nimA was first identified as an essential mitotic regulator in Aspergillus nidulans. nimA encodes a protein kinase only very distantly related to other known cell cycle regulators. Aspergillus strains expressing temperature-sensitive alleles of nimA arrest in late G2-phase at the restrictive temperature. Recent studies suggest that NIMA is a key downstream effector of Cdc2. A key question is whether NIMA is a universal cell cycle regulator, like Cdc2, or whether its role in cell cycle control is unique to Aspergillus. Recently a NimA homolog has been identified in a second filamentous fungus, Neurospora crassa. We designed PCR primers based on sequences conserved between the Aspergillus and Neurospora NimA proteins, and began a search for a homolog in Xenopus laevis oocytes. Xenopus oocytes and eggs offer a number of powerful approaches to studies of cell cycle regulators. We have identified three NIMA-related proteins, Xnek1, Xnek3, and Xnek4, and have chosen to focus on Xnek1. Xnek1 is a close relative of the mouse Nek1 protein kinase, which was fortuitously discovered in the course of studies aimed at identifying novel protein tyrosine kinases. The kinase domain of Xnek1 is 42% identical to that of NIMA, and Xnek1 and NIMA both possess long, hydrophilic C-terminal tails that contain nuclear localization sequences, predicted coiled-coil regions, PEST sequences, and multiple potential Cdc2 phosphorylation sites. We have cloned and sequenced an Xnek1 cDNA. We used this information to raise Xnek1 antisera, and established that the sera specifically immunoprecipitate a protein kinase activity that phosphorylates Ig heavy chain and, like NIMA, beta-casein. We have demonstrated that Xnek1 undergoes striking changes in activity during the first embryonic cell cycle, with its activity peaking in S-phase or G2 -phase. We suspect that Xnek1 may play a role in regulating DNA synthesis or early mitotic events. We propose to examine the regulation and function of the Xnek1 protein during meiotic and mitotic cell cycles. We shall address the following specific questions; (1) Does Xnek1 change in activity during other cell cycles? (2) How is Xnek1 activated and inactivated? (3) What biochemical and cell biological events depend upon Xnek1 function?
