Novel NEDD1 phosphorylation sites regulate γ-tubulin binding and mitotic spindle assembly

J Cell Sci. 2012 Aug 15;125(Pt 16):3745-51. doi: 10.1242/jcs.105130. Epub 2012 May 17.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalytic Domain
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Chromosomal Proteins, Non-Histone / genetics
  • Chromosomal Proteins, Non-Histone / metabolism
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Microtubule-Associated Proteins / metabolism*
  • Mitosis / physiology
  • Phosphorylation
  • Protein Binding
  • Spindle Apparatus / genetics
  • Spindle Apparatus / metabolism*
  • Tubulin / genetics
  • Tubulin / metabolism*


  • Cell Cycle Proteins
  • Cep192 protein, human
  • Chromosomal Proteins, Non-Histone
  • Microtubule-Associated Proteins
  • NEDD1 protein, human
  • Tubulin