Results Identification of GRK5-interacting Proteins by Mass Spectrometry A proteomic approach was used to identify interacting proteins of GRK5. Flag-tagged GRK5 was affinity purified from MDA- MB-231 cells stably expressing GRK5-Flag. As a control, cells stably expressing GFP were taken through the same procedure. The resulting protein complexes were electrophoresed on a 4– 20% gradient polyacrylamide gel and then stained with Coomassie Blue. Protein bands of ,300 kDa, ,230 kDa, ,130 kDa, ,85 kDa and ,55 kDa were selectively copurified with Flag- tagged GRK5 but not control beads. The gel bands were excised and analyzed by mass spectrometry as indicated in Figure 1A. The corresponding bands in the control lane were also excised and analyzed by mass spectrometry, and proteins detected in both the control lane and GRK5-immuocomplex lane were excluded from further analysis. This work led to the identification serine/arginine repetitive matrix 2 (SRRM2, 300 kDa), Myosin-9 (MYH9, 227 kDa), AP-3 complex subunit delta 1 (AP3D1, 130 kDa), damage-specific DNA binding protein 1 (DDB1, 127 kDa), heat shock protein Hsp90-alpha (Hsp90AA1, 83 kDa), heat shock protein Hsp90-beta (Hsp90AB1, 85 kDa), upstream binding transcription factor, RNA polymerase 1 (UBTF4, 89 kDa), nucleolin (NCL, 77 kDa) and serine/threonine kinase 38 (STK38, 55 kDa) as the major interacting proteins of GRK5 in MDA-MB-231 cells (Fig. 1A and Table S1). DDB1 functions as a linker to recruit substrates or substrate receptors to CUL4- ROC1 E3 ubiquitin ligase [20]. Interestingly, peptides derived from other components of DDB1-CUL4 complex were also identified in the GRK5 immunoprecipitates by mass spectrometry including CUL4B, which interacts with DDB1 and assembles ubiquitin ligase by binding to ROC1; WD40-repeat protein 22 (WDR22) and glutamate-rich WD40-repeat protein 1 (GRWD1), which contain WD40 repeat domain and constitute the substrate- specific adaptor that recruits substrates to the DDB1-CUL4 complexes through interaction with DDB1; COP9 signalosome complex subunit 7a (COPS7A), component of the COP9 signalosome complex, which cleaves NEDD8 from CUL4 and regulates the CUL4 ubiquitin ligase activity (Table S1 and S3). Similar proteomic approach was carried out in human umbilical vein endothelial Cells (HUVEC) cells transiently transfected with Flag-GRK5. SRRM2, AP3D1, Hsp90, UBTF4 and NCL were also detected in HUVEC cells by mass spectrometry (Fig. 1B, supplemental Table S1, S2 and S4). The DDB1-CLU4 complex proteins including DDB1, CUL4B, WDR22, GRWD1 and COPS7A were all detected in HUVEC cells (Fig. 1B, supplemen- tal Table S1 and S2), and two additional WD40-repeat domain containing proteins were identified in HUVEC cells including WDR12 and WDR82 (Table S2 and S4). However, STK38, which was readily detected in the GRK5 immunocomplex in MDA-MB-231 cells by mass spectrometry, was not detected in the HUVEC cells (Fig. 1A, 1B and Table S1,S2,S3,S4). As we identified several components of DDB1-CUL4 complex in both MDA-MB-231 cells and HUVEC cells by mass spectrometry, we further examined the association of GRK5 with DDB1-CUL4 ubiquitin ligase complex. GRK5 Associates with DDB1-CUL4 Ubiquitin Ligase Complex The association of GRK5 with DDB1-CUL4 complex was confirmed by immunoprecipitation and Western analysis in MDA-MB-231 cells stably expressing GRK5-Flag and in 293 T cells transiently transfected with GRK5-Flag. DDB1, CUL4A, Role of DDB1 in GRK5 Degradation and CUL4B were detected in the immunocomplex of the Flag- tagged GRK5 (Fig. 2A and 2B). The RING finger protein ROC1 was also detected in GRK5 precipitates in 293 T cells (Fig. 2B) and MDA-MB-231 cells (data not shown). Damage DNA binding protein 2 (DDB2), a known binding protein of DDB1, functioning in the global genome nucleotide excision repair, was also detected in the GRK5 precipitates (Fig. 2A and Fig. 2B). Furthermore, endogenous GRK5 was detected in DDB1 immunocomplexes in 293 T cells (Fig. 2C), demonstrat- ing association between endogenous GRK5 and endogenous DDB1 in vivo. DDB1 Targets GRK5 to CUL4 to Form a Complex To test the idea that DDB1 acts as an adapter to recruit GRK5 to CUL4 to form a complex, epitope-tagged CUL4A, DDB1, and GRK5 were expressed in 293 T cells and protein associations were analyzed by immunoprecipitation. As shown in Fig. 3A, the association of DDB1 with GRK5 could be detected in the absence of CUL4A-HA expression, and similarly, the association of DDB1 with CUL4A-HA could occur in the absence of GRK5 expression (Fig. 3A), confirming the interaction of GRK5 and CUL4A with DDB1. When CUL4A-HA, DDB1-Flag, and GRK5 were co-expressed, all three proteins were present in a complex. However, in the absence of DDB1-Flag, the association between CUL4A-HA and GRK5 was significantly attenuated (Fig. 3B). These data suggest that DDB1 is required for the association of GRK5 with CUL4A. The basal interaction of GRK5 with CUL4A-HA in the absence of DDB1-Flag expression was likely a result from co-immunoprecipitated endogenous DDB1 by CUL4A-HA, since significant amount of endogenous DDB1 could be detected in the CUL4A immunoprecipitates (Fig. 3B, panel 3). Similar results were obtained when using GRK5-HA to immunoprecipitates DDB1 and CUL4A (Fig. 3C). Furthermore, knockdown of DDB1 significantly reduced the amount of CUL4A in GRK5 immunoprecipites. However, knockdown of CUL4A had no significant effect on the association between GRK5 and endogenous DDB1 (Fig. 3D). These results further support the notion that DDB1 acts as an adaptor to link CUL4A with GRK5. Based on the overall structural organization and homology of GRKs, GRK family proteins can be divided into three subfamilies: GRK1 and GRK7; GRK2 and GRK3; and GRK4, GRK5, and GRK6 [13]. We examined the interaction of DDB1 with other members of GRK family proteins. As shown in Fig. 3E, DDB1 could be detected in the immunoprecipitates of GRK5-Flag as well as GRK6-Flag, but not in GRK2-Flag immunoprecipitates, suggesting the specific complex formation of DDB1 with GRK4 subfamily proteins. DDB1 Regulates GRK5 Ubiquitination Since DDB1 targets GRK5 to the CUL4 ubiquitin ligase complex, we tested whether DDB1 regulates GRK5 ubiquitination and degradation. 293 T cells were co-transfected with plasmids encoding Flag-tagged GRK5 and HA-tagged ubiquitin, in combination with control plasmid, DDB1, or myc-CUL4A. As shown in Fig. 4A, expression of DDB1 alone or co-expression of DDB1 with CUL4A significantly increased GRK5 ubiquitination, while expressing CUL4A alone did not affect the ubiquitination of GRK5. To examine the requirement of DDB1-CUL4 complex for GRK5 ubiquitination, 293 T cells were co-transfected with GRK5-Flag, HA-ubiquitin, and shRNA targeting DDB1 or CUL4. As shown in Fig. 4B, GRK5 ubiquitination was severely decreased by the depletion of either DDB1 or CUL4A/B. PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e43997