Abstract
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.
Highlights
Entiated cells at the tips of intestinal villi show decreased c-Src activity [1]
Our study demonstrates that the phosphorylation status of villin, which is determined by c-Src kinase activation, can be regulated by inactivation of c-Src catalytic activity by the tyrosine phosphatases, SHP-2 and PTPPEST
In Vitro Villin Can Be Tyrosine-phosphorylated by c-Src, c-Yes, and c-Fyn—We have previously reported that villin is tyrosine-phosphorylated both in vitro and in vivo [37, 41] and that phosphorylation of villin is required for its function in cell migration [30, 32]
Summary
Materials—SYF, SYF/c-Src, and Caco-2 cells (clone C2BBe1) were purchased from ATCC. HT29/19A clone was a kind gift from Dr A. SYF or SYF cells expressing full-length or mutant villin protein and/or the specific tyrosine kinase/phosphatase were extracted in a buffer containing 1% Triton X-100 and 150 mM NaCl. Tyrosine-phosphorylated villin, c-Src, c-Yes, c-Fyn, Jak, and Syk, were immunoprecipitated from the detergent-soluble extracts essentially as described before [31]. In Vitro Kinase/Phosphatase Assay—To determine if the transfected kinases were catalytically active, the kinases were immunoprecipitated from SYF/c-Src, SYF/c-Yes, SYF/c-Fyn, and SYF/Jak cells, respectively, and an in vitro kinase assay was performed as described previously in the absence or presence of the appropriate substrate, enolase (0.125 mg/ml) for the Src kinases and recombinant GST-␥c (2 g) for Jak3 [42, 43]. Recombinant villin was tyrosine-phosphorylated in vitro using recombinant c-Src kinase, recombinant Jak, or by immunoprecipitating the specific kinase from transfected/infected SYF cells, as described previously [37]. In vitro binding of tyrosine-phosphorylated villin with SH2 domain of proteins was checked using an SH2 domain array according to the manufacturer’s instructions
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