title: Novel NEDD1 phosphorylation sites regulate γ-tubulin binding and mitotic spindle assembly.
abstract: During cell division, microtubules organize a bipolar spindle to drive accurate chromosome segregation to daughter cells. Microtubules are nucleated by the γ-TuRC, a γ-tubulin complex that acts as a template for microtubules with 13 protofilaments. Cells lacking γ-TuRC core components do nucleate microtubules; however, these polymers fail to form bipolar spindles. NEDD1 is a γ-TuRC-interacting protein whose depletion, although not affecting γ-TuRC stability, causes spindle defects similar to the inhibition of its core subunits, including γ-tubulin. Several residues of NEDD1 are phosphorylated in mitosis. However, previously identified phosphorylation sites only partially regulate NEDD1 function, as NEDD1 depletion has a much stronger phenotype than mutation of these residues. Using mass spectrometry, we have identified multiple novel phosphorylated sites in the serine (S)557-S574 region of NEDD1, close to its γ-tubulin-binding domain. Serine to alanine mutations in S565-S574 inhibit the binding of NEDD1 to γ-tubulin and perturb NEDD1 mitotic function, yielding microtubule organization defects equivalent to those observed in NEDD1-depleted cells. Interestingly, additional mutations in the S557-T560 region restore the capacity of NEDD1 to bind γ-tubulin and promote bipolar spindle assembly. All together, our data suggest that the NEDD1/γ-tubulin interaction is finely tuned by multiple phosphorylation events in the S557-S574 region and is critical for spindle assembly. We also found that CEP192, a centrosomal protein similarly required for spindle formation, associates with NEDD1 and modulates its mitotic phosphorylation. Thus CEP192 may regulate spindle assembly by modulating NEDD1 function.
authors.affiliation: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5,  Canada.
authors.forename: Maria Ana
authors.initials: MA
authors.lastname: Gomez-Ferreria
authors.forename: Mikhail
authors.initials: M
authors.lastname: Bashkurov
authors.forename: Andreas O
authors.initials: AO
authors.lastname: Helbig
authors.forename: Brett
authors.initials: B
authors.lastname: Larsen
authors.forename: Tony
authors.initials: T
authors.lastname: Pawson
authors.forename: Anne-Claude
authors.initials: AC
authors.lastname: Gingras
authors.forename: Laurence
authors.initials: L
authors.lastname: Pelletier
mesh_terms: D020134:Catalytic Domain; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology; D010766:Phosphorylation; D011485:Protein Binding; D008941:Spindle Apparatus / Q000235:genetics / Q000378:metabolism*; D014404:Tubulin / Q000235:genetics / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; C526596:Cep192 protein, human; D002868:Chromosomal Proteins, Non-Histone; D008869:Microtubule-Associated Proteins; C509189:NEDD1 protein, human; D014404:Tubulin