cells. The peptides of proteins obtained in GRK5 immunocom- plex in HUVEC cells by mass spectrometry were recorded. (XLSX) Acknowledgments We thank Junjun Wang and Wenqing Yao for technical assistance. References 1. Kohout TA, Lefkowitz RJ (2003) Regulation of G protein-coupled receptor kinases and arrestins during receptor desensitization. Mol Pharmacol 63: 9–18. 2. Lefkowitz RJ (2007) Seven transmembrane receptors: something old, something new. Acta Physiol (Oxf) 190: 9–19. 3. Pronin AN, Morris AJ, Surguchov A, Benovic JL (2000) Synucleins are a novel class of substrates for G protein-coupled receptor kinases. J Biol Chem 275: 26515–26522. 4. Miller WE, Houtz DA, Nelson CD, Kolattukudy PE, Lefkowitz RJ (2003) G- protein-coupled receptor (GPCR) kinase phosphorylation and beta-arrestin recruitment regulate the constitutive signaling activity of the human cytomeg- alovirus US28 GPCR. J Biol Chem 278: 21663–21671. 5. Parameswaran N, Pao CS, Leonhard KS, Kang DS, Kratz M, et al. (2006) Arrestin-2 and G protein-coupled receptor kinase 5 interact with NFkappaB1 p105 and negatively regulate lipopolysaccharide-stimulated ERK1/2 activation in macrophages. J Biol Chem 281: 34159–34170. 6. Cant SH, Pitcher JA (2005) G protein-coupled receptor kinase 2-mediated phosphorylation of ezrin is required for G protein-coupled receptor-dependent reorganization of the actin cytoskeleton. Mol Biol Cell 16: 3088–3099. 7. Barthet G, Carrat G, Cassier E, Barker B, Gaven F, et al. (2009) Beta-arrestin1 phosphorylation by GRK5 regulates G protein-independent 5-HT4 receptor signalling. EMBO J 28: 2706–2718. 8. Chen X, Zhu H, Yuan M, Fu J, Zhou Y, et al. (2010) G-protein-coupled receptor kinase 5 phosphorylates p53 and inhibits DNA damage-induced apoptosis. J Biol Chem 285: 12823–12830. 9. Carman CV, Parent JL, Day PW, Pronin AN, Sternweis PM, et al. (1999) Selective regulation of Galpha(q/11) by an RGS domain in the G protein- coupled receptor kinase, GRK2. J Biol Chem 274: 34483–34492. 10. Sorriento D, Ciccarelli M, Santulli G, Campanile A, Altobelli GG, et al. (2008) The G-protein-coupled receptor kinase 5 inhibits NFkappaB transcriptional activity by inducing nuclear accumulation of IkappaB alpha. Proc Natl Acad Sci U S A 105: 17818–17823. 11. Jiang X, Yang P, Ma L (2009) Kinase activity-independent regulation of cyclin pathway by GRK2 is essential for zebrafish early development. Proc Natl Acad Sci U S A 106: 10183–10188. 12. Chen Y, Wang F, Long H, Wu Z, Ma L (2011) GRK5 promotes F-actin bundling and targets bundles to membrane structures to control neuronal morphogenesis. J Cell Biol 194: 905–920. 13. Premont RT, Gainetdinov RR (2007) Physiological roles of G protein-coupled receptor kinases and arrestins. Annu Rev Physiol 69: 511–534. 14. Penela P, Ruiz-Gomez A, Castano JG, Mayor F, Jr. (1998) Degradation of the G protein-coupled receptor kinase 2 by the proteasome pathway. J Biol Chem 273: 35238–35244. 15. Penela P, Elorza A, Sarnago S, Mayor F, Jr. (2001) Beta-arrestin- and c-Src- dependent degradation of G-protein-coupled receptor kinase 2. EMBO J 20: 5129–5138. 16. Elorza A, Penela P, Sarnago S, Mayor F, Jr. (2003) MAPK-dependent degradation of G protein-coupled receptor kinase 2. J Biol Chem 278: 29164– 29173. 17. Nogues L, Salcedo A, Mayor F, Jr., Penela P (2011) Multiple scaffolding functions of {beta}-arrestins in the degradation of G protein-coupled receptor kinase 2. J Biol Chem 286: 1165–1173. 18. Salcedo A, Mayor F, Jr., Penela P (2006) Mdm2 is involved in the ubiquitination and degradation of G-protein-coupled receptor kinase 2. EMBO J 25: 4752– 4762. 19. Luo J, Benovic JL (2003) G protein-coupled receptor kinase interaction with Hsp90 mediates kinase maturation. J Biol Chem 278: 50908–50914. 20. Jackson S, Xiong Y (2009) CRL4s: the CUL4-RING E3 ubiquitin ligases. Trends Biochem Sci 34: 562–570. 21. Wang H, Zhai L, Xu J, Joo HY, Jackson S, et al. (2006) Histone H3 and H4 ubiquitylation by the CUL4-DDB-ROC1 ubiquitin ligase facilitates cellular response to DNA damage. Mol Cell 22: 383–394. 22. Groisman R, Polanowska J, Kuraoka I, Sawada J, Saijo M, et al. (2003) The ubiquitin ligase activity in the DDB2 and CSA complexes is differentially regulated by the COP9 signalosome in response to DNA damage. Cell 113: 357–367. 23. Chen X, Zhang Y, Douglas L, Zhou P (2001) UV-damaged DNA-binding proteins are targets of CUL-4A-mediated ubiquitination and degradation. J Biol Chem 276: 48175–48182. 24. Sugasawa K, Okuda Y, Saijo M, Nishi R, Matsuda N, et al. (2005) UV-induced ubiquitylation of XPC protein mediated by UV-DDB-ubiquitin ligase complex. Cell 121: 387–400. Role of DDB1 in GRK5 Degradation Author Contributions Conceived and designed the experiments: YC LM. Performed the experiments: ZW YC TY QG MY. Analyzed the data: ZW YC TY LM. Contributed reagents/materials/analysis tools: QG MY. Wrote the paper: YC ZW TY. 25. Higa LA, Mihaylov IS, Banks DP, Zheng J, Zhang H (2003) Radiation-mediated proteolysis of CDT1 by CUL4-ROC1 and CSN complexes constitutes a new checkpoint. Nat Cell Biol 5: 1008–1015. 26. Hu J, McCall CM, Ohta T, Xiong Y (2004) Targeted ubiquitination of CDT1 by the DDB1-CUL4A-ROC1 ligase in response to DNA damage. Nat Cell Biol 6: 1003–1009. 27. Bondar T, Ponomarev A, Raychaudhuri P (2004) Ddb1 is required for the proteolysis of the Schizosaccharomyces pombe replication inhibitor Spd1 during S phase and after DNA damage. J Biol Chem 279: 9937–9943. 28. Wertz IE, O’Rourke KM, Zhang Z, Dornan D, Arnott D, et al. (2004) Human De-etiolated-1 regulates c-Jun by assembling a CUL4A ubiquitin ligase. Science 303: 1371–1374. 29. Horvath CM (2004) Weapons of STAT destruction. Interferon evasion by paramyxovirus V protein. Eur J Biochem 271: 4621–4628. 30. Angers S, Li T, Yi X, MacCoss MJ, Moon RT, et al. (2006) Molecular architecture and assembly of the DDB1-CUL4A ubiquitin ligase machinery. Nature 443: 590–593. 31. Abbas T, Sivaprasad U, Terai K, Amador V, Pagano M, et al. (2008) PCNA- dependent regulation of p21 ubiquitylation and degradation via the CRL4Cdt2 ubiquitin ligase complex. Genes Dev 22: 2496–2506. 32. Nishitani H, Shiomi Y, Iida H, Michishita M, Takami T, et al. (2008) CDK inhibitor p21 is degraded by a proliferating cell nuclear antigen-coupled Cul4- DDB1Cdt2 pathway during S phase and after UV irradiation. J Biol Chem 283: 29045–29052. 33. Higa LA, Banks D, Wu M, Kobayashi R, Sun H, et al. (2006) L2DTL/CDT2 interacts with the CUL4/DDB1 complex and PCNA and regulates CDT1 proteolysis in response to DNA damage. Cell Cycle 5: 1675–1680. 34. Higa LA, Wu M, Ye T, Kobayashi R, Sun H, et al. (2006) CUL4-DDB1 ubiquitin ligase interacts with multiple WD40-repeat proteins and regulates histone methylation. Nat Cell Biol 8: 1277–1283. 35. Greene TA, Alarcon S, Thomas A, Berdougo E, Doranz BJ, et al. (2011) Probenecid inhibits the human bitter taste receptor TAS2R16 and suppresses bitter perception of salicin. PLoS One 6: e20123. 36. Mundell SJ, Benovic JL (2000) Selective regulation of endogenous G protein- coupled receptors by arrestins in HEK293 cells. J Biol Chem 275: 12900–12908. 37. Loudon RP, Perussia B, Benovic JL (1996) Differentially regulated expression of the G-protein-coupled receptor kinases, betaARK and GRK6, during myelomonocytic cell development in vitro. Blood 88: 4547–4557. 38. Salim S, Eikenburg DC (2007) Role of 90-kDa heat shock protein (Hsp 90) and protein degradation in regulating neuronal levels of G protein-coupled receptor kinase 3. J Pharmacol Exp Ther 320: 1106–1112. 39. Pratt WB, Toft DO (2003) Regulation of signaling protein function and trafficking by the hsp90/hsp70-based chaperone machinery. Exp Biol Med (Maywood) 228: 111–133. 40. Bondar T, Kalinina A, Khair L, Kopanja D, Nag A, et al. (2006) Cul4A and DDB1 associate with Skp2 to target p27Kip1 for proteolysis involving the COP9 signalosome. Mol Cell Biol 26: 2531–2539. 41. Hu J, Zacharek S, He YJ, Lee H, Shumway S, et al. (2008) WD40 protein FBW5 promotes ubiquitination of tumor suppressor TSC2 by DDB1-CUL4-ROC1 ligase. Genes Dev 22: 866–871. 42. Huang J, Chen J (2008) VprBP targets Merlin to the Roc1-Cul4A-DDB1 E3 ligase complex for degradation. Oncogene 27: 4056–4064. 43. Leung-Pineda V, Huh J, Piwnica-Worms H (2009) DDB1 targets Chk1 to the Cul4 E3 ligase complex in normal cycling cells and in cells experiencing replication stress. Cancer Res 69: 2630–2637. 44. Kapetanaki MG, Guerrero-Santoro J, Bisi DC, Hsieh CL, Rapic-Otrin V, et al. (2006) The DDB1-CUL4ADDB2 ubiquitin ligase is deficient in xeroderma pigmentosum group E and targets histone H2A at UV-damaged DNA sites. Proc Natl Acad Sci U S A 103: 2588–2593. 45. Cang Y, Zhang J, Nicholas SA, Bastien J, Li B, et al. (2006) Deletion of DDB1 in mouse brain and lens leads to p53-dependent elimination of proliferating cells. Cell 127: 929–940. 46. Kim JI, Chakraborty P, Wang Z, Daaka Y (2012) G-protein coupled receptor kinase 5 regulates prostate tumor growth. J Urol 187: 322–329. 47. Blencowe BJ, Bauren G, Eldridge AG, Issner R, Nickerson JA, et al. (2000) The SRm160/300 splicing coactivator subunits. RNA 6: 111–120. 48. Sawada Y, Miura Y, Umeki K, Tamaoki T, Fujinaga K, et al. (2000) Cloning and characterization of a novel RNA-binding protein SRL300 with RS domains. Biochim Biophys Acta 1492: 191–195. 49. Hergovich A, Stegert MR, Schmitz D, Hemmings BA (2006) NDR kinases regulate essential cell processes from yeast to humans. Nat Rev Mol Cell Biol 7: 253–264. PLOS ONE | www.plosone.org 10 August 2012 | Volume 7 | Issue 8 | e43997