title: Multisite Phosphorylation of NuMA-Related LIN-5 Controls Mitotic Spindle Positioning in C. elegans.
abstract: During cell division, the mitotic spindle segregates replicated chromosomes to opposite poles of the cell, while the position of the spindle determines the plane of cleavage. Spindle positioning and chromosome segregation depend on pulling forces on microtubules extending from the centrosomes to the cell cortex. Critical in pulling force generation is the cortical anchoring of cytoplasmic dynein by a conserved ternary complex of Gα, GPR-1/2, and LIN-5 proteins in C. elegans (Gα-LGN-NuMA in mammals). Previously, we showed that the polarity kinase PKC-3 phosphorylates LIN-5 to control spindle positioning in early C. elegans embryos. Here, we investigate whether additional LIN-5 phosphorylations regulate cortical pulling forces, making use of targeted alteration of in vivo phosphorylated residues by CRISPR/Cas9-mediated genetic engineering. Four distinct in vivo phosphorylated LIN-5 residues were found to have critical functions in spindle positioning. Two of these residues form part of a 30 amino acid binding site for GPR-1, which we identified by reverse two-hybrid screening. We provide evidence for a dual-kinase mechanism, involving GSK3 phosphorylation of S659 followed by phosphorylation of S662 by casein kinase 1. These LIN-5 phosphorylations promote LIN-5-GPR-1/2 interaction and contribute to cortical pulling forces. The other two critical residues, T168 and T181, form part of a cyclin-dependent kinase consensus site and are phosphorylated by CDK1-cyclin B in vitro. We applied a novel strategy to characterize early embryonic defects in lethal T168,T181 knockin substitution mutants, and provide evidence for sequential LIN-5 N-terminal phosphorylation and dephosphorylation in dynein recruitment. Our data support that phosphorylation of multiple LIN-5 domains by different kinases contributes to a mechanism for spatiotemporal control of spindle positioning and chromosome segregation.
authors.affiliation: Developmental Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
authors.forename: Vincent
authors.initials: V
authors.lastname: Portegijs
authors.affiliation: Developmental Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
authors.forename: Lars-Eric
authors.initials: LE
authors.lastname: Fielmich
authors.affiliation: Developmental Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
authors.forename: Matilde
authors.initials: M
authors.lastname: Galli
authors.affiliation: Developmental Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands.|Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
authors.forename: Ruben
authors.identifier: http://orcid.org/0000-0001-9187-5424
authors.initials: R
authors.lastname: Schmidt
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
authors.forename: Javier
authors.identifier: http://orcid.org/0000-0003-3288-3496
authors.initials: J
authors.lastname: Muñoz
authors.affiliation: Developmental Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
authors.forename: Tim
authors.initials: T
authors.lastname: van Mourik
authors.affiliation: Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
authors.forename: Anna
authors.initials: A
authors.lastname: Akhmanova
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
authors.affiliation: Developmental Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
authors.forename: Mike
authors.identifier: http://orcid.org/0000-0003-3966-4173
authors.initials: M
authors.lastname: Boxem
authors.affiliation: Developmental Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
authors.forename: Sander
authors.initials: S
authors.lastname: van den Heuvel
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D017173:Caenorhabditis elegans / Q000196:embryology / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics* / Q000378:metabolism; D047389:Casein Kinase I / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D016764:Cell Polarity / Q000235:genetics; D056445:Cytoplasmic Dyneins / Q000235:genetics* / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000196:embryology / Q000378:metabolism; D047108:Embryonic Development / Q000235:genetics; D034521:Nuclear Matrix-Associated Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D008941:Spindle Apparatus / Q000235:genetics
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; D047389:Casein Kinase I; C115015:PKC-3 protein; D011493:Protein Kinase C; D056445:Cytoplasmic Dyneins