UBC Theses and Dissertations
Cloning and characterization of a mammalian mitotic spindle pole associated protein kinase SpaPK Cheng, Xiaoli
Passage through the cell cycle requires accurate replication of the genome and precise segregation of sister chromatids to daughter cells. Many of the proteins involved in cell cycle control are well conserved throughout evolution, from yeast to humans. The search for a human homolog of Saccharomyces cerevisiae Rad53, an important DNA damage and DNA replication checkpoint protein, led to the isolation of a gene encoding a novel protein kinase, designated SpaPK for spindle pole associated protein kinase. SpaPK, a protein of 454 amino acids with a predicted molecular mass of 52.5 kDa, is a serine/threonine protein kinase. The affinity purified recombinant SpaPK exhibits autophosphorylation activity, predominantly towards serine residues. Northern and Western blotting analyses indicated that SpaPK is expressed in various tissues and cell lines. It is also expressed at all stages of the cell cycle in proliferating cells, and is possibly phosphorylated at mitosis. During the course of this study, the SpaPK gene has been cloned independently by two other research groups and its gene product is designated ZIP kinase. The findings from their functional analyses related to apoptosis, which are different than those from this study, are discussed. In this study, characterization of the functions of SpaPK revealed that it is not a functional homolog of Rad53; instead, it plays a role in mitosis. Immunofluorescence studies showed that the subcellular localization of endogenous SpaPK is cell cycle regulated. SpaPK is first detected on duplicated centrosomes after their separation at prophase, remains on the centrosomes and centrosome-proximal part of the spindle until late anaphase, and redistributes to the spindle midzone and midbody as the cells go through cytokinesis. In addition, SpaPK is associated with microtubule organization centers in cells released from a nocodazole block and in taxol-treated mitotic cells. The subcellular localization of SpaPK is sensitive to a number of protein kinase inhibitors, indicating possible upstream regulators of SpaPK. The distinct spatial and temporal localization pattern of SpaPK suggest that this protein kinase may play a role in regulating spindle function during chromosome segregation in mammalian cells. SpaPK may also play a role at the G₂/M transition. Flow cytometry analyses showed that overexpression of wild-type or catalytically inactive recombinant SpaPK led to alteration of cell cycle progression with an accumulation of cells in G₂. Assays using Xenopus interphase extract showed that addition of purified wild-type or catalytically inactive recombinant SpaPK delays the onset of mitosis. SpaPK inhibits mitosis most likely by targeting Xenopus Cdc25C at Ser-287 whose phosphorylation is known to inhibit the activity of Cdc25C, thereby preventing the activation of Cdk1/cyclin B complex required for mitotic entry. Taken together, these data suggest that SpaPK functions as a regulator of mitotic spindle function during chromosome segregation and as a coordinator of mitotic entry at the G₂/M transition.
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