Targeted Ubiquitination and Degradation of G-Protein- Coupled Receptor Kinase 5 by the DDB1-CUL4 Ubiquitin Ligase Complex Ziyan Wu, Yuejun Chen*, Tong Yang, Qinqin Gao, Man Yuan, Lan Ma The State Key Laboratory of Medical Neurobiology and Pharmacology Research Center, Shanghai Medical College and Institutes of Brain Science, Fudan University, Shanghai, China Abstract The G protein-coupled receptor kinases (GRKs) phosphorylate agonist occupied G protein-coupled receptors (GPCRs) and desensitize GPCR-mediated signaling. Recent studies indicate they also function non-catalytically via interaction with other proteins. In this study, a proteomic approach was used to screen interacting proteins of GRK5 in MDA-MB-231 cells and HUVEC cells. Mass spectrometry analysis reveals several proteins in the GRK5 immunocomplex including damaged DNA- binding protein 1 (DDB1), an adaptor subunit of the CUL4-ROC1 E3 ubiquitin ligase complex. Co-immunoprecipitation experiments confirmed the association of GRK5 with DDB1-CUL4 complex, and reveal that DDB1 acts as an adapter to link GRK5 to CUL4 to form the complex. Overexpression of DDB1 promoted, whereas knockdown of DDB1 inhibited the ubiquitination of GRK5, and the degradation of GRK5 was reduced in cells deficient of DDB1. Furthermore, the depletion of DDB1 decreased Hsp90 inhibitor-induced GRK5 destabilization and UV irradiation-induced GRK5 degradation. Thus, our study identified potential GRK5 interacting proteins, and reveals the association of GRK5 with DDB1 in cell and the regulation of GRK5 level by DDB1-CUL4 ubiquitin ligase complex–dependent proteolysis pathway. Citation: Wu Z, Chen Y, Yang T, Gao Q, Yuan M, et al. (2012) Targeted Ubiquitination and Degradation of G-Protein-Coupled Receptor Kinase 5 by the DDB1-CUL4 Ubiquitin Ligase Complex. PLoS ONE 7(8): e43997. doi:10.1371/journal.pone.0043997 Editor: Sonja Hess, California Institute of Technology, United States of America Received March 19, 2012; Accepted July 26, 2012; Published August 27, 2012 Copyright: 2012 Wu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by the Ministry of Science and Technology grant 2009CB522006 (to LM) and National Natural Science Foundation of China grants 30830042 and 31121061 (to Lan Ma), and 30900852 (to YC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: yuejunchen@fudan.edu.cn Introduction G protein-coupled receptor (GPCR) kinases (GRKs) are a family of serine/threonine kinases that phosphorylate GPCRs and desensitize GPCR-mediated signaling. GRK-catalyzed re- ceptor phosphorylation leads to the recruitment of beta-arrestins to phosphorylated receptors, induces receptor internalization, and thus down-regulates cellular responses to extracellular signal [1,2]. Many studies indicate that GRKs are able to phosphor- ylate a variety of non-GPCR substrates such as synuclein [3], p38 [4], NF-kB1 p105 [5], ezrin [6], arrestin-2 [7], and p53 [8]. It has also been shown that GRKs can regulate signaling pathways via direct interaction with other proteins in a phos- phorylation-independent manner. GRK2 is able to interact with Gaq to regulate GPCR signaling [9]. Binding of GRK5 with IkB inhibits NF-kB-mediated transcription [10]. Our earlier research showed that the kinase activity-independent regulation of the cyclin pathway by GRK2 is essential for zebrafish early development [11] and GRK5 acts as a scaffold to promote F- actin bundling and targets bundles to membrane structures to control neuronal morphogenesis [12]. These studies implicate that GRKs, especially GRK5, may exert multiple physiological functions via various mechanisms including those independent of their kinase activities. Change in GRK protein level has been detected in a variety of human disorders including heart failure, acute myocardial in- farction, hypertension, brain ischemia, rheumatoid arthritis, Parkinson’s disease, Alzheimer’s disease and depression [13], suggesting that protein turnover plays a key role in GRK regulation. The regulation of GRK2 turnover has been studied [14,15,16,17,18]. Mdm2 plays a key role in regulation of GRK2 ubiquitination and degradation [18]. Hsp90 interacts with and stabilizes GRK2 [19]. However, little is known about regulation of other GRK subtypes. Damaged DNA-binding protein 1 (DDB1) is part of an E3 ligase complex that includes the cullin proteins CUL4A and CUL4B [20]. The DDB1–CUL4 complex is a conserved cullin-RING ubiquitin ligase, that regulates DNA repair [21,22,23,24], repli- cation [25,26,27], and transcription [28]. The DDB1–CUL4 complex can also be subverted by pathogenic viruses to benefit viral infection [29]. CUL4 assembles ubiquitin ligase by binding to ROC1, a RING protein, and to DDB1, a triple b propeller adapter protein, which functions as a linker to recruit substrates or substrate receptors to CUL4 E3 ubiquitin ligase [30]. In an effort to identify proteins that interact with GRK5, we used a proteomic approach to isolate GRK5 interacting proteins and identified several proteins in the GRK5 immunocomplex including DDB1. We further demonstrated that DDB1 acts as an adapter to link GRK5 to CUL4-ROC1 E3 ligase complex and regulates GRK5 ubiquitination and degradation. PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e43997