Role of DDB1 in GRK5 Degradation Figure 1. Identification of GRK5 interacting proteins by mass spectrometry. (A) Lysates of MDA-MB-231 cells stably expressing GFP or GRK5-Flag were immunoprecipitated with M2 affinity gel. The complexes were separated on SDS-PAGE and stained with Coomassie Blue. Six pieces of gels were excised and labled as line 1-line 6 as indicated. The corresponding control lane was also excised. These gels were analyzed by mass spectrometry. The major associated proteins identified by mass spectrometry were indicated. (B) Lysates of HUVEC cells transiently expressing GFP or GRK5-Flag were treated as in (A). The gels were analyzed by mass spectrometry. The associated proteins identified by mass spectrometry were indicated. doi:10.1371/journal.pone.0043997.g001 DDB1 Regulates GRK5 Degradation We next examined the effect of DDB1 on GRK5 degradation. 293 T cells were infected by lentivirus expressing control-shRNA or DDB1-shRNA, and the degradation of endogenous GRK5 was determined in the presence of CHX in chasing experiment. As shown in Fig. 5A, in the control cells, 50% of GRK5 was degraded in less than 3 h, whereas knockdown of DDB1 markedly attenuated the degradation of GRK5 and extended the half-life of GRK5 (,25% reduction of GRK5 in 8 h). Hsp90 has been shown to interact with GRK2 and regulate the stability of GRK2 by preventing it from proteasome-dependent degrada- tion [19]. A significant amount of Hsp90 peptides were also detected in the GRK5 immunoprecipitates by mass spectrometry (Fig. 1). We next examined the role of Hsp90 in regulating GRK5 stability. As shown in Fig. 5B, treatment of 293 T cells with geldanamycin, an Hsp90-specific inhibitor, significantly down-regulated the level of endogenous GRK5 in a dose- dependent manner, suggesting that Hsp90 regulates GRK5 stability. This is consistent with the previous report that treatment with geldanamycin resulted in about 80% down- regulation of GRK5 transiently expressed in COS-7 cells [19]. Furthermore, pretreatment of cells with MG132, a proteasome inhibitor, significantly inhibited geldanamycin-induced down- regulation of GRK5 (Fig. 5C), suggesting that GRK5 degrada- tion induced by geldanamycin was predominantly through the proteasome pathway. We further examined the role of DDB1 in Hsp90 inhibition-induced degradation of GRK5. As shown in Fig. 5D, knockdown of DDB1 significantly inhibited geldanamy- PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e43997