Phosphorylation-Dependent Binding of 14-3-3 to the Polarity Protein Par3 Regulates Cell Polarity in Mammalian Epithelia

Abstract

The mammalian homologs of the C. elegans partitioning-defective (Par) proteins have been demonstrated to be necessary for establishment of cell polarity. In mammalian epithelia, the Par3/Par6/aPKC polarity complex is localized to the tight junction and regulates its formation and positioning with respect to basolateral and apical membrane domains. Here we demonstrate a previously undescribed phosphorylation-dependent interaction between a mammalian homolog of the C. elegans polarity protein Par5, 14-3-3, and the tight junction-associated protein Par3. We identify phosphorylated serine 144 as a site of 14-3-3 binding. Expression of a Par3 mutant that contains serine 144 mutated to alanine (S144A) results in defects in epithelial cell polarity. In addition, overexpression of 14-3-3zeta results in a severe disruption of polarity, whereas overexpression of a 14-3-3 mutant that is defective in binding to phosphoproteins has no effect on cell polarity. Together, these data suggest a novel, phosphorylation-dependent mechanism that regulates the function of the Par3/Par6/aPKC polarity complex through 14-3-3 binding.

Targeted Proteomic Analysis of 14-3-3ς, a p53 Effector Commonly Silenced in Cancer

“…For example down-regulation of PAR3 was shown to prevent migration in Drosophila (37). Interestingly binding of ectopic 14-3-3 to PAR3 is known to disrupt polarity of mammalian epithelial cells (38). The inhibition of migration by 14-3-3 suggests that loss of 14-3-3 expression by epigenetic silencing or mutation of p53 in cancer cells may potentially lead to increased motility and thereby promote invasion.…”

Protein phosphatase 1 regulates the phosphorylation state of the polarity scaffold Par-3

“…By using tandem mass spectrometry we identified several known interaction partners of Par-3 ( Fig. S1B), including Par-6, aPKC (5), 14-3-3 (13,14), angiomotin (19), and DNA-PK (20). In addition, analysis of proteins migrating at a relative mass of 38,000 (M r 38,000) revealed peptides from the catalytic subunit of the serine-threonine protein phosphatase PP1.…”

“…Several residues of Par-3 have been shown to be phosphorylated by serine/threonine kinases, including the 14-3-3 binding sites S144/S885, which are substrates for Par-1 (EMK1/MARK2), and S824 of mouse Par-3, which is phosphorylated by aPKC (13,14,16). We therefore investigated whether the phosphorylation of these residues is reversed by PP1 by employing multiple reaction monitoring (MRM) (24), a mass spectrometric approach designed to quantitatively assay any selected peptides or modified peptides.…”

“…In mammalian epithelial cells Par-3 localizes to the apical junctional complex (10) and is required for the establishment of apical-basal polarity and the formation of tight junctions (TJs) (11,12). Many of the signaling events controlling cell polarity and TJ formation are regulated by the activities of serine/threonine protein kinases, notably aPKC and Par-1 (EMK1/MARK2) (4)(5)(6)(13)(14)(15)(16)(17)(18). For example, aPKC maintains the apical membrane domain by phosphorylating the basolateral determinants Lgl and Par-1, thereby releasing them from the cell cortex.…”

Regulation of epithelial cell polarity by PAR-3 depends on Girdin transcription and Girdin–Gαi3 signaling

“…In Drosophila melanogaster, cooperation of PAR-3 (which is known as Bazooka) with aPKC is indispensable for the establishment of apicobasal membrane identities (Bilder et al, 2003;Wodarz et al, 2000Wodarz et al, , 1999. Moreover, genetic manipulation in mammals, including the targeted disruption of PAR-3 in mice, has also revealed the important in vivo role of PAR-3 in tight junction formation, apical membrane development and lumen formation during epithelial morphogenesis (Chen and Macara, 2005;Hirose et al, 2006;Horikoshi et al, 2009;Hurd et al, 2003). PAR-3 interacts with the aPKC-PAR-6 complex to form the tripartite PAR-3-aPKC-PAR-6 complex (Izumi et al, 1998;Joberty et al, 2000;Lin et al, 2000;Suzuki et al, 2001), which is required for aPKC recruitment to cell-cell contacts and for apical domain development (Horikoshi et al, 2009).…”