Cross Talk between Receptor Guanylyl Cyclase C and c-src Tyrosine Kinase Regulates Colon Cancer Cell Cytostasis

Increased activation of c-src seen in colorectal cancer is an indicator of a poor clinical prognosis, suggesting that identification of downstream effectors of c-src may lead to new avenues of therapy. Guanylyl cyclase C (GC-C) is a receptor for the gastrointestinal hormones guanylin and uroguanylin and the bacterial heat-stable enterotoxin. Though activation of GC-C by its ligands elevates intracellular cyclic GMP (cGMP) levels and inhibits cell proliferation, its persistent expression in colorectal carcinomas and occult metastases makes it a marker for malignancy. We show here that GC-C is a substrate for inhibitory phosphorylation by c-src, resulting in reduced ligand-mediated cGMP production. Consequently, active c-src in colonic cells can overcome GC-C-mediated control of the cell cycle. Furthermore, docking of the c-src SH2 domain to phosphorylated GC-C results in colocalization and further activation of c-src. We therefore propose a novel feed-forward mechanism of activation of c-src that is induced by cross talk between a receptor GC and a tyrosine kinase. Our findings have important implications in understanding the molecular mechanisms involved in the progression and treatment of colorectal cancer.Colorectal carcinoma is one of the most common forms of cancer seen in the developed world (10). The incidence in developing countries appears to be somewhat lower, but with high rates of mortality similar to those seen in developed countries (43). Treatment usually involves surgery or palliative chemotherapy, and recent research focuses on investigating molecular signatures associated with colorectal tumorigenesis as well as means of early detection in order to avoid high and rapid fatalities (61). Increased activity and expression of the tyrosine kinase c-src is frequently seen in colorectal cancer (5, 12), and activation of c-src in early stages of neoplastic transformation (32) is an indicator of a poor clinical prognosis (26). In general, c-src activity is as much as 16-fold higher in cancerous cells than in the adjacent colonic mucosa (12). Information on potential molecular mechanisms for the role of c-src in colonic tumorigenesis includes evidence that c-src activity increases during mitosis of human colon carcinoma cells (39), and increased c-src activity promotes cell motility and invasion during intestinal cell migration (34).A large number of substrates for c-src have been identified in different cell types, and a consensus site for tyrosine phosphorylation by c-src has been defined (50). Prediction of these sites in proteins present in colorectal carcinoma cells may assist in identifying novel targets for c-src action. The modular structure of c-src and other members of the src family kinases (SFKs), consisting of the SH4, unique, SH3, SH2, and kinase domains, allows these kinases to interact with a diverse group of proteins, creating a highly complex signal transduction network (48, 52). Consequently, SFKs have been demonstrated to be key downstream elements in signaling pathways emerging from cell surface receptors (52).Guanylyl cyclase C (GC-C) plays an important role in maintaining fluid ion homeostasis (54) and genomic integrity in intestinal cells (29) and serves as the receptor for the diarrheagenic heat-stable enterotoxin (ST) as well as endogenous ligands guanylin and uroguanylin (57). Binding of ST to the receptor leads to increased intracellular cyclic GMP (cGMP) accumulation and activation of the cystic fibrosis transmembrane conductance regulator, resulting in fluid and ion efflux that manifests as traveler''s diarrhea (45, 54). Recently it has been noted that regions of the world with the highest incidence of enterotoxigenic Escherichia coli-associated diarrhea exhibit the lowest incidence of colon cancer (43). In this context, studies have shown that activation of GC-C by its ligands inhibits human colonic cell proliferation and adenoma formation in mice, demonstrating a critical role for GC-C in inhibiting colon cancer initiation and progression (29, 33). For example, uroguanylin treatment suppresses polyp formation in the Apc(Min/+) mouse and induces apoptosis in human colon carcinoma cells via cGMP produced by GC-C (46). Interestingly, expression of the endogenous paracrine hormones for GC-C, guanylin and uroguanylin, is lost at the initiation of transformation, and therefore this reduced signaling via GC-C may allow cells to proliferate more rapidly, thereby leading to carcinogenesis (14).Expression of GC-C is largely restricted to the luminal side of intestinal cells in humans, and consequently, monitoring GC-C expression in circulating tumor cells in the peripheral blood of patients has been suggested to be a good marker for early detection of the primary tumor and/or metastasis of colon cancer (11, 28, 36). The molecular mechanisms by which GC-C activity is regulated during the initiation and progression of carcinogenesis remain undefined, and studies to investigate whether signaling events prevalent in colorectal cancer can modulate GC-C function have not been performed. Human colorectal carcinoma cell lines express GC-C and can therefore be used to study these molecular aspects of GC-C regulation and downstream signaling events (3, 4, 22).Phosphorylation is a rapid and reversible form of covalent modification frequently found in signaling systems. In our earlier studies, we have coexpressed domains of GC-C with the tyrosine kinase EphB1/Elk and observed tyrosine phosphorylation of GC-C (7). We report here that GC-C is a substrate for c-src tyrosine kinase and identify Tyr820 as the site for phosphorylation in GC-C. Following phosphorylation, pTyr820 serves as a site for interaction with the SH2 domain of c-src, resulting in further activation of c-src. Most importantly, tyrosine phosphorylation of GC-C inhibits cGMP production and prevents the cytostatic effects of GC-C induced by ligand interaction. Our studies therefore show the existence of a new signal transduction cross talk between c-src and GC-C in colonic cells, resulting in a feed-forward mechanism to further activate c-src, with important implications in cancer cell proliferation and disease progression.