Alpha-1-syntrophin (SNTA1) and Rac1 are part of a signaling pathway via the dystrophin glycoprotein complex (DGC). Both SNTA1 and Rac1 proteins are over-expressed in various carcinomas. It is through the DGC signaling pathway that SNTA1 has been shown to act as a link between the extra cellular matrix, the internal cell signaling apparatus and the actin cytoskeleton. SNTA1 is involved in the modulation of the actin cytoskeleton and actin reorganization. Rac1 also controls actin cytoskeletal organization in the cell. In this study, we present the interplay between f-actin, SNTA1 and Rac1. We analyzed the effect of actin depolymerization on SNTA1 tyrosine phosphorylation and Rac1 activity using actin depolymerizing drugs, cytochalasin D and latrunculin A. Our results indicate a marked decrease in the tyrosine phosphorylation of SNTA1 upon actin depolymerization. Results suggest that actin depolymerization mediated loss of SNTA1 phosphorylation leads to loss of interaction between SNTA1 and Rac1, with a concomitant loss of Rac1 activation. The loss of SNTA1tyrosine phosphorylation and Rac1 activity by actin depolymerization results in increased apoptosis, decreased cell migration and decreased reactive oxygen species (ROS) levels in breast carcinoma cells. Collectively, our results present a possible role of f-actin in the SNTA1-Rac1 signaling pathway and implications of actin depolymerization on cell migration, ROS production and apoptosis.

Expression analysis of MKK6 protein in solid tumors has never been investigated. Here, we report systematic analysis of MKK6 protein in different types of human tumor samples using western blotting and immunofluorescence techniques. We observed significant increase in the expression of MKK6 in Esophageal, Stomach, and Colon cancers as compared to controls. Results were alternately confirmed by Immunofluorescence studies. Upregulation of MKK6 protein is indicative of its role in human cancers and could possibly be used as a novel diagnostic or prognostic marker in these cancers.

We have previously shown the involvement of p66shc in mediating apoptosis. Here, we demonstrate the novel mechanism of β-Amyloid-induced toxicity in the mammalian cells. β-Amyloid leads to the phosphorylation of p66shc at the serine 36 residue and activates MKK6, by mediating the phosphorylation at serine 207 residue. Treatment of cells with antioxidants blocks β-Amyloid-induced serine phosphorylation of MKK6, reactive oxygen species (ROS) generation, and hence protected cells against β-Amyloid-induced cell death. Our results indicate that serine phosphorylation of p66shc is carried out by active MKK6. MKK6 knock-down resulted in decreased serine 36 phosphorylation of p66shc. Co-immunoprecipitation results demonstrate a direct physical association between p66shc and WT MKK6, but not with its mutants. Increase in β-Amyloid-induced ROS production was observed in the presence of MKK6 and p66shc, when compared to triple mutant of MKK6 (inactive) and S36 mutant of p66shc. ROS scavengers and knock-down against p66shc, and MKK6 significantly decreased the endogenous level of active p66shc, ROS production, and cell death. Finally, we show that the MKK6-p66shc complex mediates β-Amyloid-evoked apoptotic cell death.

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