Khare Sharad, Holgren Cory, Samarel Allen M
Department of Gastroenterology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois, USA.
Am J Physiol Gastrointest Liver Physiol. 2006 Dec;291(6):G1100-12. doi: 10.1152/ajpgi.00008.2006. Epub 2006 Aug 17.
Environmental factors, including dietary fats, are implicated in colonic carcinogenesis. Dietary fats modulate secondary bile acids including deoxycholic acid (DCA) concentrations in the colon, which are thought to contribute to the nutritional-related component of colon cancer risk. Here we demonstrate, for the first time, that DCA differentially regulated the site-specific phosphorylation of focal adhesion kinase (FAK). DCA decreased adhesion of HCA-7 cells to the substratum and induced dephosphorylation of FAK at tyrosine-576/577 (Tyr-576/577) and Tyr-925. Tyrosine phosphorylation of FAK at Tyr-397 remained unaffected by DCA stimulation. Interestingly, we found that c-Src was constitutively associated with FAK and DCA actually activated Src, despite no change in FAK-397 and an inhibition of FAK-576 phosphorylation. DCA concomitantly and significantly increased association of tyrosine phosphatase ShP2 with FAK. Incubation of immunoprecipitated FAK, in vitro, with glutathione-S-transferase-ShP2 fusion protein resulted in tyrosine dephosphorylation of FAK in a concentration-dependent manner. Antisense oligodeoxynucleotides directed against ShP2 decreased ShP2 protein levels and attenuated DCA-induced FAK dephosphorylation. Inhibition of FAK by adenoviral-mediated overexpression of FAK-related nonkinase and gene silencing of Shp2 both abolished DCA's effect on cell adhesion, thus providing a possible mechanism for inside-out signaling by DCA in colon cancer cells. Our results suggest that DCA differentially regulates focal adhesion complexes and that tyrosine phosphatase ShP2 has a role in DCA signaling.
包括膳食脂肪在内的环境因素与结肠癌的发生有关。膳食脂肪可调节结肠中包括脱氧胆酸（DCA）在内的次级胆汁酸浓度，人们认为这有助于结肠癌风险中与营养相关的部分。在此，我们首次证明，DCA对粘着斑激酶（FAK）的位点特异性磷酸化具有差异性调节作用。DCA降低了HCA-7细胞与基质的粘附，并诱导FAK在酪氨酸576/577（Tyr-576/577）和酪氨酸925处去磷酸化。FAK在酪氨酸397处的酪氨酸磷酸化不受DCA刺激的影响。有趣的是，我们发现c-Src与FAK组成性结合，尽管FAK-397没有变化且FAK-576磷酸化受到抑制，但DCA实际上激活了Src。DCA同时且显著增加了酪氨酸磷酸酶ShP2与FAK的结合。将免疫沉淀的FAK与谷胱甘肽-S-转移酶-ShP2融合蛋白在体外孵育，导致FAK酪氨酸去磷酸化呈浓度依赖性。针对ShP2的反义寡脱氧核苷酸降低了ShP2蛋白水平，并减弱了DCA诱导的FAK去磷酸化。腺病毒介导的FAK相关非激酶过表达和Shp2基因沉默对FAK的抑制均消除了DCA对细胞粘附的影响，从而为DCA在结肠癌细胞中由内向外的信号传导提供了一种可能的机制。我们的结果表明，DCA对粘着斑复合物具有差异性调节作用，并且酪氨酸磷酸酶ShP2在DCA信号传导中起作用。
