Essential Function of the Polo Box of Cdc5 in Subcellular Localization and Induction of Cytokinetic Structures

Abstract

Members of the polo subfamily of protein kinases play pivotal roles in cell proliferation. In addition to the kinase domain, polo kinases have a strikingly conserved sequence in the noncatalytic C-terminal domain, termed the polo box. Here we show that the budding-yeast polo kinase Cdc5, when fused to green fluorescent protein and expressed under its endogenous promoter, localizes at spindle poles and the mother bud neck. Overexpression of Cdc5 can induce a class of cells with abnormally elongated buds in a polo box- and kinase activity-dependent manner. In addition to localizing at the spindle poles and cytokinetic neck filaments, Cdc5 induces and localizes to additional septin ring structures within the elongated buds. Without impairing kinase activity, conservative mutations in the polo box abolish the ability of Cdc5 to functionally complement the defect associated with a cdc5-1 temperature-sensitive mutation, to localize to the spindle poles and cytokinetic neck filaments, and to induce elongated cells with ectopic septin ring structures. Consistent with the polo box-dependent subcellular localization, the C-terminal domain of Cdc5, but not its polo box mutant, is sufficient for subcellular localization, and its overexpression appears to inhibit cytokinesis. These data provide evidence that the polo box is required to direct Cdc5 to specific subcellular locations and induce or organize cytokinetic structures.

ACKNOWLEDGMENTS

We are grateful to Frank J. Gonzalez, Michael Bustin, and Daniel G. Lee for critical reading of this manuscript. We also thank Philip R. Lee and G.-Y. Kim for technical support, J. V. Kilmartin for the GFP-Spc42 strain, T. N. Davis for advice in immunostaining with a GFP-Spc42 strain, J. Chant for the provision of anti-Cdc10 antibody, A. Sugino for the pKK507 and pKK625 plasmids, M. S. Longtine for pFA6a-tagging plasmids, and C. Hardy for a cdc5N209A construct.

This work was supported by NIH grant CA42580 to R.L.E. R.L.E. is an American Cancer Society Research Professor.