The Protein SET Binds the Neuronal Cdk5 Activator p35 ^nck5a and Modulates Cdk5/p35 ^nck5a 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 α-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 .