GA 12/13 Basally Regulates p53 through Mdm4 Expression Mi-Sung Kim, 1 Sang Min Lee, 1 Won Dong Kim, 1 Sung Hwan Ki, 1 Aree Moon, 2 Chang Ho Lee, 3 and Sang Geon Kim 1 1 CollegeofPharmacyandResearchInstituteofPharmaceuticalSciences,SeoulNationalUniversity; 2 CollegeofPharmacy,DuksungWomen’sUniversity;and 3 DepartmentofPharmacologyand InstituteofBiomedicalScience,CollegeofMedicine,HanyangUniversity,Seoul,Korea Abstract GA 12/13 , which belongs to the GA 12 family, participates in the regulation of diverse physiologic processes. In view of the control of GA 12/13 in cell proliferation, this study investigated the role of GA 12/13 in the regulation of p53 and mdm4. Immunoblotting and immunocytochemistry revealed that p53 was expressed in control embryonic fibroblasts and was largely localized in the nuclei. GA 12 deficiency decreased p53 levels and its DNA binding activity, accompanying p21 repression with Bcl 2 induction, whereas GA 13 deficiency exerted weak effects. GA 12 or GA 13 deficiency did not change p53 mRNA expression. ERK1/2 or Akt was not responsible for p53 repression due to GA 12 deficiency. Mdm4, a p53-stabilizing protein, was repressed by GA 12 deficiency and to a lesser extent by GA 13 deficiency, whereas mdm2, PTEN, B-catenin, ATM, and Chk2 were unaffected. p53 accumulation by proteasomal inhibition during GA 12 deficiency suggested the role of GA 12 in p53 stabilization. Constitutively active GA 12 (GA 12 QL) or GA 13 (GA 13 QL) promoted p53 accumulation with mdm4 induction in MCF10A cells. p53 accumulation by mdm4 overexpression, but no mdm4 induction by p53 overexpression, and small interfering RNA knockdown verified the regulatory role of mdm4 for p53 downstream of GA 12/13 . In control or GA 12 /GA 13 -deficient cells, genotoxic stress led to p53 accumulation. At concentrations increasing the flow cytometric pre-G 1 phase, doxorubicin or etoposide treatment caused serine phosphorylations in GA 12 À/À or GA 12/13 À/À cells, but did not induce mdm4. GA 12/13 QL transfection failed to phosphorylate p53 at serines. Our results indicate that GA 12/13 regulate basal p53 levels via mdm4, which constitutes a cell signaling pathway distinct from p53 phosphorylations elicited by genotoxic stress. (Mol Cancer Res 2007;5(5):473 – 84) Introduction Heterotrimeric GTP-binding proteins (G proteins) participate in cell signaling for the regulation of a variety of physiologic processes. The Ga subunits which define G proteins are divided into Ga s ,Ga i/o ,Ga q ,andGa 12 family members. Among the Ga families, Ga 12 members are activated by the stimulation of thrombin, TXA 2 , lysophosphatidic acid, and thyroid-stimulating hormone receptors (1). The Ga 12 family members, which consist of Ga 12 andGa 13 , regulate various intracellular effectors or cellular responses such as platelet aggregation (2), actin-stress fiber formation (3), apoptosis (4), and neurite retraction (5). In spite of the mostly overlapping functions between Ga 12 and Ga 13 , the proteins sometimes exhibit differential abilities to recruit signaling pathways for physiologic effectors (6). Ga 13 gene knockout resulted in impaired angiogenesis and intra- uterine death, whereas mice deficient in the Ga 12 gene were alive (7). G proteins and their associated proteins (e.g., h- catenin), if inappropriately activated or accumulated (8, 9), may carry transforming potential and thus play a role in tumori- genesis. In fact, constitutively active mutants of Ga 12/13 may represent oncogenic properties (10-12). Thus, it is most likely that the G proteins play roles in signaling pathways linked to the biological processes of cell survival. However, the Ga 12/13 signaling pathways for cell proliferation or survival have been inadequately defined. p53 is a well-known tumor suppressor gene that serves as a nuclear transcription factor for the regulation of target gene expression associated with cell proliferation (13). p53 is activated by various toxic stress signals, which becomes important to prevent the replication of damaged cells (e.g., oncogenic genetic lesions). p53 function is important to monitor oncogenic changes in cells or for the prevention of the growth of abnormal or damaged cells (14). Activation of p53 leads to the inhibition of cell growth through cell cycle arrest or activation of apoptosis (15, 16). Numerous studies have been conducted on p53 mutations found in tumors, which involve single amino acid mutations or the elimination of its expression (17). p53 expres- sion is altered by the exposure of cells to extracellular stimuli including hypoxia, etoposide, and g-irradiation (18, 19). p53 activity is also regulated by stabilization mediated with post- translational modifications (i.e., phosphorylation and acetyla- tion; ref. 20). The posttranslational regulatory mechanism of p53 involves mdm4 and mdm2 regulatory proteins. p53 function is positively controlled by mdm4, which does not drive p53 degradation (21). On the contrary, mdm2 targets p53 for nuclear export and proteasomal degradation. Therefore, mdm4 and mdm2 play antagonistic roles in controlling the cellular activity of p53 in the regulatory network. Received 11/30/06; revised 2/5/07; accepted 3/13/07. Grant support: Korea Science and Engineering Foundation grant funded by the Korean government (MOST; no. R01-2005-000-10596-0). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Sang Geon Kim, College of Pharmacy, Seoul National University, Sillim-dong, Kwanak-Gu, Seoul 151-742, Korea. Phone: 82-2880- 7840; Fax: 82-2872-1795. E-mail: sgk@snu.ac.kr Copyright D 2007 American Association for Cancer Research. doi:10.1158/1541-7786.MCR-06-0395 Mol Cancer Res 2007;5(5). May 2007 473 Downloaded from http://aacrjournals.org/mcr/article-pdf/5/5/473/3140805/473.pdf by guest on 11 June 2022