Tyrosine phosphorylation of cytoskeletal proteins plays an important role in the regulation of focal adhesions and stress fiber organization. In the present study we examined the role of tyrosine ...phosphatases in this process using p125FAK and paxillin as substrates. We show that tyrosine phosphatase activity in Swiss 3T3 cells was markedly increased when actin stress fibers were disassembled by cell detachment from the substratum, by serum starvation, or by cytochalasin D treatment. This activity was blocked by phenylarsine oxide, an inhibitor of a specific class of tyrosine phosphatases characterized by two vicinal thiol groups in the active site. Phenylarsine oxide treatment of serum-starved cells induced increased tyrosine phosphorylation of p125FAK and paxillin in a dose-dependent manner and induced assembly of focal adhesions and actin stress fibers, showing that inhibition of one or more phenylarsine oxide-sensitive tyrosine phosphatases is a sufficient stimulus for triggering focal adhesion and actin stress fiber formation in adherent cells.
Integrin-mediated adhesion induces several signaling pathways leading to regulation of gene transcription, control of cell cycle entry and survival from apoptosis. Here we investigate the involvement ...of the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway in integrin-mediated signaling. Plating primary human endothelial cells from umbilical cord and the human endothelial cell line ECV304 on matrix proteins or on antibody to beta1- or alphav-integrin subunits induces transient tyrosine phosphorylation of JAK2 and STAT5A. Consistent with a role for the JAK/STAT pathway in regulation of gene transcription, adhesion to matrix proteins leads to the formation of STAT5A-containing complexes with the serum-inducible element of c-fos promoter. Stable expression of a dominant negative form of STAT5A in NIH3T3 cells reduces fibronectin-induced c-fos mRNA expression, indicating the involvement of STAT5A in integrin-mediated c-fos transcription. Thus these data present a new integrin-dependent signaling mechanism involving the JAK/STAT pathway in response to cell-matrix interaction.
Melusin is a muscle-specific chaperone protein whose expression is required for a compensatory hypertrophy response to pressure overload. Here, we evaluated the consequences of melusin overexpression ...in the setting of myocardial infarction (MI) using a comprehensive multicentre approach.
Mice overexpressing melusin in the heart (TG) and wild-type controls (WT) were subjected to permanent LAD ligation and both the acute response (Day 3) and subsequent remodelling (2 weeks) were examined. Mortality in wild-type mice was significant between Days 3 and 7, primarily due to cardiac rupture, but melusin's overexpression strongly reduced mortality (43.2% in wild-type vs. 27.3% in melusin-TG, P = 0.005). At Day 3 after MI, a time point preceding the mortality peak, TG hearts had increased heat shock protein 70 expression, increased ERK1/2 signalling, reduced cardiomyocyte hyper-contractility and inflammatory cell infiltrates, and increased matricellular protein expression in the infarcted area. At 2 weeks after MI, melusin overexpression conferred a favourable adaptive remodelling characterized by reduced left ventricle dilatation and better preserved contractility in the presence of a comparable degree of hypertrophy. Adaptive remodelling in melusin TG mice was characterized by reduced apoptosis and fibrosis as well as increased cardiomyocyte contractility.
Consistent with its function as a chaperone protein, melusin overexpression exerts a dual protective action following MI reducing an array of maladaptive processes. In the early phase after MI, reduced inflammation and myocyte remodelling protect against cardiac rupture. Chronically, reduced myocyte loss and matrix remodelling, with preserved myocyte contractility, confer adaptive LV remodelling.
Adhesion of human primary skin fibroblasts and ECV304 endothelial cells to immobilized matrix proteins, β1 or αv integrin antibodies stimulates tyrosine phosphorylation of the epidermal growth factor ...(EGF) receptor. This tyrosine phosphorylation is transiently induced, reaching maximal levels 30 min after adhesion, and it occurs in the absence of receptor ligands. Similar results were observed with EGF receptor‐transfected NIH‐3T3 cells. Use of a kinase‐negative EGF receptor mutant demonstrates that the integrin‐stimulated tyrosine phosphorylation is due to activation of the receptor's intrinsic kinase activity. Integrin‐mediated EGF receptor activation leads to Erk‐1/MAP kinase induction, as shown by treatment with the specific inhibitor tyrphostin AG1478 and by expression of a dominant‐negative EGF receptor mutant. EGF receptor and Erk‐1/MAP kinase activation by integrins does not lead per se to cell proliferation, but is important for entry into S phase in response to EGF or serum. EGF receptor activation is also required for extracellular matrix‐mediated cell survival. Adhesion‐dependent MAP kinase activation and survival are regulated through EGF receptor activation in cells expressing this molecule above a threshold level (5×103 receptors per cell). These results demonstrate that integrin‐dependent EGF receptor activation is a novel signaling mechanism involved in cell survival and proliferation in response to extracellular matrix.
We recently described morgana as an essential protein able to regulate centrosome duplication and genomic stability, by inhibiting ROCK. Here we show that morgana+/− mice spontaneously develop a ...lethal myeloproliferative disease resembling human atypical chronic myeloid leukemia (aCML), preceded by ROCK hyperactivation, centrosome amplification, and cytogenetic abnormalities in the bone marrow (BM). Moreover, we found that morgana is underexpressed in the BM of patients affected by atypical CML, a disorder of poorly understood molecular basis, characterized by nonrecurrent cytogenetic abnormalities. Morgana is also underexpressed in the BM of a portion of patients affected by Philadelphia-positive CML (Ph+ CML) caused by the BCR-ABL oncogene, and in this condition, morgana underexpression predicts a worse response to imatinib, the standard treatment for Ph+ CML. Thus, morgana acts as an oncosuppressor with different modalities: (1) Morgana underexpression induces centrosome amplification and cytogenetic abnormalities, and (2) in Ph+ CML, it synergizes with BCR-ABL signaling, reducing the efficacy of imatinib treatment. Importantly, ROCK inhibition in the BM of patients underexpressing morgana restored the efficacy of imatinib to induce apoptosis, suggesting that ROCK inhibitors, combined with imatinib treatment, can overcome suboptimal responses in patients in which morgana is underexpressed.
•Morgana haploinsufficiency in mice causes a lethal and transplantable CML-like myeloid neoplasm.•Morgana is underexpressed in aCML and in a subgroup of CMLs, where it predicts a worse response to imatinib but sensitivity to ROCK inhibitors.
We recently identified p140Cap as a novel adaptor protein, expressed in epithelial‐rich tissues and phosphorylated upon cell matrix adhesion and growth factor treatment. Here, we characterise p140Cap ...as a novel Src‐binding protein, which regulates Src activation via C‐terminal Src kinase (Csk). p140Cap silencing increases cell spreading, migration rate and Src kinase activity. Accordingly, increased expression of p140Cap activates Csk, leading to inhibition of Src and downstream signalling as well as of cell motility and invasion. Moreover, cell proliferation and ‘in vivo’ breast cancer cell growth are strongly impaired by high levels of p140Cap, providing the first evidence that p140Cap is a novel negative regulator of tumour growth.
The Dbl oncogene is a putative exchange factor for the small GTPases RhoA and Cdc42, which are involved in actin polymerization into stress fibers and filopodia, respectively. We report here that, ...upon adhesion to fibronectin, Dbl-transformed NIH3T3 cells display a contracted, polygonal shape with a high number of short stress fibers. In contrast, untransformed NIH3T3 cells acquire the characteristic fibroblast morphology and organize a regular mesh of long stress fibers. We show that in Dbl-transformed and in untransformed NIH3T3 cells the different shape and actin cytoskeleton organization observed in the early steps of adhesion involves activation of distinct GTPases. Upon adhesion to fibronectin, cell morphology of Dbl-transformed NIH3T3 cells depends on activation of RhoA and not of Cdc42. In contrast Cdc42 activation is necessary to untransfected NIH3T3 cells to acquire their fibroblast shape. In both Dbl-transformed and in untransformed NIH3T3 cells a basal Rac activation is necessary to support stress fiber organization, while constitutive Rac activation promotes ruffles and lamellipodia formation. As a consequence of RhoA activation, Dbl-transformed cells show high activity of ROCK-alpha and CRIK kinases, two known RhoA effectors. In addition Dbl-transformed and NIH3T3 cells expressing the constitutive active form of RhoA are less motile on fibronectin than cells expressing constitutive active Cdc42. We conclude that in NIH3T3 cells in response to fibronectin the expression of the Dbl oncogene leads to a predominant activation of RhoA which both supports the peculiar cell shape and actin cytoskeleton organization in stress fibers and regulates cell motility.
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