title: aPKC phosphorylates NuMA-related LIN-5 to position the mitotic spindle during asymmetric division.
abstract: The position of the mitotic spindle controls the plane of cell cleavage and determines whether polarized cells divide symmetrically or asymmetrically. In animals, an evolutionarily conserved pathway of LIN-5 (homologues: Mud and NuMA), GPR-1/2 (homologues: Pins, LGN, AGS-3) and Gα mediates spindle positioning, and acts downstream of the conserved PAR-3-PAR-6-aPKC polarity complex. However, molecular interactions between polarity proteins and LIN-5-GPR-Gα remain to be identified. Here we describe a quantitative mass spectrometry approach for in vivo identification of protein kinase substrates. Applying this strategy to Caenorhabditis elegans embryos, we found that depletion of the polarity kinase PKC-3 results in markedly decreased levels of phosphorylation of a cluster of four LIN-5 serine residues. These residues are directly phosphorylated by PKC-3 in vitro. Phospho-LIN-5 co-localizes with PKC-3 at the anterior cell cortex and temporally coincides with a switch from anterior- to posterior-directed spindle movements in the one-cell embryo. LIN-5 mutations that prevent phosphorylation increase the extent of anterior-directed spindle movements, whereas phosphomimetic mutations decrease spindle migration. Our results indicate that anterior-located PKC-3 inhibits cortical microtubule pulling forces through direct phosphorylation of LIN-5. This molecular interaction between polarity and spindle-positioning proteins may be used broadly in cell cleavage plane determination.
authors.affiliation: Developmental Biology, Utrecht University, 3584 CH Utrecht, The Netherlands.
authors.forename: Matilde
authors.initials: M
authors.lastname: Galli
authors.forename: Javier
authors.initials: J
authors.lastname: Muñoz
authors.forename: Vincent
authors.initials: V
authors.lastname: Portegijs
authors.forename: Mike
authors.initials: M
authors.lastname: Boxem
authors.forename: Stephan W
authors.initials: SW
authors.lastname: Grill
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
authors.forename: Sander
authors.initials: S
authors.lastname: van den Heuvel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000196:embryology / Q000235:genetics / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division; D016764:Cell Polarity; D004591:Electrophoresis, Polyacrylamide Gel; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D047468:Immunoprecipitation; D013058:Mass Spectrometry / Q000379:methods; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation; D034521:Nuclear Matrix-Associated Proteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012694:Serine / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism*
chemical_list: D029742:Caenorhabditis elegans Proteins; D018797:Cell Cycle Proteins; C475458:G protein regulator 1, C elegans; C475459:G protein regulator 2, C elegans; D034521:Nuclear Matrix-Associated Proteins; C404808:lin-5 protein, C elegans; D049452:Green Fluorescent Proteins; D012694:Serine; C115015:PKC-3 protein; D011493:Protein Kinase C