Abstract
Phosphorylation of the polarity protein Par-3 by the serine/threonine kinases aPKCzeta/iota and Par-1 (EMK1/MARK2) regulates various aspects of epithelial cell polarity, but little is known about the mechanisms by which these posttranslational modifications are reversed. We find that the serine/threonine protein phosphatase PP1 (predominantly the alpha isoform) binds Par-3, which localizes to tight junctions in MDCKII cells. PP1alpha can associate with multiple sites on Par-3 while retaining its phosphatase activity. By using a quantitative mass spectrometry-based technique, multiple reaction monitoring, we show that PP1alpha specifically dephosphorylates Ser-144 and Ser-824 of mouse Par-3, as well as a peptide encompassing Ser-885. Consistent with these observations, PP1alpha regulates the binding of 14-3-3 proteins and the atypical protein kinase C (aPKC) zeta to Par-3. Furthermore, the induced expression of a catalytically inactive mutant of PP1alpha severely delays the formation of functional tight junctions in MDCKII cells. Collectively, these results show that Par-3 functions as a scaffold, coordinating both serine/threonine kinases and the PP1alpha phosphatase, thereby providing dynamic control of the phosphorylation events that regulate the Par-3/aPKC complex.