Identification of substrate specificity determinants for the cell cycle-regulated NIMA protein kinase.
NIMA is a cell cycle-regulated protein kinase required for the G2/M transition in the filamentous fungus Aspergillus nidulans. Previous biochemical characterization of the recombinant enzyme indicated that NIMA is a protein serine/threonine specific kinase with beta-casein being 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, 8769-8776). However, substrate specificity or physiologically relevant substrates for NIMA remained unknown. In search for a peptide substrate for this enzyme, we screened an assembled library of synthetic peptides that each contained a phosphorylation site for a known protein kinase and found an excellent peptide substrate for NIMA, phospholemman 42-72 (PLM(42-72)). NIMA kinase phosphorylated PLM(42-72) uniquely and stoichiometrically on Ser63 with a Vmax of 1.4 mumol/min/mg and apparent Km of 20.0 microM. These kinetic constants were about 10-fold higher and 3-fold lower than those for beta-casein, respectively. A detailed analysis of substrate specificity determinants using synthetic peptide analogs of PLM(42-72) indicated that Phe-Arg-Xaa-Ser/Thr represents the optimal primary sequence for NIMA kinase phosphorylation. Replacement of the Arg at P-2 with Ala resulted in a 6-fold increase in Km and 2-fold decrease in Vmax, while substitution of the Phe at P-3 with Ala abolished NIMA phosphorylation. These results reveal the unique nature of substrate recognition by the NIMA kinase and should prove valuable in the search for biologically relevant NIMA substrates.
Lu
K P
KP
Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710.
Kemp
B E
BE
Means
A R
AR
D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000166:cytology / Q000201:enzymology*; D002364:Caseins / Q000378:metabolism; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D016195:G2 Phase; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008938:Mitosis; D008969:Molecular Sequence Data; D000072380:NIMA-Related Kinase 1; D000072379:NIMA-Related Kinases; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D010449:Peptide Mapping; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010750:Phosphoproteins / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013379:Substrate Specificity
D002364:Caseins; D018797:Cell Cycle Proteins; D008565:Membrane Proteins; D009842:Oligopeptides; D010455:Peptides; D010748:Phosphopeptides; D010750:Phosphoproteins; C069141:phospholemman; D000072380:NIMA-Related Kinase 1; D000072379:NIMA-Related Kinases; C442331:NIMA-related kinase 6; D017346:Protein Serine-Threonine Kinases