Fig. 4. Nmyc1 phosphorylation is regulated by GSK3 and the PI3K pathway in CGNPs, independent of Shh signaling. (A) CGNP cultures were treated with Shh, cyclopamine and/or the proteosome inhibitor lactacystin, which effectively blocks ongoing turnover of endogenous Nmyc1 (compare lanes 3 and 5 to lanes 2 and 4). Lysates were blotted to determine levels of T50-phosphorylated Nmyc1 relative to total Nmyc1 and results representative of three independent experiments are shown. Tubulin immunoreactivity indicates equivalent loading of the lanes. The level of T50-phosphorylated Nmyc1 relative to total Nmyc1 was comparable regardless of treatment with Shh or cyclopamine, indicating that Shh signaling has neither positive (destabilizing) nor negative (stabilizing) effects on endogenous Nmyc1 phosphorylation. (B) GSK3 activity is required for endogenous Nmyc1 phosphorylation. Shh-treated CGNP cultures were exposed to LiCl (left) or a commercially available GSK3 inhibitor (right) for the indicated length of time. Both methods for blocking endogenous GSK3 activity effectively reduced levels of Nmyc1 phosphorylation. (C) Activity of PI3K is required for stabilizing Nmyc1. CGNP cultures were infected as indicated. After 24 hours, the cells were treated (3 hours) with wortmannin, a PI3K inhibitor. In the presence of wortmannin, levels of wild-type retroviral Nmyc1 were sharply reduced. By contrast, levels of Nmyc1T50A and Nmyc1S54A were unaffected, ruling out a general effect of wortmannin treatment on protein synthesis. These results also show that the non-phosphorylatable mutants do not depend on the PI3K pathway for stabilization. (D) IGF, a prominent activator of PI3K in CGNPs, stabilizes endogenous Nmyc1. CGNPs cultured in N2 (insulin)-containing medium were treated with (lanes 2-5) or without Shh (lane 1) for 24 hours. At this time, medium in lanes 2-4 was withdrawn and cells were washed extensively and treated with fresh medium containing Shh (lanes 2-5), N2 (lanes 2, 5), no N2 or IGF1 (lane 3), IGF1 (20 ng ml^–1, lane 4), or LY294002 (10 µM, lane 5). After 3 hours, protein lysates were prepared and western blotted for Nmyc1, phsophorylated Akt, and phosphorylated GSK3. Lack of N2 or IGF1 resulted in downregulation of the PI3K pathway, and decreased Nmyc1 protein. N2 and IGF1 were each capable of supporting PI3K pathway activity and Nmyc1 protein levels. LY294002 inhibited the PI3K pathway and promoted destabilization of endogenous Nmyc1.