Isolation and characterization of Neurospora crassa nucleoside diphosphate kinase NDK-1.

We have previously reported that phosphorylation of a 15-kDa protein increased after blue-light irradiation in Neurospora crassa. In this study, the 15-kDa protein was purified using four columns; DEAE-cellulose, Blue-Sepharose, SP-Sepharose and Mono Q. The 15-kDa protein was shown to be homologous with nucleoside diphosphate kinase by amino acid sequencing and was also shown to possess nucleoside diphosphate kinase activity. A gene encoding N. crassa nucleoside diphosphate kinase, ndk-1, was isolated from the mycelial cDNA and genomic libraries. The deduced amino acid sequence of NDK-1 was identical to that of the 15-kDa protein. Northern blot analysis suggested that WC-1 and WC-2, the key factors of blue-light signal transduction in N. crassa, did not regulate NDK-1 at the transcriptional level. NDK-1 also showed rapid autophosphorylation activity and protein kinase activity against myelin basic protein with a Km value of 0.36 mM. These results suggest that NDK-1 acts as a signal transducer by phosphorylating proteins.