Isolation of a Functional Homolog of the Cell Cycle-specific NIMA Protein Kinase of Aspergillus nidulans and Functional Analysis of Conserved Residues

doi:10.1074/jbc.270.30.18110

Isolation of a Functional Homolog of the Cell Cycle-specific NIMA Protein Kinase of Aspergillus nidulans and Functional Analysis of Conserved Residues (*)

From the (1) Weis Center For Research, Geisinger Clinic, Danville, Pennsylvania 17822-2617, the
Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6, and
(2) National Center for Agricultural Utilization Research, United States Department of Agriculture, Peoria, Illinois 61604

^§ To whom correspondence should be addressed:
Weis Center for Research, Geisinger Clinic, 100 N. Academy Ave., Danville, PA 17822-2617
. Tel.: 717-271-6677; Fax: 717-271-6701; SAO{at}SMTP.GEISINGER.EDU

Abstract

To investigate the degree of conservation of the cell cycle-specific NIMA protein kinase of Aspergillus nidulans, and to help direct its functional analysis, we cloned a homolog (designated nim-1) from Neurospora crassa. Over the catalytic domain NIM-1 is 75% identical to NIMA, but overall the identity drops to 52%. nim-1 was able to functionally complement nimA5 in A. nidulans. Mutational analysis of potential activating phosphorylation sites found in NIMA, NIM-1, and related protein kinases was performed on NIMA. Mutation of threonine 199 (conserved in all NIMA-related kinases) inhibited NIMA β-casein kinase activity and abolished its in vivo function. This site conforms to a minimal consensus phosphorylation site for NIMA (FXXT) and is analogous to the autophosphorylation site of cyclic-AMP-dependent protein kinases. However, mutation of a unique cysteine residue found only in the catalytic site of NIMA and NIM-1 had no effect on NIMA kinase activity or function. Three temperature-sensitive alleles of nimA that cause arrest in G₂ were sequenced and shown to generate three different amino acid substitutions. None of the mutations prevented accumulation of NIMA protein during G₂ arrest, but all prevented the p34/cyclin B-dependent phosphorylation of NIMA normally seen during mitotic initiation even though p34/cyclin B H1 kinase activity was fully activated.

Footnotes

↵* This work was supported by the Geisinger Foundation and by National Institutes of Health Grant GM42564 (to S. A. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) L42573[GenBank Link].
↵¹ The abbreviations used are:

kb

kilobase(s)

PAGE

polyacrylamide gel electrophoresis.
↵²X. S. Ye and S. A. Osmani, manuscript in preparation.
- Received March 3, 1995.
- Revision received May 22, 1995.