Identification of substrate specificity determinants for the cell cycle-regulated NIMA protein kinase.

Journal Article (Journal Article)

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.

Full Text

Duke Authors

Cited Authors

  • Lu, KP; Kemp, BE; Means, AR

Published Date

  • March 4, 1994

Published In

Volume / Issue

  • 269 / 9

Start / End Page

  • 6603 - 6607

PubMed ID

  • 8120013

International Standard Serial Number (ISSN)

  • 0021-9258


  • eng

Conference Location

  • United States