Isolation of a functional homolog of the cell cycle-specific NIMA protein kinase of Aspergillus nidulans and functional analysis of conserved residues.
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 beta-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 p34cdc2/cyclin B-dependent phosphorylation of NIMA normally seen during mitotic initiation even though p34cdc2/cyclin B H1 kinase activity was fully activated.
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Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822-2617, USA.
Xu
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Wu
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Vierula
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O'Donnell
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Ye
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Osmani
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D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000166:cytology / Q000201:enzymology*; D001483:Base Sequence; D002384:Catalysis; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003545:Cysteine / Q000235:genetics; D004252:DNA Mutational Analysis; D004271:DNA, Fungal; D004789:Enzyme Activation; D008938:Mitosis; D008969:Molecular Sequence Data; D000072380:NIMA-Related Kinase 1; D000072379:NIMA-Related Kinases; D009492:Neurospora crassa / Q000235:genetics; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature
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; D003545:Cysteine