Src tyrosine kinase phosphorylation of nuclear receptor HNF4α correlates with isoform-specific loss of HNF4α in human colon cancer

Src tyrosine kinase has long been implicated in colon cancer but much remains to be learned about its substrates. The nuclear receptor hepatocyte nuclear factor 4 alpha (HNF4 alpha) has just recently been implicated in colon cancer but its role is poorly defined. Here we show that c-Src phosphorylates human HNF4 alpha on three tyrosines in an interdependent and isoform-specific fashion. The initial phosphorylation site is a Tyr residue (Y14) present in the N-terminal A/B domain of P1-but not P2-driven HNF4 alpha. Phospho-Y14 interacts with the Src SH2 domain, leading to the phosphorylation of two additional tyrosines in the ligand binding domain (LBD) in P1-HNF4 alpha. Phosphomimetic mutants in the LBD decrease P1-HNF4 alpha protein stability, nuclear localization and transactivation function. Immunohistochemical analysis of approximately 450 human colon cancer specimens (Stage III) reveals that P1-HNF4 alpha is either lost or localized in the cytoplasm in approximately 80% of tumors, and that staining for active Src correlates with those events in a subset of samples. Finally, three SNPs in the human HNF4 alpha protein, two of which are in the HNF4 alpha F domain that interacts with the Src SH3 domain, increase phosphorylation by Src and decrease HNF4 alpha protein stability and function, suggesting that individuals with those variants may be more susceptible to Src-mediated effects. This newly identified interaction between Src kinase and HNF4 alpha has important implications for colon and other cancers.