IDENTIFICATION OF SUBSTRATE-SPECIFICITY DETERMINANTS FOR THE CELL-CYCLE-REGULATED NIMA PROTEIN-KINASE

NIMA is a cell cycle-regulated protein kinase required for the G(2)/M transition in the filamentous fungus Aspergillus nidulans. Previous bi ochemical characterization of the recombinant enzyme indicated that NI MA is a protein serine/threonine specific kinase with beta-casein bein g the best substrate from the many proteins and peptides tested (Lu, K . P., Osmani, S. A, and Means, A R. (1993) J. Biol. Chem. 268, 8769-87 76). However, substrate specificity or physiologically relevant substr ates for NIMA remained unknown. In search for a peptide substrate for this enzyme, we screened an assembled library of synthetic peptides th at each contained a phosphorylation site for a known protein kinase an d found an excellent peptide substrate for NIMA, phospholemman 42-72 ( PLM(42-72)). NIMA kinase phosphorylated PLM(42-72) uniquely and stoich iometrically on Ser(63) with a V-max of 1.4 mu mol/min/mg and apparent K-m of 20.0 mu M. These kinetic constants were about 10-fold higher a nd 3-fold lower than those for beta-casein, respectively. A detailed a nalysis of substrate specificity determinants using synthetic peptide analogs of PLM(42-72) indicated that Phe-Arg-Xaa-Ser/Thr represents th e optimal primary sequence for NIMA kinase phosphorylation. Replacemen t of the Arg at P-2 with Ala resulted in a 6-fold increase in K-m and 2-fold decrease in V-max, while substitution of the Phe at P-3 with Al a abolished NIMA phosphorylation. These results reveal the unique natu re of substrate recognition by the NIMA kinase and should prove valuab le in the search for biologically relevant NIMA substrates.