Identifying novel substrates for mouse Cdk5 kinase using the yeast Saccharomyces cerevisiae.
Among the mammalian Cdk family members, Cdk5, activated by the binding of p35, plays an important role in the control of neurogenesis, and its deregulation is thought to be one of the causes of neurodegenerative diseases. Overproduction of Cdk5 and p35 in yeast cells causes growth arrest, probably because of hyperphosphorylation of yeast proteins. We screened mouse brain cDNA that could recover the growth of yeast cells overproducing Cdk5 and p35, hoping that such cDNA encodes a substrate or inhibitor of Cdk5. Mouse brain cDNA library was introduced into a yeast strain in which Cdk5, p35 and mouse cDNA were over-expressed under the control of the GAL promoter, and cDNA plasmids were isolated from the transformants that recovered growth on galactose medium. The analysis of those plasmids revealed that they harbored cDNA that encodes neuronal proteins including SCLIP and CRMP-1, and those with unknown function. We found that Cdk5 could phosphorylate SCLIP and CRMP-1 in vitro and the two proteins in cultured cells showed a mobility shift depending on Cdk5 activity and the presence of specific Ser or Thr residues, indicating that SCLIP and CRMP-1 are likely substrates for Cdk5 in vitro and in cultured cells. Further screening with these systems will enable us to identify more novel substrates and regulators of Cdk5/p35, which will lead to the exploration of Cdk5 function in diverse cellular systems.
Horiuchi
Youko
Y
Department of Microbiology and Immunology, Keio University School of Medicine, Shinjuku, Tokyo 160-8582, Japan.
Asada
Akiko
A
Hisanaga
Shin-ichi
S
Toh-e
Akio
A
Nishizawa
Masafumi
M
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D051360:Cyclin-Dependent Kinase 5 / Q000378:metabolism*; D018076:DNA, Complementary; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D050777:Stathmin; D013379:Substrate Specificity; D014170:Transformation, Genetic; D000098855:Collapsin Response Mediator Protein 1
D051360:Cyclin-Dependent Kinase 5; D018076:DNA, Complementary; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D010770:Phosphotransferases; D011993:Recombinant Fusion Proteins; D050777:Stathmin; C492236:Cdk5r1 protein, mouse; C502872:Crmp1 protein, mouse; D000098855:Collapsin Response Mediator Protein 1; C491793:Stmn3 protein, mouse