Par3 Controls Epithelial Spindle Orientation by aPKC-Mediated Phosphorylation of Apical Pins

• Published in: Current biology 2010-10, Vol.20 (20), p.1809-1818
• Main Authors: Hao, Yi, Du, Quansheng, Chen, Xinyu, Zheng, Zhen, Balsbaugh, Jeremy L., Maitra, Sushmit, Shabanowitz, Jeffrey, Hunt, Donald F., Macara, Ian G.
• Format: Article
• Language: English
• Subjects: Adaptor Proteins, Signal Transducing; Animals; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Cell Line; Cell Polarity - physiology; Chromatography, Liquid; Dogs; Epithelial Cells - physiology; Humans; Immunoprecipitation; Intracellular Signaling Peptides and Proteins - metabolism; Membrane Proteins - genetics; Membrane Proteins - metabolism; Microscopy, Fluorescence; Microtubules - metabolism; Microtubules - physiology; Mutagenesis, Site-Directed; Phosphorylation; Protein Kinase C - metabolism; Spindle Apparatus - physiology; Tandem Mass Spectrometry
• ISSN: 0960-9822; 1879-0445
• DOI: 10.1016/j.cub.2010.09.032

Formation of epithelial sheets requires that cell division occurs in the plane of the sheet. During mitosis, spindle poles align so the astral microtubules contact the lateral cortex. Confinement of the mammalian Pins protein to the lateral cortex is essential for this process. Defects in signaling through Cdc42 and atypical protein kinase C (aPKC) also cause spindle misorientation. When epithelial cysts are grown in 3D cultures, misorientation creates multiple lumens. We now show that silencing of the polarity protein Par3 causes spindle misorientation in Madin-Darby canine kidney cell cysts. Silencing of Par3 also disrupts aPKC association with the apical cortex, but expression of an apically tethered aPKC rescues normal lumen formation. During mitosis, Pins is mislocalized to the apical surface in the absence of Par3 or by inhibition of aPKC. Active aPKC increases Pins phosphorylation on Ser401, which recruits 14-3-3 protein. 14-3-3 binding inhibits association of Pins with Gαi, through which Pins attaches to the cortex. A Pins S401A mutant mislocalizes over the cell cortex and causes spindle orientation and lumen defects. The Par3 and aPKC polarity proteins ensure correct spindle pole orientation during epithelial cell division by excluding Pins from the apical cortex. Apical aPKC phosphorylates Pins, which results in the recruitment of 14-3-3 and inhibition of binding to Gαi, so the Pins falls off the cortex. In the absence of a functional exclusion mechanism, astral microtubules can associate with Pins over the entire epithelial cortex, resulting in randomized spindle pole orientation. ► Par3 is required for spindle pole orientation in mitotic epithelial cells ► Par3 functions to recruit aPKC to the apical surface, which in turn excludes Pins ► aPKC mediates the phosphorylation of Pins on S401, which recruits 14-3-3 ► 14-3-3 binding reduces Pins association with Gαi at the cell cortex.