Properties and regulation of the cell cycle-specific NIMA protein kinase of Aspergillus nidulans.
NIMA is the protein product of the nimA gene of the filamentous fungus Aspergillus nidulans, required for progression of cells from G2 into mitosis. The protein kinase activity of NIMA, assayed by phosphorylation of beta-casein, varies during the nuclear division cycle, reaching a maximum in late G2 and M. To investigate the biochemical properties of this cell cycle-regulated protein kinase, we have expressed nimA cDNA that encodes full-length NIMA in Escherichia coli as a fusion product with glutathione S-transferase. Purified NIMA phosphorylated beta-casein, with a Km of 38 microM and Vmax of 156 nmol/min/mg. NIMA also demonstrated a Km of 69 microM for ATP. Both recombinant and cellular NIMA kinases behaved as oligomers on gel filtration chromatography, and their kinase activities were strongly inhibited by various salts. By using both protein and peptide substrates, NIMA demonstrated a serine/threonine-specific protein kinase activity. Cellular NIMA exists as a phosphoprotein, and bacterially expressed NIMA was also phosphorylated on multiple serine/threonine residues. Some of these phosphorylations appeared essential for NIMA activity as the enzyme could be dephosphorylated and inactivated in vitro by protein serine/threonine phosphatases. Use of a kinase-negative mutant of NIMA revealed that the NIMA enzyme undergoes autophosphorylation when expressed at high concentrations in bacteria. Taken together, these data suggest that cellular mechanisms may exist to regulate the phosphorylation state and activity of the NIMA protein kinase during the nuclear division cycle in A. nidulans.
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Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710.
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D001233:Aspergillus nidulans / Q000166:cytology / Q000201:enzymology*; D001483:Base Sequence; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D015966:Gene Expression Regulation, Fungal; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D000072380:NIMA-Related Kinase 1; D000072379:NIMA-Related Kinases; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D000072380:NIMA-Related Kinase 1; D000072379:NIMA-Related Kinases; C442331:NIMA-related kinase 6; D017346:Protein Serine-Threonine Kinases