ORIGINAL ARTICLE Protein serine/threonine phosphatase-1 dephosphorylates p53 at Ser-15 and Ser-37 to modulate its transcriptional and apoptotic activities DW-CLi 1,2 ,J-PLiu 1 ,PCSchmid 1 ,RSchlosser 1 ,HFeng 2 ,W-BLiu 2 ,QYan 2 ,LGong 2 ,S-MSun 2 , M Deng 2 andYLiu 2 1 The Hormel Institute, University of Minnesota, Austin, MN, USA; and 2 College of Life Sciences, Hunan Normal University, Changsha, Hunan, China We have previously demonstrated that the serine/threo- nineproteinphosphatase-1(PP-1)playsanimportantrole in promoting cell survival. However, the molecular mechanisms by which PP-1 promotes survival remain largelyunknown.Inthepresentstudy,weprovideevidence to show that PP-1 can directly dephosphorylate a master regulator of apoptosis, p53, to negatively modulate its transcriptional and apoptotic activities, and thus to promote cell survival. As a transcriptional factor, the function of p53 can be greatly regulated by phosphoryla- tionanddephosphorylation.Whilethekinasesresponsible for phosphorylation of the 17 serine/threonine sites have been identified, the dephosphorylation of these sites remains largely unknown. In the present study, we demonstrate that PP-1 can dephosphorylate p53 at Ser- 15 and Ser-37 through co-immunoprecipitation, in vitro and in vivo dephosphorylation assays, overexpression and silence of the gene encoding the catalytic subunit for PP- 1. We further show that mutations mimicking constitutive dephosphorylationorphosphorylationofp53atthesesites attenuate or enhance its transcriptional activity, respec- tively. As a result of the changed p53 activity, expression of the downstream apoptosis-related genes such as bcl-2 and bax is accordingly altered and the apoptotic events areeitherlargelyabrogatedorenhanced.Thus,ourresults demonstratethatPP-1directlydephosphorylatesp53,and dephosphorylation of p53 has as important impact on its functions as phosphorylation does. In addition, our results revealthatoneofthemolecularmechanismsbywhichPP- 1 promotes cell survival is to dephosphorylate p53, and thus negatively regulate p53-dependent death pathway. Oncogene (2006) 25, 3006–3022.doi:10.1038/sj.onc.1209334; published online 27 February 2006 Keywords: p53; PP-1; dephosphorylation; apoptosis; RNAi; phosphorylation and signal transduction Introduction The reversible phosphorylation and dephosphorylation at the serine and threonine residues on proteins play important roles in regulating gene expression (Hunter andKarin,1992),cellcycleprogression(Pawson,1995), andapoptosis(GjertsenandDoskeland,1995;Franklin and McCubrey, 2000; Klumpp and Krieglstein, 2002). In eukaryotes, dephosphorylation at the serine/ threoninesitesislargelyexecutedbyfourmajorprotein phosphatases: phosphatase-1 (PP-1), phosphatase-2A (PP-2A), phosphatase-2B (PP-2B) and phosphatase-2C (PP-2C) (Cohen, 1989; Mumby and Walter, 1993), although other protein phosphatases, including phos- phatase-4 (PP-4), phosphatase-5 (PP-5), phosphatase-6 (PP-6)andphosphatase-7(PP-7),alsocontributetothis process (Brewis et al., 1993; Bastians and Ponstingl, 1994; Chen et al., 1994; Chinkers, 1994; Huang and Honkananen, 1998). The majority of intracellular protein phosphatase activity has been attributed to PP-1 and PP-2A (Cohen, 1989). Both phosphatases are important in promoting survival because inhibition of the PP-1 and PP-2A activities by either okadaic acid or calyculin A leads to apoptosis of many different typesofcells(GjertsenandDoskeland,1995).However, the molecular mechanisms by which PP-1 and PP-2A promote survival remain largely unknown. Here, we present evidence to show that PP-1 can directly depho- sphorylate p53 at two important phosphorylation sites to negatively regulate its transcriptional activity and apoptotic ability, and thus promote cell survival. The tumor suppressor p53 is a master regulator of apoptosisinmanytypesofcells(ShenandWhite,2001; BargonettiandManfredi,2002;VousdenandLu,2002; Manfredi, 2003; Oren, 2003). Loss of functional p53 is sufficienttoinactivatetheapoptoticmachineryinmany typesoftumorcellsaswellasnontumorcells(Shenand White, 2001). As a master regulator of apoptosis in different cells, p53 regulates apoptosis by two mechan- isms. First, it acts as a transcriptional factor to regu- late expression of many genes involved in apoptosis (Miyashita et al., 1994; Buckbinder et al., 1995; Miyashita and Reed, 1995; Wen-Schaub et al., 1995; Israeli et al., 1997; Polyak et al., 1997; Wu et al., 1997; El-Deiry, 1998). Second, it can activate Bax in mitochondria to antagonize the antiapoptotic ability of Received 23 June 2005; revised 11 November 2005; accepted 14 November 2005; published online 27 February 2006 Correspondence: Dr DW-C Li, The Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912, USA. E-mail: dwli1688@hotmail.com Oncogene (2006) 25, 3006–3022 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc