Role of DDB1 in GRK5 Degradation Figure 2. GRK5 associates with DDB1-CUL4 ubiquitin ligase complex. (A,B) MDA-MB-231 cells stably expressing GFP or GRK5-Flag (A), or 293 T cells transiently transfected with GFP or GRK5-Flag (B) were lysed, immunoprecipitated with M2 affinity gel, and analyzed by Western blotting with the indicated antibodies. (C) Endogenous DDB1 complexes were immunoprecipitated from untransfected 293 T cell lysate using the anti-DDB1 antibody and analyzed by Western blotting with GRK5 antibody. Mouse IgG was included as a control. * indicates mouse IgG. doi:10.1371/journal.pone.0043997.g002 cin-induced down-regulation of GRK5 in 293 T cells, suggesting that DDB1 mediates Hsp90 inhibition-induced proteasome- dependent degradation of GRK5. DDB1 Mediates UV Irradiation-induced GRK5 Degradation We further explored the potential role of the DDB1-CUL4 ubiquitin ligase in GRK5 degradation in 293 T cells. It has been shown that DDB1-CUL4 ubiquitin ligase promotes degradation of many proteins including p21 [31,32], CDT1 [26,33,34] and DDB2 [24] following ultraviolet light (UV) irradiation. The response of GRK5 following DNA damage with UV irradiation was examined. The cellular GRK5 level was significantly down- regulated following UV treatment with as little as 20 J/m2 UV, whereas GRK2 levels remained largely unchanged under the same condition (Fig. 6A and 6B). Remarkably, knock down of DDB1 effectively prevented UV-induced GRK5 degradation, suggesting that UV irradiation-induced degradation of GRK5 is mediated by DDB1 (Fig. 6C). The effect of GPCR activation on the stability of GRK5 was also examined. As shown in Fig. 6D, treatment of cells with isoproterenol to activate endogenous b2-adrenergic receptors in 293 T cells [35,36] had no significant effect on GRK5 protein level (data not shown) or UV irradiation-induced GRK5 degradation (Fig. 6D). Discussion In the current study, a proteomic approach was used to screen GRK5 interacting proteins in MDA-MB-231 cells and HUVEC cells. Several proteins were detected in the GRK5 immunocom- plex including SRRM2, MYH9, AP3D1, DDB1, Hsp90, UBTF4, NCL and STK38. Interestingly, other components of DDB1-CUL4 ubiquitin ligase complex including CUL4B, WDR22, GRWD1 and COPS7A, were also detected in the GRK5 immunocomplex in both MDA-MB-231 cells and HUVEC cells. We further provided evidence that GRK5 forms a complex with DDB1-CUL4-ROC1 E3 ubiquitin ligase and DDB1 acts as an adaptor to link GRK5 to CUL4 to form the complex. DDB1 regulates the ubiquitination and degradation of GRK5. Furthermore, depletion of DDB1 inhibited Hsp90 inhibitor-induced GRK5 destabilization and UV irradiation- induced GRK5 degradation. Thus, our immunoprecipitation- mass spectrometry data provide useful information about GRK5 interacting proteins in cells, and our results reveal DDB1 as a key regulator of GRK5 stability. Altered GRK protein expression and/or activity have profound effects on cell signaling and physiological functions, and changed GRK expression has been observed in a variety of human disorders [13]. The mechanisms that govern GRK2 cellular levels have recently been addressed. GRK2 is rapidly degraded via the proteasome pathway. The ubiquitination and turnover of GRK2 are stimulated by b2-adrenergic receptor activation [14], through a mechanism involving GRK2 phosphorylation by c-Src or MAPK in a beta-arrestin-dependent manner [15,16]. Mdm2 is subsequently identified as the key E3 ubiquitin ligase involved in GRK2 ubiquitination and degradation [18]. However, how the stability of other GRK subtypes is controlled remains largely unknown. In the current study, we identified DDB1-CUL4 complex as the key ubiquitin ligase responsible for GRK5 ubiquitination and degradation. Several lines of evidence support the notion that DDB1 serves as a linker to target GRK5 to DDB1- CUL4 E3 ligase for GRK5 ubiquitination and degradation. First, DDB1 was detected in GRK5 immunoprecipitates of lysates from different cell lines. Second, a pool of endogenous GRK5 and DDB1 can be found in the same molecular complex, as indicated by co-immunoprecipitation. Moreover, overexpression or knock- down of the protein reveals DDB1 is an adapter linking GRK5 to DDB1-CUL4 complex. Third, GRK5 ubiquitination and degra- dation is significantly impaired in DDB1/CUL4-knockdown cells. Finally, we found that degradation of GRK5 induced by both Hsp90 inhibitor and UV-irradiation could be both inhibited in DDB1 deficient cells. Thus this may serve as a new regulation mechanism for GRK5 stability. It will be interesting to further explore if the DDB1-mediated GRK5 degradation is DDB1/ GRK5 interaction-dependent, or if there is a direct interaction between GRK5 and DDB1. We also show that DDB1 preferentially associates with GRK4 family proteins. DDB1 could be observed in both GRK5 and GRK6, but not GRK2 immunoprecipitates. GRK2 has been shown to interact with Mdm2 [18]. These results suggest that different GRK subtypes may form complexes with diverse E3 ubiquitin ligases. In addition, Penela et al. reported that GRK2 has PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e43997