Protein kinase D1 inhibition interferes with mitosis progression

Experimental Cell Research, 2008

The protein kinase D (PKD) family consists of three serine/threonine protein kinases involved in the regulation of fundamental biological processes in response to their activation and intracellular redistribution. Although a substantial amount of information is available describing the mechanisms regulating the activation and intracellular distribution of the PKD isozymes during interphase, nothing is known of their activation status, localization and role during mitosis. The results presented in this study indicate that during mitosis, PKD3 and PKD are phosphorylated at Ser 731 and Ser 744 within their activation loop by a mechanism that requires protein kinase C. Mitosis-associated PKD3 Ser 731 and PKD Ser 744 phosphorylation is related to the catalytic activation of these kinases as evidenced by in vivo phosphorylation of histone deacetylase 5, a substrate of PKD and PKD3. Activation loop-phosphorylated PKD3 and PKD, as well as PKD2, associate with centrosomes, spindles and midbody suggesting that these activated kinases establish dynamic interactions with the mitotic apparatus. Thus, this study reveals a connection between the PKD isozymes and cell division, suggesting a novel role for this family of serine/threonine kinases.

Journal of Biological Chemistry, 2004

This review article focuses on protein kinases regulating the onset and transition through mitosis. The essay begins by introducing the structural features of the protein kinase catalytic domain and emphasizing the mechanism of enzymatic activation of this class of proteins. Next follows a short historical perspective on cell division and a description of our current understanding of mitosis. In the central part of the review I examine the four major kinases that set the stage for mitosis, which consist of Cdk1, Polo-like 1, Nek2 and Aurora kinases. For each entry dealt with, I focus particularly on studies that have linked DNA damage response pathways to inhibition of kinase activity, and I evaluate the conclusions drawn. Finally, I examine protein kinases initially described in the context of different cell cycle transitions and only later proposed to be involved in the control of mitosis.

Frontiers in Cell and Developmental Biology

Molecular Cell, 2005

Centrosome separation is critical for bipolar spindle formation and the accurate segregation of chromosomes during mammalian cell mitosis. Kinesin-5 (Eg5) is a microtubule motor essential for centrosome separation, and Tiam1 and its substrate Rac antagonize Eg5-dependent centrosome separation in early mitosis promoting efficient chromosome congression. Here we identify S1466 of Tiam1 as a novel Cdk1 site whose phosphorylation is required for the mitotic function of Tiam1. We find that this phosphorylation of Tiam1 is required for the activation of group I p21-activated kinases (Paks) on centrosomes in prophase. Further, we show that both Pak1 and Pak2 counteract centrosome separation in a kinase-dependent manner and demonstrate that they act downstream of Tiam1. We also show that depletion of Pak1/2 allows cells to escape monopolar arrest by Eg5 inhibition, highlighting the potential importance of this signalling pathway for the development of Eg5 inhibitors as cancer therapeutics.

Molecular cancer research : MCR, 2017

To form a proper mitotic spindle, centrosomes must be duplicated and driven poleward in a timely and controlled fashion. Improper timing of centrosome separation and errors in mitotic spindle assembly may lead to chromosome instability, a hallmark of cancer. Protein Kinase C epsilon (PKCε) has recently emerged as a regulator of several cell cycle processes associated with the resolution of mitotic catenation during the metaphase-anaphase transition and in regulating the abscission checkpoint. However, an engagement of PKCε in earlier (pre)mitotic events has not been addressed. Here, we now establish that PKCε controls prophase-to-metaphase progression by coordinating centrosome migration and mitotic spindle assembly in transformed cells. This control is exerted through cytoplasmic dynein function. Importantly, it is also demonstrated that the PKCε dependency of mitotic spindle organization is correlated with the non-functionality of the TOPO2A-dependent G2 checkpoint, a characterist...

Nature Cell Biology, 2008

Separation of duplicated centrosomes (spindle-pole bodies or SPBs in yeast) is a crucial step in the biogenesis of the mitotic spindle. In vertebrates, centrosome separation requires the BimC family kinesin Eg5 and the activities of Cdk1 and polo kinase; however, the roles of these kinases are not fully understood. In Saccharomyces cerevisiae, SPB separation also requires activated Cdk1 and the plus-end kinesins Cin8 (homologous to vertebrate Eg5) and Kip1. Here we report that polo kinase has a role in the separation of SPBs. We show that adequate accumulation of Cin8 and Kip1 requires inactivation of the anaphase-promoting complex-activator Cdh1 through sequential phosphorylation by Cdk1 and polo kinase. In this process, Cdk1 functions as a priming kinase in that Cdk1-mediated phosphorylation creates a binding site for polo kinase, which further phosphorylates Cdh1. Thus, Cdh1 inactivation through the synergistic action of Cdk1 and polo kinase provides a new model for inactivation of cell-cycle effectors. Centrosomes function as microtubule organizing centres (MTOC) and are responsible for the bipolar nature of the mitotic spindle 1. Misregulation of the centrosome cycle can produce an abnormal number of centrosomes and multi-polar spindles, and cause chromosome missegregation resulting in aneuploidy. Centrosomal abnormalities and aneuploidy are common in many types of cancers 2. In mammalian cells, centrosomes consist of a pair of centrioles and the surrounding dense fibrillar mass known as the pericentriolar material 3. A cell inherits one centrosome from its progenitor but replicates it as it progresses through the cell cycle. The duplicated centrosomes remain tethered to each other by a linker until the time of mitotic entry, when the linkage between the sister centrosomes is severed and they move away from each other, eventually positioning themselves face-to-face, separated by an interdigitated array of microtubules. The structure and duplication of centrosomes have been studied in some detail; however, the process by which they separate is not well understood.

Current Biology, 2004

Introduction 1 Research Institute of Molecular Pathology Dr. Bohr-Gasse 7 Sister chromatid separation and the subsequent forma-1030 Vienna tion of two genetically identical daughter cells depend Austria on the symmetrical attachment of all chromosomes to 2 Centro de Investigaciones Bioló gicas the mitotic spindle, a process called chromosome bior-Consejo Superior de Investigaciones Científicas ientation. The assembly of bipolar spindles is preceded Ramiro de Maeztu, 9 by the movement of centrosomes toward opposite poles 28040 Madrid of the cell, where these structures function as nucleation Spain sites for microtubule polymerization. In prometaphase, 3 European Molecular Biology Laboratory microtubules emanating from the spindle poles establish Meyerhofstrasse 1 contacts with kinetochores on chromosomes through a 69117 Heidelberg stochastic "search and capture" mechanism [1]. The Germany binding of microtubules to kinetochores is reversible as long as physical tension at the kinetochore is low. Once both sister kinetochores of one chromosome have been Summary captured by microtubules from opposite spindle poles (amphitelic attachments), spindle pulling forces gener-Background: The stable association of chromosomes ate tension across sister kinetochores, and this tension with both poles of the mitotic spindle (biorientation) deis thought to stabilize the microtubule-kinetochore interpends on spindle pulling forces. These forces create actions [2, 3]. The stabilization of microtubule-kinetotension across sister kinetochores and are thought to chore interactions allows the kinetochore to become stabilize microtubule-kinetochore interactions and to sieventually fully occupied with microtubules in the case lence the spindle checkpoint. Polo-like kinase 1 (Plk1) of mammalian cells with a total of 15-30 microtubules has been implicated in regulating centrosome maturaper kinetochore (reviewed in [4]). tion, mitotic entry, sister chromatid cohesion, the ana-Sister chromatid separation in anaphase is initiated phase-promoting complex/cyclosome (APC/C), and cyonly once all chromosomes have been attached to both tokinesis, but it is unknown if Plk1 controls chromosome poles of the spindle with a full complement of microtubiorientation. bules [5, 6]. Before this metaphase stage has been Results: We have analyzed Plk1 functions in synchroreached,

Cell Cycle, 2008