SILAC APMS analysis of GFP tagged TSC2 in Hela Cells

HSP90 and its co-chaperon R2TP are involved to assemble and stabilize many macro-molecular complexes involved in cell proliferation. R2TP complex is composed of RUVBL1 and RUVBL2 AAA+ ATPases and two adapter proteins RPAP3 and PIH1D1. The N-terminal domain of PIH1D1 was described to possess a basic pocket that binds phosphorylated peptides on some of the R2TP clients. Subunits of TSC complex (Tuberous SClerosis) have previously been described in proteomic analysis of proteins immune-precipitated with PIH1D1 N-terminal domain. TSC complex is a regulator of mTOR activity. It is composed of TSC1, TSC2 and TBC1D7 subunits. Here, we show that (i) TSC1 and TSC2 are both substrates of HSP90, (ii) the HSP90/R2TP system has multiple interaction with TSC complex subunits and (iii) it is functionally involved in the assembly of the TSC complex. We found a direct interaction of TSC1 with the phospho-binding pocket of PIH1D1 N-terminal domain regardless of phosphorylationbut in a non-canonical phosphorylation independent manner[XM1] . TSC2 and TBC1D7 would also interact with R2TP but independently of PIH1D1. Taken together, these data suggest that R2TP acts as a platform for the independent recruitment of TSC subunits before the assembly of the TSC complex