The mitotic checkpoint kinase NEK2A regulates kinetochore microtubule attachment stability

Abstract

Loss or gain of whole chromosome, the form of chromosome instability commonly associated with cancers is thought to arise from aberrant chromosome segregation during cell division. Chromosome segregation in mitosis is orchestrated by the interaction of kinetochores with spindle microtubules. Our studies show that NEK2A is a kinetochore-associated protein kinase essential for faithful chromosome segregation. However, it was unclear how NEK2A ensures accurate chromosome segregation in mitosis. Here we show that NEK2A-mediated Hec1 (highly expressed in cancer) phosphorylation is essential for faithful kinetochore microtubule attachments in mitosis. Using phospho-specific antibody, our studies show that NEK2A phosphorylates Hec1 at Ser165 during mitosis. Although such phosphorylation is not required for assembly of Hec1 to the kinetochore, expression of non-phosphorylatable mutant Hec1 S165 perturbed chromosome congression and resulted in a dramatic increase in microtubule attachment errors, including syntelic and monotelic attachments. Our in vitro reconstitution experiment demonstrated that Hec1 binds to microtubule in low affinity and phosphorylation by NEK2A, which prevents aberrant kinetochore-microtubule connections in vivo, increases the affinity of the Ndc80 complex for microtubules in vitro. Thus, our studies illustrate a novel regulatory mechanism in which NEK2A kinase operates a faithful chromosome attachment to spindle microtubule, which prevents chromosome instability during cell division.

Toxoplasma gondii Virulence Factor ROP18 Inhibits the Host NF-κB Pathway by Promoting p65 Degradation

“…In Vitro Phosphorylation Assay-The His-tagged full-length p65 and its truncates (p65-N1 and p65-C) were expressed in Escherichia coli strain BL21 (DE3) and purified by using Ni 2ϩ -Sepharose beads (Qiagen) as previously described (23,24). For in vitro phosphorylation assay, anti-GFP immunoprecipitates from 293T cells transiently expressing ROP18-GFP were incubated with purified recombinant full-length p65 and its truncates (200 g each) in kinase buffer (25 mM HEPES, pH 7.2, 1 mM DTT, 50 mM NaCl, 2 mM EGTA, 5 mM MgSO 4 ) with 50 M ATP and 0.5 Ci of [ 32 P]ATP.…”

Role of Nek2 on centrosome duplication and aneuploidy in breast cancer cells

“…We also found a higher Alternatively, Nek2 and pRb signaling could be interconnected through the mitotic kinetochore protein Hec1. Previous work has shown that Nek2 may participate in chromosome condensation through its interactions with Hec1 (30), and Nek2-dependent phosphorylaton of Hec1 is essential for kinetochore attachments to microtubules during mitosis (37). Hec1 is functionally linked to the pRb pathway because disruption of pRb activity leads to Hec1 overexpression, inducing defects in chromosome segregation and mitosis (38).…”

Phosphorylation of Microtubule-binding Protein Hec1 by Mitotic Kinase Aurora B Specifies Spindle Checkpoint Kinase Mps1 Signaling at the Kinetochore

“…Previous studies indicated the kinetochore localization of Mps1 depends on the key outer kinetochore protein Hec1 and Aurora B kinase (16 -18). In addition, Nek2A kinase phosphorylates Hec1 and strengthens Hec1 interaction with kinetochore microtubule (19). However, the underlying molecular mechanism remains to be elucidated.…”