Phosphorylation and dephosphorylation of Ser852 and Ser889 control the clustering, localization and function of PAR3

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ABSTRACT

Cell polarity is essential for various asymmetric cellular events, and the partitioning defective (PAR) protein PAR3 (encoded by PARD3 in mammals) plays a unique role as a cellular landmark to establish polarity. In epithelial cells, PAR3 localizes at the subapical border, such as the tight junction in vertebrates, and functions as an apical determinant. Although we know a great deal about the regulators of PAR3 localization, how PAR3 is concentrated and localized to a specific membrane domain remains an important question to be clarified. In this study, we demonstrate that ASPP2 (also known as TP53BP2), which controls PAR3 localization, links PAR3 and protein phosphatase 1 (PP1). The ASPP2–PP1 complex dephosphorylates a novel phosphorylation site, Ser852, of PAR3. Furthermore, Ser852- or Ser889-unphosphorylatable PAR3 mutants form protein clusters, and ectopically localize to the lateral membrane. Concomitance of clustering and ectopic localization suggests that PAR3 localization is a consequence of local clustering. We also demonstrate that unphosphorylatable forms of PAR3 exhibited a low molecular turnover and failed to coordinate rapid reconstruction of the tight junction, supporting that both the phosphorylated and dephosphorylated states are essential for the functional integrity of PAR3.

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Atypical protein kinase C cooperates with PAR-3 to establish embryonic polarity in Caenorhabditis elegans.

K Kemphues, Y. Izumi, B J Miwa … (1998)

Asymmetric cell divisions, critically important to specify cell types in the development of multicellular organisms, require polarized distribution of cytoplasmic components and the proper alignment of the mitotic apparatus. In Caenorhabditis elegans, the maternally expressed protein, PAR-3, is localized to one pole of asymmetrically dividing blastomeres and is required for these asymmetric divisions. In this paper, we report that an atypical protein kinase C (PKC-3) is essential for proper asymmetric cell divisions and co-localizes with PAR-3. Embryos depleted of PKC-3 by RNA interference die showing Par-like phenotypes including defects in early asymmetric divisions and mislocalized germline-specific granules (P granules). The defective phenotypes of PKC-3-depleted embryos are similar to those exhibited by mutants for par-3 and another par gene, par-6. Direct interaction of PKC-3 with PAR-3 is shown by in vitro binding analysis. This result is reinforced by the observation that PKC-3 and PAR-3 co-localize in vivo. Furthermore, PKC-3 and PAR-3 show mutual dependence on each other and on three of the other par genes for their localization. We conclude that PKC-3 plays an indispensable role in establishing embryonic polarity through interaction with PAR-3.

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Involvement of ASIP/PAR-3 in the promotion of epithelial tight junction formation.

Hidetake Kurihara, Tomonori Hirose, Keiko Mizuno … (2002)

The mammalian protein ASIP/PAR-3 interacts with atypical protein kinase C isotypes (aPKC) and shows overall sequence similarity to the invertebrate proteins C. elegans PAR-3 and Drosophila Bazooka, which are crucial for the establishment of polarity in various cells. The physical interaction between ASIP/PAR-3 and aPKC is also conserved in C. elegans PAR-3 and PKC-3 and in Drosophila Bazooka and DaPKC. In mammals, ASIP/PAR-3 colocalizes with aPKC and concentrates at the tight junctions of epithelial cells, but the biological meaning of ASIP/PAR-3 in tight junctions remains to be clarified. In the present study, we show that ASIP/PAR-3 staining distributes to the subapical domain of epithelial cell-cell junctions, including epithelial cells with less-developed tight junctions, in clear contrast with ZO-1, another tight-junction-associated protein, the staining of which is stronger in cells with well-developed tight junctions. Consistently, immunogold electron microscopy revealed that ASIP/PAR-3 concentrates at the apical edge of tight junctions, whereas ZO-1 distributes alongside tight junctions. To clarify the meaning of this characteristic localization of ASIP, we analyzed the effects of overexpressed ASIP/PAR-3 on tight junction formation in cultured epithelial MDCK cells. The induced overexpression of ASIP/PAR-3, but not its deletion mutant lacking the aPKC-binding sequence, promotes cell-cell contact-induced tight junction formation in MDCK cells when evaluated on the basis of transepithelial electrical resistance and occludin insolubilization. The significance of the aPKC-binding sequence in tight junction formation is also supported by the finding that the conserved PKC-phosphorylation site within this sequence, ASIP-Ser827, is phosphorylated at the most apical tip of cell-cell contacts during the initial phase of tight junction formation in MDCK cells. Together, our present data suggest that ASIP/PAR-3 regulates epithelial tight junction formation positively through interaction with aPKC.