FIGURE 7.
Phosphorylation of TSC2 Thr1203 by ROCK both in vivo and in vitro. A, phosphorylation of TSC2 but not TSC2T1203A during anchorage (Anc) deprivation in REFs expressing activated ROCK1. Left panel, validation of the specificity of the anti-phospho-RRX(S/T) antibody for the detection of TSC2 Thr1203 phosphorylation. Eker-Cdc6-aRK-iTsc2i4 and Eker-Cdc6-aRK-iTsc2i4A cells were cultured for 3 days in the absence of doxycycline and lysed. TSC2 was immunoprecipitated with the anti-TSC2 antibody and immunoblotted with the anti-phospho-RRX(S/T) (p-RRXT) antibody or the anti-TSC2 antibody. Right panel, logarithmically proliferating REF-Cdc6 cells were cultured in methylcellulose medium for 36 h with sampling every 12 h. Cells were lysed, immunoprecipitated for TSC2, and immunoblotted with the anti-phospho-RRX(S/T) or the anti-TSC2 antibody. In parallel, the levels of LIMK1, phosphorylated LIMK1 Thr508, S6K1, and phosphorylated S6K1 Thr389 in the cell lysates were determined by immunoblotting. B, treatment with the ROCK inhibitor Y2 abolishes TSC2 Thr1203 phosphorylation in vivo. Logarithmically proliferating REF-Cdc6 cells were cultured in anchorage-furnished culture dishes for 24 h with or without 20 μm Y2. Cells were harvested every 12 h and analyzed as in A. C, in vitro TSC2 Thr1203 phosphorylation by E. coli-expressed active ROCK1. An E. coli-expressed, truncated (trunc.) active ROCK1 (aRK) and GST-TSC2i4 or -TSC2i4A fragments were incubated in a reaction buffer containing ATP with or without Y2. Phosphorylation of GST-TSC2i4 at Thr1203 was detected with the anti-phospho-RRX(S/T) antibody.