Identification and characterization of novel Par1b substrates that contribute to epithelial cell polarity

Abstract

Non-stratifying epithelia display either a columnar or hepatocytic polarity phenotype which differs in the positioning of their luminal domains, the distribution of luminal surfaces during cell divisions, and the mechanisms of luminal protein targeting in the biosynthetic pathway. Our group discovered that overexpression of the serine/threonine kinase Par1b in kidney-derived MDCK cells induced a switch from columnar to a hepatocytic polarity. Conversely, Par1binhibition in a hepatocytic cell line promoted multiple aspects of the columnar polarity. These observations suggest that Par1b might be a key determinant for the branching of the two epithelial phenotypes. To identify relevant Par1b substrates, we conducted an unbiased substrate screen based on an engineered Par1b kinase that transfers thio-phosphate from ATP-analogues to substrates and thus acts as an affinity tag to allow their specific isolation. Importantly, engineered Par1b phenocopied the polarity switch observed with Par1b overexpression in MDCK cells. We identified 9 known substrates and 59 novel substrate candidates and their putative Par1b-phosphorylation sites. Of 19 candidates tested, we validated 17 as bona fide substrates, attesting to the screen's accuracy. Our characterization of two new substrates, BORG5 and VIPAR/SPE-39, supports the hypothesis that multiple Par1b targets, functioning in distinct cellular pathways, contribute to the epithelial polarity switch. We found that phosphorylation of the Par1b site in the Cdc42-effector protein BORG5 antagonized its ability to stimulate cell spreading, a process we have previously linked to Par1b's role in lumen-and mitotic spindle position. On the other hand, a Par1b-phoshosphomimetic form of VIPAR increased the size of the hepatocytic-like lumina in Par1b-MDCK cells. VIPAR is a component of the vesicle tethering HOPS complex, suggesting the involvement of protein trafficking in Par1b's regulation of epithelial lumen. Thus, our roster of epithelial Par1b substrates promises to be a starting point for the elucidation of the many aspects of a complex signaling phenotype that yields the unique polarity of hepatocytes.