Role of DDB1 in GRK5 Degradation Figure 3. DDB1 associates with GRK5 and targets GRK5 to CUL4. (A–C) 293 T cells were transiently co-transfected with indicated plasmids alone or co-expressing both or all of them. The cell lysates were immunoprecipitated with M2 affinity gel (A) or anti-HA agarose (B and C) and then the immunoprecipitates and cell lysate input were analyzed by Western blotting with the indicated antibodies. (D) 293 T cells were co-transfected with GRK5-Flag and indicated shRNA plasmids. Cells were immunoprecipitated with M2 affinity gel. The immunoprecipitates and cell lysate input were analyzed by Western blotting with the indicated antibodies. (E) 293 T cells expressing Flag-GRK2, Flag-GRK5, Flag-GRK6, or control plasmid were lysed and immunoprecipitated with M2 affinity gel. The immunoprecipitates and the cell lysate input were analyzed by Western blotting with the indicated antibodies. doi:10.1371/journal.pone.0043997.g003 a half-life of about 1 h in C6 glioma and Jurkat cells [14,15]. In this study, we observed that in 293 T cells, endogenous GRK5, but not GRK2, was rapidly degraded after CHX treatment. Our results are consistent with previous reports showing that the half-life of GRK2 in HL60 cells is 20–24 h [19,37]. This may reflect difference in degradation mechanisms of GRK2 among these cell types. Hsp90 has been shown to interact with GRK2 and GRK3, and regulates the stability of GRK2 and GRK3 [19,38]. Inhibition of Hsp90 results in rapid, proteasome-dependent, down-regulation of GRK2 and GRK3 [19,38]. In this study, we found that inhibition of Hsp90 resulted in proteasome-dependent degradation of GRK5. These studies and our data implicate that Hsp90-mediated stabilization and proteasome-dependent degradation may play a general role in regulation of the stability of GRK family proteins. Hsp90 has also been reported to interact with and participate in the regulation of other kinases, such as Erb2, Akt/PKB, and Raf-1 [39]. Inhibition of Hsp90 by geldanamycin results in enhanced degradation of these proteins [39]. Therefore, stabilization of client protein seems to be a general function of Hsp90. We further provide the evidence that the degradation of endogenous GRK5 induced by Hsp90 inhibition was mediated by DDB1. Depletion of DDB1 inhibited geldanamycin-induced degradation of GRK5. These results suggest that disrupting the interaction between GRK5 and Hsp90 induces DDB1-CUL4 complex-mediated ubiquitination-proteasome dependent degradation of GRK5, and put forward the notion that the stability of GRK5 was counter-regulated through interaction with DDB1-CUL4 complex and Hsp90. DDB1-CUL4 complex controls ubiquitination and stability of many cellular substrates including p21 [31,32], DDB2 [24], Cdt1 PLOS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e43997