Only the CDK inhibitor, roscovitine, led to net dephosphorylation of SAD-A as indicated by a shift toward the 76 kDa form (Figure 7A and data not shown). We also treated cultured DRG neurons with roscovitine and found a similar although less pronounced shift in SAD-A mobility (Figure 7A). These results suggest that selleck chemicals llc CDKs are physiological regulators of CTD phosphorylation. To test this idea directly, we coexpressed either wild-type or catalytically inactive CDK5 with the p35 coactivator and SAD-AWT or SAD-A18A. Expression of active but not inactive CDK5 caused SAD-AWT protein to migrate exclusively at 85 kDa, while migration of SAD-A18A

was only slightly affected (Figure 7B). Moreover, whereas expression of active CDK5 completely

eliminated ALT phosphorylation of SAD-AWT, ALT phosphorylation of SAD-A18A was largely resistant to CDK5-mediated inhibition (Figure 7B). The fact that the SAD-A18A mutant is not completely refractory to the inhibitory effects of CDK5 suggests that there may be other check details residues involved in mediating SAD-A inhibition. Thus, CDK5 can phosphorylate SAD-A in the CTD, preventing activating phosphorylation at the ALT. These results reveal a mechanism in which activation of SAD kinase by canonical activation loop phosphorylation is inhibited by phosphorylation of distal sites in the CTD. To ask which phosphorylation sites in the SAD-A CTDs are important for inhibition of SAD activation, we divided them into two groups and mutated each separately: the 13 sites in the PXX[S/T]P motifs N-terminal to the D box (aa 428–468, mutant called SAD-A13A) or the 5 sites (4 of which are [S/T]P) C-terminal to the D box (aa 490–513, Fossariinae mutant called SAD-A5A; Figure S6A), We expressed the mutants in 293T cells with CDK5 and examined the effects on SAD ALT phosphorylation. CDK5 activation suppressed SAD ALT phosphorylation of both the SAD-A13A and SAD-A5A mutants (Figure S6B). We conclude that no single phosphorylation event in the CTD regulates SAD activity, but rather phosphorylation of residues throughout

the SAD CTD is sufficient to block SAD ALT phosphorylation. What signaling pathway does NT-3 use to regulate phosphorylation of the SAD CTD? We analyzed SAD-A immunoprecipitates from untreated and NT-3 treated cells using the anti-p[S/T]P antibody. Consistent with results presented above (Figure 6C), SAD-A protein from untreated cells was strongly phosphorylated at [S/T]P sites, whereas SAD-A protein from NT-3 treated cells lacked [S/T]P phosphorylation (Figure 7C). Thus, NT-3/TrkC signaling induces net SAD-A CTD dephosphorylation. Inhibitors of MEK1/2 or PLCγ decreased NT-3 dependent SAD-A CTD dephosphorylation in TrkC+ HeLa cells (Figure 7D), indicating that both of these pathways are capable of regulating SAD-A CTD dephsophorylation in response to NT-3.