Proteome analysis of human mesothelial cells during epithelial to mesenchymal transitions induced by shed menstrual effluent

Peritoneal endometriosis is the result of ectopic implantation and growth of endometrium tissue that has been regurgitated into the abdominal cavity during menstruation. We have previously shown that menstrual effluent induces epithelial to mesenchymal transitions (EMT) in mesothelial cells, which results in cell retraction and exposure of submesothelial extracellular matrix. Since endometrial tissue preferentially adheres to the extracellular matrix, adhesion of endometrial tissue to the peritoneum is facilitated. The EMT were shown to be associated with differential expression and phosphorylation of mesothelial proteins. Using radiolabeling and proteomics we detected changes in protein expression and phosphorylation that occur in mesothelial cells during the EMT process. The identity of 73 proteins, which were obtained from 324 analyzed spots, was confirmed. The expression of 35 proteins involved in organization of the cytoskeleton, signal transduction, regulation of the redox state, and production of ATP, was altered during the EMT process. Four of the identified proteins were differentially phosphorylated: annexin-1, an actin-binding protein and a substrate for receptor tyrosine kinases; tropomyosin-alpha, a regulator of actin filament stability and cell shape; elongation factor 1 delta; ATP synthase beta-chain. In conclusion, factors from menstrual effluent induce specific changes in the expression and phosphorylation status of structural, regulatory and metabolic proteins relevant to the complex process of EMT in mesothelial cells.     

Role of extracellular matrix-cell interaction and epidermal growth factor (EGF) on EGF-receptors and actin cytoskeleton arrangement in infantile pituitary cells

Epidermal growth factor (EGF) induces changes in cell morphology, actin cytoskeleton, and adhesion processes in cultured infantile pituitary cells. The extracellular matrix, through integrin engagement, collaborates with growth factors in cell signaling. We have examined the participation of collagen I/III and collagen plus fibronectin in the EGF response of infantile pituitary cells with respect to their cell morphology and actin cytoskeleton. As a comparison, we have used poly-lysine as a substrate. Infantile cells elicit the EGF response when they are associated with extracellular matrix proteins, but no response can be obtained with poly-lysine as the substrate. Cells acquire a flattened shape and organize their actin filaments and vinculin as in focal adhesions. Because the EGF receptor (EGFR) is linked to the actin cytoskeleton in other cells structuring a microdomain in cell signaling, we have investigated this association and substrate adhesion participation in infantile pituitary cells. The proportion of EGFR associated with the actin cytoskeleton is approximately 31%; no difference has been observed between the substrates used. Cells in suspension show actin-associated EGFR, suggesting an association independent of cell adhesion. However, no colocalization of EGFRs with actin fibers has been observed, suggesting an indirect association. Compared with β₁-integrin, which is linked to actin fibers through structural proteins, EGFR binds more strongly with the actin cytoskeleton. This study thus shows cell adhesion dependence on the EGF effect in the actin cytoskeleton arrangement; this is probably favored by the actin fiber/EGFR association that facilitates the cell signaling pathways for actin cytoskeleton organization in infantile pituitary cells.This work was supported by the National Council of Science and Technology of México (grant 44619, and a fellowship to C.T.).     

Epidermal growth factor receptor-dependent regulation of integrin-mediated signaling and cell cycle entry in epithelial cells

Integrin-mediated adhesion of epithelial cells to extracellular matrix (ECM) proteins induces prolonged tyrosine phosphorylation and partial activation of epidermal growth factor receptor (EGFR) in an integrin-dependent and EGFR ligand-independent manner. Integrin-mediated activation of EGFR in epithelial cells is required for multiple signal transduction events previously shown to be induced by cell adhesion to matrix proteins, including tyrosine phosphorylation of Shc, Cbl, and phospholipase Cgamma, and activation of the Ras/Erk and phosphatidylinositol 3'-kinase/Akt signaling pathways. In contrast, activation of focal adhesion kinase, Src, and protein kinase C, adhesion to matrix proteins, cell spreading, migration, and actin cytoskeletal rearrangements are induced independently of EGFR kinase activity. The ability of integrins to induce the activation of EGFR and its subsequent regulation of Erk and Akt activation permitted adhesion-dependent induction of cyclin D1 and p21, Rb phosphorylation, and activation of cdk4 in epithelial cells in the absence of exogenous growth factors. Adhesion of epithelial cells to the ECM failed to efficiently induce degradation of p27, to induce cdk2 activity, or to induce Myc and cyclin A synthesis; subsequently, cells did not progress into S phase. Treatment of ECM-adherent cells with EGF, or overexpression of EGFR or Myc, resulted in restoration of late-G(1) cell cycle events and progression into S phase. These results indicate that partial activation of EGFR by integrin receptors plays an important role in mediating events triggered by epithelial cell attachment to ECM; EGFR is necessary for activation of multiple integrin-induced signaling enzymes and sufficient for early events in G(1) cell cycle progression. Furthermore, these findings suggest that EGFR or Myc overexpression may provoke ligand-independent proliferation in matrix-attached cells in vivo and could contribute to carcinoma development.     

Abl kinases are required for invadopodia formation and chemokine-induced invasion

The Abl tyrosine kinases, Abl and Arg, play a role in the regulation of the actin cytoskeleton by modulating cell-cell adhesion and cell motility. Deregulation of both the actin cytoskeleton and Abl kinases have been implicated in cancers. Abl kinase activity is elevated in a number of metastatic cancers and these kinases are activated downstream of several oncogenic growth factor receptor signaling pathways. However, the role of Abl kinases in regulation of the actin cytoskeleton during tumor progression and invasion remains elusive. Here we identify the Abl kinases as essential regulators of invadopodia assembly and function. We show that Abl kinases are activated downstream of the chemokine receptor, CXCR4, and are required for cancer cell invasion and matrix degradation induced by SDF1α, serum growth factors, and activated Src kinase. Moreover, Abl kinases are readily detected at invadopodia assembly sites and their inhibition prevents the assembly of actin and cortactin into organized invadopodia structures. We show that active Abl kinases form complexes with membrane type-1 matrix metalloproteinase (MT1-MMP), a critical invadopodia component required for matrix degradation. Further, loss of Abl kinase signaling induces internalization of MT1-MMP from the cell surface, promotes its accumulation in the perinuclear compartment and inhibits MT1-MMP tyrosine phosphorylation. Our findings reveal that Abl kinase signaling plays a critical role in invadopodia formation and function, and have far-reaching implications for the treatment of metastatic carcinomas.     

Oxidative stress activates both Src-kinases and their negative regulator Csk and induces phosphorylation of two targeting proteins for Csk: caveolin-1 and paxillin

Csk negatively regulates Src family kinases (SFKs). In lymphocytes, Csk is constitutively active, and is transiently inactivated in response to extracellular stimuli, allowing activation of SFKs. In contrast, both SFKs and Csk were inactive in unstimulated mouse embryonic fibroblasts, and both were activated in response to oxidative stress. Csk modulated the oxidative stress-induced, but not the basal SFK activity in these cells. These data indicate that Csk may be more important for the return of Src-kinases to the basal state than for the maintenance of basal activity in some cell types. Csk must be targeted to its SFK substrates through an SH2-domain-mediated interaction with a phosphoprotein. Our data indicate that caveolin-1 is one of these targeting proteins. SFKs bind to caveolin-1 and phosphorylate it in response to oxidative stress and insulin. Csk binds specifically to the phosphorylated caveolin-1 and attenuates its stress-induced phosphorylation. Importantly, phosphocaveolin was one of two major phosphoproteins associated with Csk after incubation with peroxide or insulin. Paxillin was the other. Activation/rapid attenuation of SFKs by Csk is required for actin remodeling. Caveolin-1 is phosphorylated at the ends of actin fibers at points of contact between the actin cytoskeleton and the plasma membrane, where it could in part mediate this attenuation.     

Polymeric Immunoglobulin Receptor-mediated Invasion of Streptococcus pneumoniae into Host Cells Requires a Coordinate Signaling of SRC Family of Protein-tyrosine Kinases,ERK, and c-Jun N-terminal Kinase

Streptococcus pneumoniae are commensals of the human nasopharynx with the capacity to invade mucosal respiratory cells. PspC, a pneumococcal surface protein, interacts with the human polymeric immunoglobulin receptor (pIgR) to promote bacterial adherence to and invasion into epithelial cells. Internalization of pneumococci requires the coordinated action of actin cytoskeleton rearrangements and the retrograde machinery of pIgR. Here, we demonstrate the involvement of Src protein-tyrosine kinases (PTKs), focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) but not p38 mitogen-activated protein kinases (MAPK) in pneumococcal invasion via pIgR. Pharmacological inhibitors of PTKs and MAPKs and genetic interference with Src PTK and FAK functions caused a significant reduction of pIgR-mediated pneumococcal invasion but did not influence bacterial adhesion to host cells. Furthermore, pneumococcal ingestion by host cells induces activation of ERK1/2 and JNK. In agreement with activated JNK, its target molecule and DNA-binding protein c-Jun was phosphorylated. We also show that functionally active Src PTK is essential for activation of ERK1/2 upon pneumococcal infections. In conclusion, these data illustrate the importance of a coordinated signaling between Src PTKs, ERK1/2, and JNK during PspC-pIgR-mediated uptake of pneumococci by host epithelial cells.     

Adenovirus E4 open reading frame 4-induced apoptosis involves dysregulation of Src family kinases

The adenoviral early region 4 open reading frame 4 (E4orf4) death factor induces p53-independent apoptosis in many cell types and appears to kill selectively transformed cells. Here we show that expression of E4orf4 in transformed epithelial cells results in early caspase-independent membrane blebbing, associated with changes in the organization of focal adhesions and actin cytoskeleton. Evidence that E4orf4 can associate with and modulate Src family kinase activity, inhibiting Src-dependent phosphorylation of focal adhesion kinase (FAK) and paxillin while increasing phosphorylation of cortactin and some other cellular proteins, is presented. Furthermore, E4orf4 dramatically inhibited the ability of FAK and c-src to cooperate in induction of tyrosine phosphorylation of cellular substrates, suggesting that E4orf4 can interfere with the formation of a signaling complex at focal adhesion sites. Consistent with a functional role for E4orf4-Src interaction, overexpression of activated c-src dramatically potentiated E4orf4-induced membrane blebbing and apoptosis, whereas kinase dead c-src constructs inhibited E4orf4 effects on cell morphology and death. Moreover treatment of E4orf4-expressing cells with PP2, a selective Src kinase inhibitor, led to inhibition of E4orf4-dependent membrane blebbing and later to a marked decrease in E4orf4-induced nuclear condensation. Taken together, these observations indicate that expression of adenovirus 2 E4orf4 can initiate caspase-independent extranuclear manifestations of apoptosis through a modulation of Src family kinases and that these are involved in signaling E4orf4-dependent apoptosis. This study also suggests that Src family kinases are likely to play a role in the cytoplasmic execution of apoptotic programs.     

Src redox regulation: there is more than meets the eye

Src-family kinases are critically involved in the control of cytoskeleton organization and in the generation of integrin-dependent signaling responses, inducing tyrosine phosphorylation of many signaling and cytoskeletal proteins. Activity of the Src family of tyrosine kinases is tightly controlled by inhibitory phosphorylation of a carboxy-terminal tyrosine residue, inducing an inactive conformation through binding with its SH2 domain. Dephosphorylation of C-ter tyrosine, as well as its deletion of substitution with phenylalanine in oncogenic Src kinases, leads to autophosphorylation at a tyrosine in the activation loop, thereby leading to enhanced Src activity. Beside this phophorylation/dephosphorylation circuitry, cysteine oxidation has been recently reported as a further mechanism of enzyme activation. Mounting evidence describes Src activation via its redox regulation as a key outcome in several circumstances, including growth factor and cytokines signaling, integrin-mediated cell adhesion and motility, membrane receptor cross-talk as well in cell transformation and tumor progression. Among the plethora of data involving Src kinase in physiological and pathophysiological processes, this review will give emphasis to the redox component of the regulation of this master kinase.     

Role of Src-family kinases in formation of the cortical actin cap at the dorsal cell surface

Protein-tyrosine phosphorylation is regulated by protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs). Src-family tyrosine kinases (SFKs) participate in the regulation of the actin cytoskeleton. Actin filaments can be accumulated in a cap at the dorsal cell surface, which is called the cortical actin cap. Here, we show that SFKs play an important role in formation of the cortical actin cap. HeLa cells normally exhibit the cortical actin cap, one of the major sites of tyrosine phosphorylation. The cortical actin cap is disrupted by SFK inhibitors or overexpression of the Lyn SH3 domain. Csk-knockout cells form the cortical actin cap when the level of tyrosine phosphorylation is increased by Na₃VO₄, a PTP inhibitor, and the formation of the cortical actin cap is inhibited by SFK inactivation with re-introduction of Csk. SYF cells lacking SFKs minimally exhibit the cortical actin cap even in the presence of Na₃VO₄, and transfection with Lyn restores the cortical actin cap in the presence of Na₃VO₄. Disruption of the cortical actin cap by dominant-negative Cdc42 causes loss of tyrosine phosphorylation at the cell top. These results suggest that SFK(s) is involved in formation of the cortical actin cap, which may serve as a platform of tyrosine phosphorylation signaling.     

Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

Background

Proteinase-activated receptors (PARs; PAR_1–4) that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR₄, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR₄ stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT) which contributes to the increase in myofibroblast population.

Methods

EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA) for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells).

Results

Stimulation of PAR with thrombin (1 U/ml) or a synthetic PAR₄ agonist peptide (AYPGKF-NH₂, 100 μM) for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR₄ stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β). Western blot analyses of PAR₄-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR₄-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR) kinase and Src. PAR₄-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR₄ stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor.

Conclusion

PAR₄ stimulation of alveolar epithelial cells induced epithelial-mesenchymal transition (EMT) as monitored by cell shapes, and epithelial or myofibroblast marker at least partly through EGFR transactivation via receptor-linked Src activation.     

Potential molecular mechanism for c-Src kinase-mediated regulation of intestinal cell migration

The ubiquitously expressed Src tyrosine kinases (c-Src, c-Yes, and c-Fyn) regulate intestinal cell growth and differentiation. Src activity is also elevated in the majority of malignant and premalignant tumors of the colon. The development of fibroblasts with the three ubiquitously expressed kinases deleted (SYF cells) has identified the role of Src proteins in the regulation of actin dynamics associated with increased cell migration and invasion. Despite this, unexpectedly nothing is known about the role of the individual Src kinases on intestinal cell cytoskeleton and/or cell migration. We have previously reported that villin, an epithelial cell-specific actin-modifying protein that regulates actin reorganization, cell morphology, cell migration, cell invasion, and apoptosis, is tyrosine-phosphorylated. In this report using the SYF cells reconstituted individually with c-Src, c-Yes, c-Fyn, and wild type or phosphorylation site mutants of villin, we demonstrate for the first time the absolute requirement for c-Src in villin-induced regulation of cell migration. The other major finding of our study is that contrary to previous reports, the nonreceptor tyrosine kinase, Jak3 (Janus kinase 3), does not regulate phosphorylation of villin or villin-induced cell migration and is, in fact, not expressed in intestinal epithelial cells. Further, we identify SHP-2 and PTP-PEST (protein-tyrosine phosphatase proline-, glutamate-, serine-, and threonine-rich sequence) as negative regulators of c-Src kinase and demonstrate a new function for these phosphatases in intestinal cell migration. Together, these data suggest that in colorectal carcinogenesis, elevation of c-Src or down-regulation of SHP-2 and/or PTP-PEST may promote cancer metastases and invasion by regulating villin-induced cell migration and cell invasion.     

Reggies/flotillins regulate E-cadherin-mediated cell contact formation by affecting EGFR trafficking

The reggie/flotillin proteins are implicated in membrane trafficking and, together with the cellular prion protein (PrP), in the recruitment of E-cadherin to cell contact sites. Here, we demonstrate that reggies, as well as PrP down-regulation, in epithelial A431 cells cause overlapping processes and abnormal formation of adherens junctions (AJs). This defect in cell adhesion results from reggie effects on Src tyrosine kinases and epidermal growth factor receptor (EGFR): loss of reggies reduces Src activation and EGFR phosphorylation at residues targeted by Src and c-cbl and leads to increased surface exposure of EGFR by blocking its internalization. The prolonged EGFR signaling at the plasma membrane enhances cell motility and macropinocytosis, by which junction-associated E-cadherin is internalized and recycled back to AJs. Accordingly, blockage of EGFR signaling or macropinocytosis in reggie-deficient cells restores normal AJ formation. Thus, by promoting EGFR internalization, reggies restrict the EGFR signaling involved in E-cadherin macropinocytosis and recycling and regulate AJ formation and dynamics and thereby cell adhesion.     

Signaling through focal adhesion kinase

Focal adhesion kinase (FAK) is a nonreceptor protein-tyrosine kinase implicated in controlling cellular responses to the engagement of cell-surface integrins, including cell spreading and migration, survival and proliferation. Aberrant FAK signaling may contribute to the process of cell transformation by certain oncoproteins, including v-Src. Progress toward elucidating the events leading to FAK activation following integrin-mediated cell adhesion, as well as events downstream of FAK, has come through the identification of FAK phosphorylation sites and interacting proteins. A signaling partnership is formed between FAK and Src-family kinases, leading to tyrosine phosphorylation of FAK and associated ‘docking’ proteins Cas and paxillin. Subsequent recruitment of proteins containing Src homology 2 domains, including Grb2 and c-Crk, to the complex is likely to trigger adhesion-induced cellular responses, including changes to the actin cytoskeleton and activation of the Ras-MAP kinase pathway.     

Distinct roles for ROCK1 and ROCK2 in the regulation of cell detachment

This study, using mouse embryonic fibroblast (MEF) cells derived from ROCK1^−/− and ROCK2^−/− mice, is designed to dissect roles for ROCK1 and ROCK2 in regulating actin cytoskeleton reorganization induced by doxorubicin, a chemotherapeutic drug. ROCK1^−/− MEFs exhibited improved actin cytoskeleton stability characterized by attenuated periphery actomyosin ring formation and preserved central stress fibers, associated with decreased myosin light chain 2 (MLC2) phosphorylation but preserved cofilin phosphorylation. These effects resulted in a significant reduction in cell shrinkage, detachment, and predetachment apoptosis. In contrast, ROCK2^−/− MEFs showed increased periphery membrane folding and impaired cell adhesion, associated with reduced phosphorylation of both MLC2 and cofilin. Treatment with inhibitor of myosin (blebbistatin), inhibitor of actin polymerization (cytochalasin D), and ROCK pan-inhibitor (Y27632) confirmed the contributions of actomyosin contraction and stress fiber instability to stress-induced actin cytoskeleton reorganization. These results support a novel concept that ROCK1 is involved in destabilizing actin cytoskeleton through regulating MLC2 phosphorylation and peripheral actomyosin contraction, whereas ROCK2 is required for stabilizing actin cytoskeleton through regulating cofilin phosphorylation. Consequently, ROCK1 and ROCK2 can be functional different in regulating stress-induced stress fiber disassembly and cell detachment.     

Wiskott-Aldrich syndrome protein is associated with the adapter protein Grb2 and the epidermal growth factor receptor in living cells.

Src homology domains [i.e., Src homology domain 2 (SH2) and Src homology domain 3 (SH3)] play a critical role in linking receptor tyrosine kinases to downstream signaling networks. A well-defined function of the SH3-SH2-SH3 adapter Grb2 is to link receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), to the p21ras-signaling pathway. Grb2 has also been implicated to play a role in growth factor-regulated actin assembly and receptor endocytosis, although the underlying mechanisms remain unclear. In this study, we show that Grb2 interacts through its SH3 domains with the human Wiskott-Aldrich syndrome protein (WASp), which plays a role in regulation of the actin cytoskeleton. We find that WASp is expressed in a variety of cell types and is exclusively cytoplasmic. Although the N-terminal SH3 domain of Grb2 binds significantly stronger than the C-terminal SH3 domain to WASp, full-length Grb2 shows the strongest binding. Both phosphorylation of WASp and its interaction with Grb2, as well as with another adapter protein Nck, remain constitutive in serum-starved or epidermal growth factor-stimulated cells. WASp coimmunoprecipitates with the activated EGFR after epidermal growth factor stimulation. Purified glutathione S-transferase-full-length-Grb2 fusion protein, but not the individual domains of Grb2, enhances the association of WASp with the EGFR, suggesting that Grb2 mediates the association of WASp with EGFR. This study suggests that Grb2 translocates WASp from the cytoplasm to the plasma membrane and the Grb2-WASp complex may play a role in linking receptor tyrosine kinases to the actin cytoskeleton.     

Role of Ca2+ in diperoxovanadate-induced cytoskeletal remodeling and endothelial cell barrier function

Diperoxovanadate (DPV), a potent inhibitor of protein tyrosine phosphatases and activator of tyrosine kinases, alters endothelial barrier function via signaling pathways that are incompletely understood. One potential pathway is Src kinase-mediated tyrosine phosphorylation of proteins such as cortactin that regulate endothelial cell (EC) cytoskeleton assembly. As DPV modulates endothelial cell signaling via protein tyrosine phosphorylation, we determined the role of DPV-induced intracellular free calcium concentration ([Ca2+]i) in activation of Src kinase, cytoskeletal remodeling, and barrier function in bovine pulmonary artery endothelial cells (BPAECs). DPV in a dose- and time-dependent fashion increased [Ca2+]i, which was partially blocked by the calcium channel blockers nifedipine and Gd3+. Treatment of cells with thapsigargin released Ca2+ from the endoplasmic reticulum, and subsequent addition of DPV caused no further change in [Ca2+]i. These data suggest that DPV-induced [Ca2+]i includes Ca release from the endoplasmic reticulum and Ca influx through store-operated calcium entry. Furthermore, DPV induced an increase in protein tyrosine phosphorylation, phosphorylation of Src and cortactin, actin remodeling, and altered transendothelial electrical resistance in BPAECs. These DPV-mediated effects were significantly attenuated by BAPTA (25 microM), a chelator of [Ca2+]i. Immunofluorescence studies reveal that the DPV-mediated colocalization of cortactin with peripheral actin was also prevented by BAPTA. Chelation of extracellular Ca2+ by EGTA had marginal effects on DPV-induced phosphorylation of Src and cortactin; actin stress fibers formation, however, affected EC barrier function. These data suggest that DPV-induced changes in [Ca2+]i regulate endothelial barrier function using signaling pathways that involve Src and cytoskeleton remodeling.     

ATP-Mediated Transactivation of the Epidermal Growth Factor Receptor in Airway Epithelial Cells Involves DUOX1-Dependent Oxidation of Src and ADAM17

The respiratory epithelium is subject to continuous environmental stress and its responses to injury or infection are largely mediated by transactivation of the epidermal growth factor receptor (EGFR) and downstream signaling cascades. Based on previous studies indicating involvement of ATP-dependent activation of the NADPH oxidase homolog DUOX1 in epithelial wound responses, the present studies were performed to elucidate the mechanisms by which DUOX1-derived H₂O₂ participates in ATP-dependent redox signaling and EGFR transactivation. ATP-mediated EGFR transactivation in airway epithelial cells was found to involve purinergic P2Y₂ receptor stimulation, and both ligand-dependent mechanisms as well as ligand-independent EGFR activation by the non-receptor tyrosine kinase Src. Activation of Src was also essential for ATP-dependent activation of the sheddase ADAM17, which is responsible for liberation and activation of EGFR ligands. Activation of P2Y₂R results in recruitment of Src and DUOX1 into a signaling complex, and transient siRNA silencing or stable shRNA transfection established a critical role for DUOX1 in ATP-dependent activation of Src, ADAM17, EGFR, and downstream wound responses. Using thiol-specific biotin labeling strategies, we determined that ATP-dependent EGFR transactivation was associated with DUOX1-dependent oxidation of cysteine residues within Src as well as ADAM17. In aggregate, our findings demonstrate that DUOX1 plays a central role in overall epithelial defense responses to infection or injury, by mediating oxidative activation of Src and ADAM17 in response to ATP-dependent P2Y₂R activation as a proximal step in EGFR transactivation and downstream signaling.     

Phosphorylation of VASP by AMPK alters actin binding and occurs at a novel site

Vasodilator-stimulated phosphoprotein (VASP) is an actin regulatory protein that functions in adhesion and migration. In epithelial cells, VASP participates in cell–cell adhesion. At the molecular level, VASP drives actin bundling and polymerization. VASP activity is primarily regulated by phosphorylation. Three physiologically relevant phosphorylation sites significantly reduce actin regulatory activity and are targeted by several kinases, most notable Abl and protein kinases A and G (PKA and PKG). AMP-dependent kinase (AMPK) is best characterized as a cellular sensor of ATP depletion, but also alters actin dynamics in epithelial cells and participates in cell polarity pathways downstream of LKB1. While little is known about how AMPK direct changes in actin dynamics, AMPK has been shown to phosphorylate VASP at one of these three well-characterized PKA/PKG phosphorylation sites. Here we show that phosphorylation of VASP by AMPK occurs at a novel site, serine 322, and that phosphorylation at this site alters actin filament binding. We also show that inhibition of AMPK activity results in the accumulation of VASP at cell–cell adhesions and a concomitant increase in cell–cell adhesion.     

Chlamydia trachomatis tarp is phosphorylated by src family tyrosine kinases

The translocated actin recruiting phosphoprotein (Tarp) is injected into the cytosol shortly after Chlamydia trachomatis attachment to a target cell and subsequently phosphorylated by an unidentified tyrosine kinase. A role for Tarp phosphorylation in bacterial entry is unknown. In this study, recombinant C. trachomatis Tarp was employed to identify the host cell kinase(s) required for phosphorylation. Each tyrosine rich repeat of L2 Tarp harbors a sequence similar to a Src and Abl kinase consensus target. Furthermore, purified p60-src, Yes, Fyn, and Abl kinases were able to phosphorylate Tarp. Mutagenesis of potential tyrosines within a single tyrosine rich repeat peptide indicated that both Src and Abl kinases phosphorylate the same residues suggesting that C. trachomatis Tarp may serve as a substrate for multiple host cell kinases. Surprisingly, chemical inhibition of Src and Abl kinases prevented Tarp phosphorylation in culture and had no measurable effect on bacterial entry into host cells.     

Coordinate interactions of Csk, Src, and Syk kinases with [alpha]IIb[beta]3 initiate integrin signaling to the cytoskeleton

Integrins regulate cell adhesion and motility through tyrosine kinases, but initiation of this process is poorly understood. We find here that Src associates constitutively with integrin alphaIIbbeta3 in platelets. Platelet adhesion to fibrinogen caused a rapid increase in alphaIIbbeta3-associated Src activity, and active Src localized to filopodia and cell edges. Csk, which negatively regulates Src by phosphorylating Tyr-529, was also constitutively associated with alphaIIbbeta3. However, fibrinogen binding caused Csk to dissociate from alphaIIbbeta3, concomitant with dephosphorylation of Src Tyr-529 and phosphorylation of Src activation loop Tyr-418. In contrast to the behavior of Src and Csk, Syk was associated with alphaIIbbeta3 only after fibrinogen binding. Platelets multiply deficient in Src, Hck, Fgr, and Lyn, or normal platelets treated with Src kinase inhibitors failed to spread on fibrinogen. Inhibition of Src kinases blocked Syk activation and inhibited phosphorylation of Syk substrates (Vav1, Vav3, SLP-76) implicated in cytoskeletal regulation. Syk-deficient platelets exhibited Src activation upon adhesion to fibrinogen, but no spreading or phosphorylation of Vav1, Vav3, and SLP-76. These studies establish that platelet spreading on fibrinogen requires sequential activation of Src and Syk in proximity to alphaIIbbeta3, thus providing a paradigm for initiation of integrin signaling to the actin cytoskeleton.