aPKC phosphorylates NuMA-related LIN-5 to position the mitotic spindle during asymmetric division

Nat Cell Biol. 2011 Aug 21;13(9):1132-8. doi: 10.1038/ncb2315.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Caenorhabditis elegans / embryology
  • Caenorhabditis elegans / genetics
  • Caenorhabditis elegans / metabolism
  • Caenorhabditis elegans Proteins / genetics
  • Caenorhabditis elegans Proteins / metabolism*
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Cell Division*
  • Cell Polarity
  • Electrophoresis, Polyacrylamide Gel
  • Embryo, Nonmammalian / cytology
  • Embryo, Nonmammalian / embryology
  • Embryo, Nonmammalian / metabolism
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Immunoprecipitation
  • Mass Spectrometry / methods
  • Microscopy, Confocal
  • Molecular Sequence Data
  • Mutation
  • Nuclear Matrix-Associated Proteins / metabolism
  • Phosphorylation
  • Protein Binding
  • Protein Kinase C / genetics
  • Protein Kinase C / metabolism*
  • RNA Interference
  • Serine / metabolism
  • Spindle Apparatus / metabolism*

Substances

  • Caenorhabditis elegans Proteins
  • Cell Cycle Proteins
  • G protein regulator 1, C elegans
  • G protein regulator 2, C elegans
  • Nuclear Matrix-Associated Proteins
  • lin-5 protein, C elegans
  • Green Fluorescent Proteins
  • Serine
  • PKC-3 protein
  • Protein Kinase C