Role of DDB1 in GRK5 Degradation Figure 4. DDB1 regulates GRK5 ubiquitination. (A) 293 T cells were co-transfected with GRK5-Flag, HA-Ub, DDB1, and Myc-CUL4A in various combinations as indicated. Cell lysate was immunoprecipitated with M2 affinity gel. The GRK5-ubiquitin complex was detected with anti-HA antibody after immunoblotting. (B) 293 T cells were co-transfected with GRK5-Flag, HA-Ub, control RNAi, DDB1RNAi, CUL4A RNAi, and CUL4B RNAi in various combinations as indicated. Cells were pretreated with 10 mM MG132 for 4 h, and GRK5-ubiquitin complex was examined as in A. doi:10.1371/journal.pone.0043997.g004 [26,33,34], p27 [40], TSC2 [41], Merlin [42], Chk1 [43], c-Jun [28], and histones [21,44], through DDB1 bridged interaction of substrates with CUL4 E3 ubiquitin ligase. p21 and Cdt1 are the most established substrates of DDB1-CUL4 ubiquitin ligase complex after UV irradiation. The degradation of p21 and Cdt1 following UV irradiation, which promotes DNA repair and suppresses replication licensing, is critical for the cell to respond to DNA damage. Interestingly, in this study, we found that GRK5, but not GRK2, is degraded after UV irradiation, and DDB1 mediates UV irradiation-induced GRK5 degradation. The identification of GRK5 as a DDB1-CUL4 complex target following UV irradiation adds it to the growing list of cellular substrates targeted to CUL4 by DDB1. The physiological consequence of UV irradiation-induced GRK5 degradation remains to be explored. The current study is aimed to explore the possible regulation of GRK5 by the DDB1-CUL4 ubiquitin ligase complex. However, the association of GRK5 with DDB1-CUL4 complex may also result in the phosphorylation of DDB1 and/or Cul4 by GRK5, and thus regulates the function of DDB1-CUL4 ubiquitin ligase complex itself. In fact, cells deficient of GRK5 or DDB1 exhibit similar phenotypes, such as apoptosis and cell growth retardation [8,45,46]. It would be interesting to determine whether GRK5 regulates the function of DDB1-CUL4 ubiquitin ligase complex and the ubiquitination of other substrates of DDB1-CUL4 complex, and investigate whether DDB1-CUL4 complex is involved in GRK5 deficiency-induced cell growth retardation in future studies. We also identified other new interacting proteins of GRK5, including SRRM2 and STK38, by immunoprecipitation-mass spectrometry in MDA-MB-231 cells. SRRM2, which has been shown to be involved in pre-mRNA splicing [47,48], is the most abundant among proteins identified by mass spectrometry in GRK5-immunocomplex in both MDA-MB-231 cells and HU- VEC cells (supplemental Table S1 and S2). These results implicate possible function of GRK5 in mRNA splicing. Another interesting interacting protein of GRK5 is STK38 which is identified in GRK5 immunocomplex in cancer cell lines (MDA-MB-231 cells), but not in normal cells (HUVEC). STK38, also known as NDR1, is a serine-threonine protein kinase of AGC kinase subfamily, it has been shown to regulate many essential processes including cell division [49], proliferation [49], centrosome duplication [50] and apoptosis [51]. Interestingly, GRK5 has also been shown to be involved in proliferation and apoptosis [8,46,52]. It will be of interest to examine the possible role of interaction between GRK5 and STK38 in regulating these processes. Materials and Methods Reagents and Antibodies Dulbecco’s modified Eagle’s and Leibovitz’s L-15 media were purchased from Invitrogen. Fetal bovine serum (FBS) was obtained from Hyclone. Mouse anti-DDB1 antibody, rabbit anti-ROC1 antibody, and mouse antibody against Myc epitope PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e43997