Inhibition of protein kinase C ζ 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 ζ (PKCζ) is localized at the mitotic spindle during mitosis and plays a role in stable kinetochore-microtubule attachment. Striking staining for PKCζ was observed at the mitotic spindle and spindle poles in cells at prometaphase and metaphase. PKCζ molecules at these stages were phosphorylated at Thr-410, as detected by a phosphospecific antibody. PKCζ was also detected at the spindle midzone and the midbody during anaphase and telophase, respectively, and PKCζ at these stages was no longer phosphorylated at Thr-410. The polarity determinants Par3 and Par6, which are known to associate with PKCζ, were also localized to the spindles and spindle poles at prometaphase and metaphase. Knockdown of PKCζ by RNA interference affected normal chromosome alignment leading to generation of cells with aberrant nuclei. A specific PKCζ inhibitor strongly blocked the formation of cold-sensitive stable kinetochore microtubules, and thus prevented microtubule-kinetochore attachment. Treatment of cells with the PKCζ 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 PKCζ inhibitor eventually resulted in cell death. These results suggest a critical role of PKCζ in spindle microtubule-kinetochore attachment and subsequent chromosomal separation.

Introduction

Kinetochores are structures that form the interface between the chromosomes and the microtubules of the mitotic spindle [1], [2], [3]. Kinetochores function as the attachment site of the chromosome to the spindle microtubules and monitor the attachment. When abnormal attachment of chromosomes to the spindles is detected, the mitotic checkpoint is activated. Chromosome separation is achieved by the dynamic instability of spindle microtubules, which alternates between phases of growth and shrinkage. The highly dynamic nature of microtubule behavior is integrated with the kinetochore function to move and segregate chromosomes. Many proteins have been identified that localize and function in the kinetochores. These kinetochore-associated proteins include those in chromatid pairing such as Survivin and Aurora B [4], [5], those in kinetochore assembly, such as CENP-A, C, G, H [6], and those in regulating microtubule attachment and dynamics as well as checkpoint signaling such as Mad1, Mad2, Bub1, BubR1, dynein and CENP-E [1], [7], [8].

Cytoplasmic dynein is a large, multisubunit ATPase that moves along microtubules toward their minus-ends [9]. Dynein transports and localizes membranous organelles, and is a transient kinetochore component whose binding is regulated by microtubule attachment [10], [11], [12]. Dynein is recruited to kinetochores at prometaphase, but gradually loses the attachment from metaphase to anaphase [13]. Dynein and CENP-E are the only known kinetochore proteins with demonstrated motor activity. Dynein may contribute to kinetochore microtubule capture and chromosome movement [3].

Protein kinase C (PKC) is involved in intracellular signal transduction, cell proliferation, apoptosis, cell cycle and polarity determination [14], [15]. The PKC family consists of 11 different serine/threonine kinases that are divided into three subfamilies depending on their structure similarity and cofactor requirements. The conventional PKCs are diacylglycerol (DAG)-, phospholipids- and calcium-dependent, and include PKC α, βI, βII and γ. Novel PKCs are DAG- and phospholipid-dependent, but calcium-independent, and include PKCε, η, μ, θ and δ. The third group is the atypical PKC (aPKC) isoforms that are both DAG- and calcium-independent, and includes PKCζ and human PKCι/mouse PKCλ. Recently, an isoform with a new PKCζ catalytic domain, designated PKMζ, was described to be specific to the brain [16]. Another newly identified PKCζ member, PKCζII, which is a truncated form of PKCζ, is functionally involved in cell polarity through inhibition of tight junction formation [17].

Most studies on PKCζ functions are focused on the development of polarity in mammalian cells and lower eukaryotic cells [14], [15]. Recently, PKCζ has been reported to associate with meiotic spindles. Inhibition of PKCζ, but not other PKC isoforms results in rapid disruption of meiotic spindles in mouse eggs, indicating a role of PKCζ in regulating spindle organization and stability during mouse oocyte meiosis [18], [19]. PKCζ is also localized to the mitotic apparatus in primary cell cultures of shark rectal gland and CHO cells [20]. However, the biological functions of PKCζ in mitotic cells are still unknown.

We have previously identified PKCζ as a protein which binds to the guanine nucleotide exchange factor ECT2 [21]. ECT2 regulates cytokinesis in the steps of contractile ring formation and abscission [22], [23]. However, ECT2 is localized to the mitotic spindle during mitosis [23], [24]. In this report, we show that PKCζ is also localized to the mitotic spindle and the spindle poles in HeLa cells. Knockdown of PKCζ by RNA interference caused abnormal kinetochore attachment to the mitotic spindle in prometaphase. Treatment of cells with an PKCζ-specific inhibitor caused disruption of the mitotic spindle. We propose a new role of PKCζ in spindle attachment to kinetochores in mitosis.