Inhibition of protein kinase C zeta blocks the attachment of stable microtubules to kinetochores leading to abnormal chromosome alignment

Abstract

The attachment of spindle microtubules to kinetochores is crucial for accurate segregation of chromosomes to daughter cells during mitosis. While a growing number of proteins involving this step are being identified, its molecular mechanisms are still not clear. Here we show that protein kinase C zeta (PKCzeta) is localized at the mitotic spindle during mitosis and plays a role in stable kinetochore-microtubule attachment. Striking staining for PKCzeta was observed at the mitotic spindle and spindle poles in cells at prometaphase and metaphase. PKCzeta molecules at these stages were phosphorylated at Thr-410, as detected by a phosphospecific antibody. PKCzeta was also detected at the spindle midzone and the midbody during anaphase and telophase, respectively, and PKCzeta at these stages was no longer phosphorylated at Thr-410. The polarity determinants Par3 and Par6, which are known to associate with PKCzeta, were also localized to the spindles and spindle poles at prometaphase and metaphase. Knockdown of PKCzeta by RNA interference affected normal chromosome alignment leading to generation of cells with aberrant nuclei. A specific PKCzeta inhibitor strongly blocked the formation of cold-sensitive stable kinetochore microtubules, and thus prevented microtubule-kinetochore attachment. Treatment of cells with the PKCzeta inhibitor also dislocated the minus-end directed motor protein dynein from kinetochores, but not the mitotic checkpoint proteins Mad2 and CENP-E. Prolonged exposure to the PKCzeta inhibitor eventually resulted in cell death. These results suggest a critical role of PKCzeta in spindle microtubule-kinetochore attachment and subsequent chromosomal separation.