Scientific Abstract

Proposal No.  IBD-0026
Principal Investigator: Laurie L. Shekels, Ph.D.
Applicant Organization: University of Minnesota (Minneapolis, U.S.A.)
Project Title:  Does phosphorylation play a role in the function of the MUC3-related mucins?
Period of Award:  November 1, 2002 - February 29, 2004

It has been recognized for many years that there is a genetic predisposition to inflammatory bowel disease.  This has led to gene linkage studies that have identified susceptibility loci on numerous chromosomes.  Of particular interest to our lab is the demonstration via microsatellite marker studies that a predisposition to inflammatory bowel disease is carried on chromosome 7q22.  Several related genes are found within this region - the MUC3A, MUC3B, MUC12 and MUC17 mucin genes.  These genes encode for cell surface glycoproteins expressed along the intestinal tract.  Alterations in mucin content and properties have been found in inflammatory bowel disease, however, these reports focused solely on changes in the type and quantity of mucin present.

This proposal begins investigation on the functional properties of the MUC3 related mucins and any genetic alterations that may alter their biological role.  The MUC3-related mucins are membrane-bound and characterized by two EGF-like domains in their extracellular domain and numerous serines, threonines and tyrosines in the cytoplasmic domain.  These hydroxyl residues may serve as sites for phosphorylation.  Two other membrane bound mucins, MUC1 and MUC4, are capable of associating with other proteins and phosphorylation of the MUC1 cytoplasmic tail modulates its interactions.  Both protein kinase C and EGF receptor tyrosine kinase activities have been shown to be enhanced in tissue from ulcerative colitis tissue.  An additional tyrosine kinase whose activity has been associated with malignant transformation is the cellular oncogene c-src.  Elevated c-src activity is found in the premalignant colonic epithelia of individuals with ulcerative colitis.  From these reports it is apparent that phosphorylation plays a role in colitis and the progression to colon cancer.  The intriguing sequence data identifying EGF-like domains and possible phosphorylation sites in the MUC3-related mucins, combined with the reported interactions of the MUC1 and MUC4 mucins and the phosphorylation of MUC1, have prompted the question of whether or not the MUC3-related mucins associate with other proteins and are phosphorylated.

This proposal will identify interactions of the cytoplasmic domain using the yeast two hybrid system.  Phosphorylation will be examined with recombinant proteins corresponding to the cytoplasmic domain and cell lysates from intestinal cell lines.  The site of phosphorylation will be determined using anti-phosphoamino acid antibodies and mass spectrometry.  Protein interactions may be modulated either positively or negatively by phosphorylation.  Therefore, any interactions identified by the yeast two hybrid will be assessed for the effect of phosphorylation and protein interactions dependent on phosphorylation will also be examined by screening of an expression cDNA library.  DNA samples of patients with IBD and controls will be evaluated for gene polymorphisms of the carboxyl terminal exons and, more specifically, for differences in the sequence surrounding potential sites of phosphorylation.  This project examines MUC3-related mucin function in a novel setting suggesting that they may actively play a role in the inflammatory and reparative process through signaling and protein-protein interactions.  Insight into the functional activities of these mucin proteins and genetic alterations will provide additional knowledge about susceptibility, pathogenesis and therapy of IBD.