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

Abstract

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 4α (HNF4α) has just recently been implicated in colon cancer but its role is poorly defined. Here we show that c-Src phosphorylates human HNF4α 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 HNF4α. Phospho-Y14 interacts with the Src SH2 domain, leading to the phosphorylation of two additional tyrosines in the ligand binding domain (LBD) in P1-HNF4α. Phosphomimetic mutants in the LBD decrease P1-HNF4α protein stability, nuclear localization and transactivation function. Immunohistochemical analysis of approximately 450 human colon cancer specimens (Stage III) reveals that P1-HNF4α 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 HNF4α protein, two of which are in the HNF4α F domain that interacts with the Src SH3 domain, increase phosphorylation by Src and decrease HNF4α 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 HNF4α has important implications for colon and other cancers.HNF4 isoforms | SH2 SH3 domain | SNP | Src kinase | tyrosine phosphorylation C olon cancer, the third most common malignancy in the United States, is a multifactorial disease that is influenced by both genetics and the environment (1, 2). c-Src is a nonreceptor tyrosine kinase that is strongly implicated in the development, growth, progression, and metastasis of several human cancers (3). In colon cancer, Src activation is associated with the early stages (4, 5) as well as progression and metastasis (6-8). Despite this long association with colon cancer, much remains to be learned about Src substrates (9).Hepatocyte nuclear factor 4alpha (HNF4α) (NR2A1) is a highly conserved member of the nuclear receptor superfamily with a recently identified endogenous ligand (linoleic acid) that binds in a reversible fashion (10, 11). HNF4α is best known for its role as a master regulator of liver-specific gene expression and as a key player in beta cells of the pancreas where it is mutated in an inherited form of type 2 diabetes (12)(13)(14). HNF4α is also expressed in kidney, stomach, and intestine; several recent papers also show an important role for HNF4α in the colon (15-20). There are two different promoters (P1 and P2) of HNF4A that are utilized in a temporal and tissue-specific fashion (11) (Fig. S1). While only P1-driven HNF4α (P1-HNF4α) is expressed in the adult liver, both P1-and P2-driven HNF4α (P2-HNF4α) are expressed in the adult intestine and colon (21, 22). Expression of P1-HNF4α is decreased in several human cancers including hepatocellular, gastric, renal, and...

Systematic Characterization and Prediction of Post-Translational Modification Cross-Talk *

“…A small portion of the cross-talk PTMs were located far from each other; for instance, the phosphorylation on Y14 of HNF4␣ was distant from its cross-talk partners (i.e. phosphorylations on Y286 and Y288) (47). However, 130 of 193 (67.4%) PTM cross-talks in our data were located within 20 amino acids.…”

Differential Effects of Hepatocyte Nuclear Factor 4α Isoforms on Tumor Growth and T-Cell Factor 4/AP-1 Interactions in Human Colorectal Cancer Cells

“…Immunohistochemical staining for HNF4␣ in liver, colon, and stomach cancers showed that there is a dysregulation of the HNF4␣ isoforms, with P2-HNF4␣ typically being expressed at higher levels than P1-HNF4␣ (18,34,35). Additionally, in a large cohort of 450 human colon cancer samples, we found a loss of nuclear P1-HNF4␣, which we attributed to Src tyrosine kinase preferentially phosphorylating P1-but not P2-driven HNF4␣ (17). The Cancer Genome Atlas (TCGA) recently identified the region encompassing HNF4A (20q13.12) as being one of several amplified loci in over 255 human colon cancers (36) and found an overexpression of the HNF4␣ protein in a subset of those samples (37).…”

“…Mutations in the HNF4A gene or HNF4␣ binding sites have been linked to various human diseases, including an inherited form of type 2 diabetes (maturity-onset diabetes of the young 1 [MODY1]) and hemophilia (6,16). Recently, HNF4␣ was shown to be involved in colon cancer, but its precise role remains elusive (11,12,17,18).…”

Activation of Src tyrosine kinase in esophageal carcinoma cells in different regulatory environments and corresponding occurrence mechanism

“…Due to historical reasons, research on the Src Family Kinase Src (SFKS) in carcinoma majorly focused on the fibroblasts of birds and mammals and human hematopoietic cells (Ma et al, 2012). Recently, many studies show that the abnormal overexpression of Src tyrosine kinase is associated with the occurrence of multiple epithelial-originated tumors, i.e., human colon carcinoma, breast carcinoma, and nonsmall cell lung carcinoma (Chellappa et al, 2012;Lv and Tian, 2012).…”