The Protein SET Binds the Neuronal Cdk5 Activator p35 and Modulates Cdk5/p35 Activity

Abstract

The neuronal Cdk5 kinase is composed of the catalytic subunit Cdk5 and the activator protein p35 nck5a or its isoform, p39nck5ai . 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 ␣-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 .Cdk5 is distinct from other cyclin-dependent kinases by virtue of its functions in post-mitotic neurons, but not in proliferating cells. Although Cdk5 is ubiquitously expressed, Cdk5-associated kinase activity has been primarily demonstrated in central nervous system neurons. In such neurons, Cdk5 is associated with p35 nck5a or a p35 nck5a isoform (p39 nck5ai ), two Cdk5 activators with restricted expression in central nervous system neurons (1-3). In a recent report, p35 nck5a was also found in muscle cells at the neuromuscular junction (4). Besides the full-length protein of p35 nck5a , a proteolytic fragment called p25 nck5a exists in central nervous system neurons. The p25 nck5a protein is generated when the N-terminal 98 amino acids are removed from p35 nck5a (1, 5-7). Moreover, p25 nck5a is fully functional in terms of Cdk5 activation (8). In association with p35 nck5a /p25 nck5a and p39 nck5ai , Cdk5 exhibits a variety of functions in neuronal differentiation and neurocytoskeleton dynamics as well as neuronal degeneration and cell death (9 -13).Despite little homology between p35 nck5a and cyclins at the primary sequence level, it was proposed that p35 nck5a forms a core structure similar to that of cyclins to support Cdk5 enzyme activity (14 -16). The minimal region required for Cdk5 binding and activation was localized to a region in the C-terminal half of p35 nck5a as well as in p25 nck5a (16,17). Moreover, Cdk5/ p35 nck5a shows many distinct regulatory properties. Cdk5 is highly activated upon association with p35 nck5a /p25 nck5a , and the activation process is not regulated by the cyclin-dependent kinase-activating kinase Cdk7/cyclin H (8, 16). Moreover, upregulation of Cdk5/p35 nck5a activity was observed when Cdk5 was phosphorylated at Tyr-15 by the cellular tyrosine kinase c-Abl in complex ...

Glyceraldehyde 3-phosphate dehydrogenase is a SET-binding protein and regulates cyclin B-cdk1 activity

“…It reverses the p21 Cip1 inhibitory effect on cyclin E-cdk2 activity (Estanyol et al, 1999), inhibits cyclin B-cdk1 activity (Kellogg et al, 1995;Canela et al, 2003) and enhances the activity of p35 nck5a -cdk5 (Qu et al, 2002).…”

Regulation of S6 Kinase 1 Activation by Phosphorylation at Ser-411

“…S6K1 mutations, S411A, S411D, S411A/S424A, T389E, and T389E/S411A, were created by site-directed mutagenesis. The constructs of Cdk5 and p35 were described previously (30,31).…”

“…Protein Binding Assay-Recombinant proteins tagged with GST or His 6 were expressed in Escherichia coli BL21(DE3) and were purified as described in earlier reports (30,31). To test the binding of S6K1 to p35 fragments, 1 g of GST-p35 fragments or GST was incubated with 0.5 g of His 6 -S6K1 for 1 h at 4°C in 500 l of 25 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol, 100 mM NaCl, 0.2% Triton X-100, and the protease inhibitor mixture (Roche Applied Science).…”

A 24-Residue Peptide (p5), Derived from p35, the Cdk5 Neuronal Activator, Specifically Inhibits Cdk5-p25 Hyperactivity and Tau Hyperphosphorylation

“…A fundamental structural difference between the two activators offers a possible explanation; p25, a truncated version of p35, lacks the myristoylated p10 N-terminal domain of the intact p35. The p10 region is responsible for interaction of p35 with several cellular proteins, such as importins, Munc18, calmodulin, microtubules, and protein kinase CK2 (31,(53)(54)(55). Microtubules, for example, bind p35 due to its p10 domain, whereas they do not interact with the p25 due to the loss of the p10 region of the p35 truncated fragment (31).…”