The protein SET binds the neuronal Cdk5 activator p35nck5a and modulates Cdk5/p35nck5a activity.
The neuronal Cdk5 kinase is composed of the catalytic subunit Cdk5 and the activator protein p35(nck5a) or its isoform, p39(nck5ai). To identify novel p35(nck5a)- and p39(nck5ai)-binding proteins, fragments of p35(nck5a) and p39(nck5ai) were utilized in affinity isolation of binding proteins from rat brain homogenates, and the isolated proteins were identified using mass spectrometry. With this approach, the nuclear protein SET was shown to interact with the N-terminal regions of p35(nck5a) and p39(nck5ai). Our detailed characterization showed that the SET protein formed a complex with Cdk5/p35(nck5a) through its binding to p35(nck5a). The p35(nck5a)-interacting region was mapped to a predicted alpha-helix in SET. When cotransfected into COS-7 cells, SET and p35(nck5a) displayed overlapping intracellular distribution in the nucleus. The nuclear co-localization was corroborated by immunostaining data of endogenous SET and Cdk5/p35(nck5a) from cultured cortical neurons. Finally, we demonstrated that the activity of Cdk5/p35(nck5a), but not that of Cdk5/p25(nck5a), was enhanced upon binding to the SET protein. The tail region of SET, which is rich in acidic residues, is required for the stimulatory effect on Cdk5/p35(nck5a).
Qu
Dianbo
D
Proteomics Group, Institute of Molecular and Cell Biology, National University of Singapore, 30 Medical Drive, Singapore 117609.
Li
Qing
Q
Lim
Hui-Ying
HY
Cheung
Nam Sang
NS
Li
Rong
R
Wang
Jerry H
JH
Qi
Robert Z
RZ
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D002478:Cells, Cultured; D002868:Chromosomal Proteins, Non-Histone; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D056488:Histone Chaperones; D006801:Humans; D007150:Immunohistochemistry; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism* / Q000502:physiology*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions; D014157:Transcription Factors; D014162:Transfection
D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D056488:Histone Chaperones; D009419:Nerve Tissue Proteins; D011506:Proteins; D011994:Recombinant Proteins; C513473:SET protein, human; D014157:Transcription Factors; C089495:neuronal Cdk5 activator (p25-p35); D005982:Glutathione Transferase; D051360:Cyclin-Dependent Kinase 5; C495905:CDK5 protein, human; C495906:Cdk5 protein, mouse; C495907:Cdk5 protein, rat; D018844:Cyclin-Dependent Kinases