin-mediated migration and invasion of breast epithelial cells through collagen and disrupted differentiation into tubule structures, whereas dominant negative R-Ras had opposite effects. These results imply novel roles for R-Ras and TC21 in promoting a transformed phenotype and in the basal migration and polarization of these cells. Importantly, R-Ras induced an increase in cellular adhesion and migration on collagen but not fibronectin, suggesting that R-Ras signals to specific integrins. This was further supported by experiments in which R-Ras enhanced the migration of cells expressing integrin chimeras containing the alpha2, but not the alpha5, cytoplasmic domain. In addition, a transdominant inhibition previously noted only between integrin beta cytoplasmic domains was observed for the alpha2 cytoplasmic domain; alpha2beta1-mediated migration was inhibited by the expression of excess alpha2 but not alpha5 cytoplasmic domain-containing chimeras, suggesting the existence of limiting factors that bind the integrin alpha subunit. Using pharmacological inhibitors, we found that R-Ras induced migration on collagen through a combination of phosphatidylinositol 3-kinase and protein kinase C, but not MAPK, which is distinct from the other Ras family members, Rac, Cdc42, and N- and K-Ras. Thus, R-Ras communicates with specific integrin alpha cytoplasmic domains through a unique combination of signaling pathways to promote cell migration and invasion.     

Reduced cell adhesion during mitosis by threonine phosphorylation of beta1 integrin

Cell shape and adhesion of cultured mammalian cells change dramatically during mitosis, however, how cell cycle-dependent alterations in cell adhesion are regulated remain to be elucidated. We show here that normal human mammary epithelial (HME) cells which became less adhesive and adopted the rounded morphology during the G(2)/M phase of the cell cycle significantly reduced their dependence on beta1 integrin-mediated adhesion to laminin, by using function blocking antibody to beta1 integrin. In G(2)/M cells, both total and cell surface expressions of beta1 integrin were comparable with those in G(1) cells but it was phosphorylated at threonines 788-789 within its cytoplasmic domain and coimmunoprecipitated Ca(2+)/calmodulin-dependent protein kinase (CaMK) II. The threonine phosphorylated beta1 integrin significantly reduced its intracellular linkage with actin, with no significant reduction in the actin expression. In contrast, beta1 integrin in G(1) cells was not threonine phosphorylated but formed a link with actin and coimmunoprecipitated the core enzyme of the serine/threonine protein phosphatase (PP) 2A. The results suggest that reduced beta1 integrin-mediated cell adhesion of HME cells to the substratum during mitosis may be induced by beta1 integrin phosphorylation at threonines 788-789 and its reduced ability to link with the actin cytoskeleton.     

Drosophila integrin-linked kinase is required at sites of integrin adhesion to link the cytoskeleton to the plasma membrane

Integrin-linked kinase (ILK) was identified by its interaction with the cytoplasmic tail of human beta1 integrin and previous data suggest that ILK is a component of diverse signaling pathways, including integrin, Wnt, and protein kinase B. Here we show that the absence of ILK function in Drosophila causes defects similar to loss of integrin adhesion, but not similar to loss of these signaling pathways. ILK mutations cause embryonic lethality and defects in muscle attachment, and clones of cells lacking ILK in the adult wing fail to adhere, forming wing blisters. Consistent with this, an ILK-green fluorescent protein fusion protein colocalizes with the position-specific integrins at sites of integrin function: muscle attachment sites and the basal junctions of the wing epithelium. Surprisingly, mutations in the kinase domain shown to inactivate the kinase activity of human ILK do not show any phenotype in Drosophila, suggesting a kinase-independent function for ILK. The muscle detachment in ILK mutants is associated with detachment of the actin filaments from the muscle ends, unlike integrin mutants, in which the primary defect is detachment of the plasma membrane from the extracellular matrix. Our data suggest that ILK is a component of the structure linking the cytoskeleton and the plasma membrane at sites of integrin-mediated adhesion.     

The LIM-only protein DRAL/FHL2 binds to the cytoplasmic domain of several alpha and beta integrin chains and is recruited to adhesion complexes

LIM proteins contain one or more double zinc finger structures (LIM domains) mediating specific contacts between proteins that participate in the formation of multiprotein complexes. We report that the LIM-only protein DRAL/FHL2, with four and a half LIM domains, can associate with alpha(3A), alpha(3B), alpha(7A), and several beta integrin subunits as shown in yeast two-hybrid assays as well as after overexpression in human cells. The amino acid sequence immediately following the conserved membrane-proximal region in the integrin alpha subunits or the C-terminal region with the conserved NXXY motif of the integrin beta subunits are critical for binding DRAL/FHL2. Furthermore, the DRAL/FHL2 associates with itself and with other molecules that bind to the cytoplasmic domain of integrin alpha subunits. Deletion analysis of DRAL/FHL2 revealed that particular LIM domains or LIM domain combinations bind the different proteins. These results, together with the fact that full-length DRAL/FHL2 is found in cell adhesion complexes, suggest that it is an adaptor/docking protein involved in integrin signaling pathways.     

Integrin cross-talk in endothelial cells is regulated by protein kinase A and protein phosphatase 1

In endothelial cells (ECs) beta1 integrin function-blocking antibodies inhibit alphavbeta3 integrin-mediated adhesion to a recombinant alpha4-laminin fragment (ralpha4LN fragment). beta1 integrin sequestration of talin is not the mechanism by which beta1 integrin modulates alphavbeta3 integrin ligand binding. Rather, treatment of the ECs with beta1 integrin function-blocking antibodies enhances cAMP-dependent protein kinase (PKA) activity and increases beta3 integrin serine phosphorylation. The PKA inhibitor H-89 abrogates the effect of beta1 integrin function-blocking antibodies on beta3 integrin serine phosphorylation and EC-ralpha4LN fragment binding. beta3 integrin contains a serine residue at position 752. To confirm the importance of this residue in alphavbeta3 integrin-ralpha4LN fragment binding, we mutated it to alanine (beta3S752A) or aspartic acid (beta3S752D). Chinese hamster ovary (CHO) cells expressing wild type or beta3S752A integrin attach robustly to ligand. CHO cells expressing beta3S752D integrin do not. Because the beta3 cytoplasmic tail lacks a PKA consensus site, it is unlikely that PKA acts directly on beta3 integrin. Instead, we have tested an hypothesis that PKA regulates beta3 integrin serine phosphorylation indirectly through phosphorylation of inhibitor-1, which, when phosphorylated, inhibits protein phosphatase 1 (PP1). Treatment of ECs with beta1 integrin function-blocking antibodies significantly increases phosphorylation of inhibitor-1. Furthermore, blocking PP1 activity pharmacologically inhibits alphavbeta3-mediated cell adhesion to the ralpha4LN fragment when both PKA and beta1 integrin function are inhibited. Concomitantly, there is an increase in serine phosphorylation of the beta3 integrin cytoplasmic tail. These results indicate a novel mechanism by which beta1 integrin negatively modulates alphavbeta3 integrin-ligand binding via activation of PKA and inhibition of PP1 activity.     

Osteopontin induces AP-1-mediated secretion of urokinase-type plasminogen activator through c-Src-dependent epidermal growth factor receptor transactivation in breast cancer cells

We have recently reported that osteopontin (OPN) stimulates cell motility and nuclear factor kappaB-mediated secretion of urokinase-type plasminogen activator (uPA) through phosphatidylinositol 3-kinase/Akt signaling pathways in breast cancer cells (Das, R., Mahabeleshwar, G. H., and Kundu, G. C. (2003) J. Biol. Chem. 278, 28593-28606). However, the role(s) of OPN on AP-1-mediated uPA secretion and cell motility and the involvement of c-Src/epidermal growth factor receptor (EGFR) in these processes in breast cancer cells are not well defined. In this study we report that OPN induces alpha(v)beta(3) integrin-mediated c-Src kinase activity in both highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. Ligation of OPN with alpha(v)beta(3) integrin induces kinase activity and tyrosine phosphorylation of EGFR in MDA-MB-231 and wild type EGFR-transfected MCF-7 cells, and this was inhibited by the dominant negative form of c-Src (dn c-Src) indicating that c-Src kinase plays a crucial role in this process. OPN induces association between alpha(v)beta(3) integrin and EGFR on the cell membrane in a macromolecular form with c-Src. Furthermore, OPN induces alpha(v)beta(3) integrin/EGFR-mediated ERK1/2 phosphorylation and AP-1 activation. Moreover, dn c-Src also suppressed the OPN-induced phosphatidylinositol (PI) 3-kinase activity in these cells indicating that c-Src acts as master switch in regulating MEK/ERK1/2 and phosphatidylinositol 3-kinase/Akt signaling pathways. OPN-induced ERK phosphorylation, AP-1 activation, uPA secretion, and cell motility were suppressed when cells were transfected with dn c-Src or pretreated with alpha(v)beta(3) integrin antibody, c-Src kinase inhibitor (pp2), EGFR tyrosine kinase inhibitor (PD153035), and MEK-1 inhibitor (PD98059). To our knowledge, this is the first report that OPN induces alpha(v)beta(3) integrin-mediated AP-1 activity and uPA secretion by activating c-Src/EGFR/ERK signaling pathways and further demonstrates a functional molecular link between OPN-induced integrin/c-Src-dependent EGFR phosphorylation and ERK/AP-1-mediated uPA secretion, and all of these ultimately control the motility of breast cancer cells.     

The role of protein tyrosine phosphorylation in integrin-mediated gene induction in monocytes

Integrin-mediated cell adhesion, or cross-linking of integrins using antibodies, often results in the enhanced tyrosine phosphorylation of certain intracellular proteins, suggesting that integrins may play a role in signal transduction processes. In fibroblasts, platelets, and carcinoma cells, a novel tyrosine kinase termed pp125FAK has been implicated in integrin-mediated tyrosine phosphorylation. In some cell types, integrin ligation or cell adhesion has also been shown to result in the increased expression of certain genes. Although it seems reasonable to hypothesize that integrin-mediated tyrosine phosphorylation and integrin-mediated gene induction are related, until now, there has been no direct evidence supporting this hypothesis. In the current report, we explore the relationship between integrin- mediated tyrosine phosphorylation and gene induction in human monocytes. We demonstrate that monocyte adherence to tissue culture dishes or to extracellular matrix proteins is followed by a rapid and profound increase in tyrosine phosphorylation, with the predominant phosphorylated component being a protein of 76 kD (pp76). Tyrosine phosphorylation of pp76 and other monocyte proteins can also be triggered by incubation of monocytes with antibodies to the integrin beta 1 subunit, or by F(ab')2 fragments of such antibodies, but not by F(ab) fragments. The ligation of beta 1 integrins with antibodies or F(ab')2 fragments also induces the expression of immediate-early (IE) genes such as IL-1 beta. When adhering monocytes are treated with the tyrosine kinase inhibitors genistein or herbimycin, both phosphorylation of pp76 and induction of IL-1 beta message are blocked in a dose-dependent fashion. Similarly, treatment with genistein or herbimycin can block tyrosine phosphorylation of pp76 and IL-1 beta message induction mediated by ligation of beta 1 integrin with antibodies. These observations suggest that protein tyrosine phosphorylation is an important aspect of integrin-mediated IE gene induction in monocytes. The cytoplasmic tyrosine kinase pp125FAK, although important in integrin signaling in other cell types, seems not to play a role in monocytes because this protein could not be detected in these cells.     

Integrin alpha 2 beta 1 promotes activation of protein phosphatase 2A and dephosphorylation of Akt and glycogen synthase kinase 3 beta

Serine/threonine kinase Akt is a downstream effector protein of phosphatidylinositol-3-kinase (PI-3K). Many integrins can function as positive modulators of the PI-3K/Akt pathway. Integrin alpha 2 beta 1 is a collagen receptor that has been shown to induce specific signals distinct from those activated by other integrins. Here, we found that, in contrast what was found for cells adherent to fibronectin, alpha 2 beta 1-mediated cell adhesion to collagen leads to dephosphorylation of Akt and glycogen synthase kinase 3 beta (GSK3 beta) and concomitantly to the induction of protein serine/threonine phosphatase 2A (PP2A) activity. PP2A activation can be inhibited by mutation in the alpha 2 cytoplasmic domain and by a function-blocking anti-alpha 2 antibody. Akt can be coprecipitated with PP2A, and coexpression of Akt with PP2Ac (catalytic subunit) inhibits Akt kinase activity. Integrin alpha 2 beta 1-related activation of PP2A is dependent on Cdc42. These results indicate that cell adhesion to collagen modulates Akt activity via the alpha 2 beta 1-induced activation of PP2A.     

Regulation of protein phosphatase 2A-mediated recruitment of IQGAP1 to beta1 integrin by EGF through activation of Ca2+/calmodulin-dependent protein kinase II

Maintenance of beta1 integrin-mediated cell adhesion in quiescent human mammary epithelial (HME) cells requires protein phosphatase (PP) 2A for not only dephosphorylation of beta1 integrin but also recruitment of IQGAP1 to Rac-bound beta1 integrin. However, how PP2A-dependent regulatory machinery of cell adhesion responds to EGF remains to be elucidated. We report here that phosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII) at threonine 286 was involved in the beta1 integrin complex that consisted of PP2A, Rac, and IQGAP1 in quiescent HME cells. Stimulation of the cells with EGF concomitantly induced an increase in intracellular Ca2+, activation of CaMKII, and dissociation of PP2A-IQGAP1-CaMKII from beta1 integrin-Rac. Because the activation of CaMKII and dissociation of PP2A-IQGAP1-CaMKII were blocked by either Ca2+-chelator or CaMKII inhibitor, we therefore propose that EGF has the ability to abrogate the PP2A function in the maintenance of beta1 integrin-mediated cell adhesion by dissociation of PP2A-IQGAP1-CaMKII from beta1 integrin-Rac through activation of CaMKII.     

Control of TCR-mediated activation of beta 1 integrins by the ZAP-70 tyrosine kinase interdomain B region and the linker for activation of T cells adapter protein

One of the earliest functional responses of T lymphocytes to extracellular signals that activate the Ag-specific CD3/TCR complex is a rapid, but reversible, increase in the functional activity of integrin adhesion receptors. Previous studies have implicated the tyrosine kinase zeta-associated protein of 70 kDa (ZAP-70) and the lipid kinase phosphatidylinositol 3-kinase, in the activation of beta(1) integrins by the CD3/TCR complex. In this report, we use human ZAP-70-deficient Jurkat T cells to demonstrate that the kinase activity of ZAP-70 is required for CD3/TCR-mediated increases in beta(1) integrin-mediated adhesion and activation of phosphatidylinositol 3-kinase. A tyrosine to phenylalanine substitution at position 315 in the interdomain B of ZAP-70 inhibits these responses, whereas a similar substitution at position 292 enhances these downstream signals. These mutations in the ZAP-70 interdomain B region also specifically affect CD3/TCR-mediated tyrosine phosphorylation of residues 171 and 191 in the cytoplasmic domain of the linker for activation of T cells (LAT) adapter protein. CD3/TCR signaling to beta(1) integrins is defective in LAT-deficient Jurkat T cells, and can be restored with expression of wild-type LAT. Mutant LAT constructs with tyrosine to phenylalanine substitutions at position 171 and/or position 191 do not restore CD3/TCR-mediated activation of beta(1) integrins in LAT-deficient T cells. Thus, these studies demonstrate that the interdomain B region of ZAP-70 regulates beta(1) integrin activation by the CD3/TCR via control of tyrosine phosphorylation of tyrosine residues 171 and 191 in the LAT cytoplasmic domain.     

Integrin-linked kinase stabilizes myotendinous junctions and protects muscle from stress-induced damage

Skeletal muscle expresses high levels of integrin-linked kinase (ILK), predominantly at myotendinous junctions (MTJs) and costameres. ILK binds the cytoplasmic domain of beta1 integrin and mediates phosphorylation of protein kinase B (PKB)/Akt, which in turn plays a central role during skeletal muscle regeneration. We show that mice with a skeletal muscle-restricted deletion of ILK develop a mild progressive muscular dystrophy mainly restricted to the MTJs with detachment of basement membranes and accumulation of extracellular matrix. Endurance exercise training enhances the defects at MTJs, leads to disturbed subsarcolemmal myofiber architecture, and abrogates phosphorylation of Ser473 as well as phosphorylation of Thr308 of PKB/Akt. The reduction in PKB/Akt activation is accompanied by an impaired insulin-like growth factor 1 receptor (IGF-1R) activation. Coimmunoprecipitation experiments reveal that the beta1 integrin subunit is associated with the IGF-1R in muscle cells. Our data identify the beta1 integrin-ILK complex as an important component of IGF-1R/insulin receptor substrate signaling to PKB/Akt during mechanical stress in skeletal muscle.     

CD98-mediated links between amino acid transport and beta 1 integrin distribution in polarized columnar epithelia

In non-polarized cells, CD98 has been shown to both influence beta(1) integrins and heterodimerize with LAT-2, which confers amino acid transport capability on the LAT-2/CD98 heterodimer. Since LAT-2 is most heavily expressed in intestine and CD98 associates with the beta(1) integrin splice form selectively found in such epithelia, we investigated the relationship and polarity of these proteins using the intestinal epithelial model Caco2-BBE. CD98 was found to selectively coimmunoprecipitate with both LAT-2 and beta(1) integrin, and, logically, all three proteins were polarized to the same (basolateral) domain. Furthermore, expression of CD98 in polarized epithelia lacking human CD98 (MDCK cells) disrupted beta(1) integrin surface distribution and cytoskeletal architecture, suggesting that CD98 can influence integrin function. Expression of a CD98 mutant lacking the specific residues conferring LAT-2 binding similarly affected cells, confirming that the latter effect was not due to LAT-2 sequestration. Use of CD98 truncation mutants suggest that a 10-amino acid domain located at the putative cytoplasmic tail/transmembrane domain interface was necessary and sufficient to induce the phenotype change. We conclude that the CD98/LAT-2 amino acid transporter is polarized to the same domain on which beta(1) integrin resides. CD98 appears to associate with beta(1) integrin and, in doing so, may influence its function as revealed by disruption of the outside-in signaling that confers cytoskeletal organization. Furthermore, such findings suggest a link between classic transport events and a critical element of barrier function: integrin-mediated influences on cytoskeletal organization.     

Integrin alpha3beta1 (CD 49c/29) is a cellular receptor for Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) entry into the target cells

Human herpesvirus-8 (HHV-8) is implicated in the pathogenesis of Kaposi's sarcoma. HHV-8 envelope glycoprotein B possesses the RGD motif known to interact with integrin molecules, and HHV-8 infectivity was inhibited by RGD peptides, antibodies against RGD-dependent alpha3 and beta1 integrins, and by soluble alpha3beta1 integrin. Expression of human alpha3 integrin increased the infectivity of virus for Chinese hamster ovary cells. Anti-gB antibodies immunoprecipitated the virus-alpha3 and -beta1 complexes, and virus binding studies suggest a role for alpha3beta1 in HHV-8 entry. Further, HHV-8 infection induced the integrin-mediated activation of focal adhesion kinase (FAK). These findings implicate a role for alpha3beta1 integrin and the associated signaling pathways in HHV-8 entry into the target cells.     

Butyrate-treated colonic Caco-2 cells exhibit defective integrin-mediated signaling together with increased apoptosis and differentiation

We previously reported that the enterocytic differentiation of human colonic Caco-2 cells correlated with alterations in integrin signaling. We now investigated whether differentiation and apoptosis of Caco-2 cells induced by the short-chain fatty acid butyrate (NaBT) was associated with alterations in the integrin-mediated signaling pathway with special interest in the expression and activity of focal adhesion kinase (FAK), of the downstream phosphatidylinositol 3'-kinase (PI 3-kinase)-Akt pathway and in the role of the nuclear factor kappaB (NF-kappaB). NaBT increased the level of sucrase. It induced apoptosis as shown by: (1) decreased Bcl-2 and Bcl-X(L) proteins and increased Bax protein; (2) activation of caspase-3; and (3) increased shedding of apoptotic cells in the medium. This effect was associated with defective integrin-mediated signaling as shown by: (1) down-regulation of beta1 integrin expression; 2) decreased FAK expression and tyrosine phosphorylation; (3) concerted alterations in cytoskeletal and structural focal adhesions proteins (talin, ezrin); and (4) decreased FAK ability to associate with PI 3-kinase. However, in Caco-2 cells, beta1-mediated signaling failed to be activated downstream of FAK and PI 3-kinase at the level of Akt. Transfection studies show that NaBT treatment of Caco-2 cells promoted a significant activation of the NF-kappaB which was probably involved in the NaBT-induced apoptosis. Our results indicate that the prodifferentiating agent NaBT induced apoptosis of Caco-2 cells probably through NF-kappaB activation together with a defective beta1 integrin-FAK-PI 3-kinase pathways signaling.     

The protein-tyrosine phosphatase TCPTP regulates epidermal growth factor receptor-mediated and phosphatidylinositol 3-kinase-dependent signaling.

In this study we have investigated the down-regulation of epidermal growth factor (EGF) receptor signaling by protein-tyrosine phosphatases (PTPs) in COS1 cells. The 45-kDa variant of the PTP TCPTP (TC45) exits the nucleus upon EGF receptor activation and recognizes the EGF receptor as a cellular substrate. We report that TC45 inhibits the EGF-dependent activation of the c-Jun N-terminal kinase, but does not alter the activation of extracellular signal-regulated kinase 2. These data demonstrate that TC45 can regulate selectively mitogen-activated protein kinase signaling pathways emanating from the EGF receptor. In EGF receptor-mediated signaling, the protein kinase PKB/Akt and the mitogen-activated protein kinase c-Jun N-terminal kinase, but not extracellular signal-regulated kinase 2, function downstream of phosphatidylinositol 3-kinase (PI 3-kinase). We have found that TC45 and the TC45-D182A mutant, which is capable of forming stable complexes with TC45 substrates, inhibit almost completely the EGF-dependent activation of PI 3-kinase and PKB/Akt. TC45 and TC45-D182A act upstream of PI 3-kinase, most likely by inhibiting the recruitment of the p85 regulatory subunit of PI 3-kinase by the EGF receptor. Recent studies have indicated that the EGF receptor can be activated in the absence of EGF following integrin ligation. We find that the integrin-mediated activation of PKB/Akt in COS1 cells is abrogated by the specific EGF receptor protein-tyrosine kinase inhibitor tyrphostin AG1478, and that TC45 and TC45-D182A can inhibit activation of PKB/Akt following the attachment of COS1 cells to fibronectin. Thus, TC45 may serve as a negative regulator of growth factor or integrin-induced, EGF receptor-mediated PI 3-kinase signaling.