Mitotic Regulation by NEK Kinase Networks

Abstract

Genetic studies in yeast and Drosophila led to identification of cyclin-dependent kinases (CDKs), Polo-like kinases (PLKs) and Aurora kinases as essential regulators of mitosis. These enzymes have since been found in the majority of eukaryotes and their cell cycle-related functions characterized in great detail. However, genetic studies in another fungal species, Aspergillus nidulans, identified a distinct family of protein kinases, the NEKs, that are also widely conserved and have key roles in the cell cycle, but which remain less well studied. Nevertheless, it is now clear that multiple NEK family members act in networks to regulate specific events of mitosis, including centrosome separation, spindle assembly and cytokinesis. Here, we describe our current understanding of how the NEK kinases contribute to these processes, particularly through targeted phosphorylation of proteins associated with the microtubule cytoskeleton. We also present the latest findings on molecular events that control the activation state of the NEKs and how these are revealing novel modes of enzymatic regulation relevant not only to other kinases but also to pathological mechanisms of disease.

Keywords: centrosome; cilia; microtubule; mitosis; protein kinase.

Figure 1

Kinase-mediated pathways regulating centrosome disjunction. (A) An immunofluorescence micrograph of a human U2OS osteosarcoma cell stained with acetylated-tubulin antibodies to detect centrioles (red), C-Nap1 antibodies to detect the centrosome linker (green) and Hoechst 33258 to detect DNA (blue). This illustrates how the centrosome appears as a paired structure that sits in the cytoplasm close to the nucleus in most interphase cells. (B) A schematic cartoon showing how the centrosome linker is thought to extend between the proximal ends of the two parental centrioles throughout interphase, both before (G1) and during (S/G2) the process of centriole duplication. The best-characterized linker proteins are C-Nap1, which associates with proximal ends of the centrioles, and rootletin, which forms connecting filaments between the centrioles. Additional proteins known to localize to the linker are indicated. (C) The centrosome linker undergoes disassembly at the onset of mitosis as a result of activation of Nek2 and phosphorylation of linker proteins. The activation of Nek2 is tightly controlled by a network of other kinases and phosphatases as indicated.

Figure 2

The Nek9-Nek6-Nek7 module. A schematic cartoon of the timeline of activation of the Nek9-Nek6-Nek7 signaling module and its known substrates shown with respect to the different stages of mitosis. During prophase Nek9 phosphorylates the γ-TuRC adapter NEDD1. This contributes to NEDD1 recruitment to the centrosomes and the maturation of these organelles needed for robust microtubule nucleation and spindle formation. Simultaneously, through activation of Nek6 and Nek7, Nek9 controls recruitment of the kinesin Eg5 to the centrosomes that leads to their separation. Nek6 and Nek7 are also involved in nuclear envelope breakdown (NEBD) through phosphorylation of the Nup98 nucleoporin, and subsequently in spindle organization through phosphorylation of Hsp72. Finally, Nek6 and Nek7 are involved in the control of cytokinesis via regulation of the kinesins, Mklp2 and Kif14. Note that some of the substrates attributed to Nek6 and Nek7 may be specific for one of these two kinases (e.g., Hsp72 and Mklp2 for Nek6, and Kif14 for Nek7). Meta, Ana, and Telo, metaphase, anaphase and telophase.

Figure 3

Proposed activation pathway of NEK7. The four amino acids that form the R-spine of Nek7 are shown as gray hexagons. On the left, inactive Nek7 is shown with a condensed R-spine in which the side chain of Tyr97 (Y) is located in the interior of the protein. This is a stable conformation that prevents formation of a productive kinase active site. In the center, dimeric Nek9 is shown interacting with two molecules of Nek7, bringing them together in a back-to-back conformation that destabilizes the inactive conformation, promoting Nek7 autophosphorylation. Nek9 can also activate Nek7 through direct phosphorylation of the activation loop. On the right, Nek7 is shown in a catalytically active state with phosphorylation on Ser195 and, we predict, alignment of the R-spine residues.