Protein serine/threonine phosphatase-1 dephosphorylates p53 at Ser-15 and Ser-37 to modulate its transcriptional and apoptotic activities

Abstract

We have previously demonstrated that the serine/threonine protein phosphatase-1 (PP-1) plays an important role in promoting cell survival. However, the molecular mechanisms by which PP-1 promotes survival remain largely unknown. In the present study, we provide evidence 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 phosphorylation and dephosphorylation. While the kinases responsible 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 dephosphorylation or phosphorylation of p53 at these sites attenuate or enhance its transcriptional activity, respectively. 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 are either largely abrogated or enhanced. Thus, our results demonstrate that PP-1 directly dephosphorylates p53, and dephosphorylation of p53 has as important impact on its functions as phosphorylation does. In addition, our results reveal that one of the molecular mechanisms by which PP-1 promotes cell survival is to dephosphorylate p53, and thus negatively regulate p53-dependent death pathway.

Dual-specificity phosphatase 26 is a novel p53 phosphatase and inhibits p53 tumor suppressor functions in human neuroblastoma

“…Many serine residues within the N-and C-terminal regions of p53 are phosphorylated in response to stress conditions such as hypoxia and DNA damage (Kruse and Gu, 2009). Both Ser20 and Ser37 phosphorylation on the p53 protein has been shown to have an important role in p53 stability and downstream apoptotic function (Chehnab et al, 1999;Unger et al, 1999;Li et al, 2006;Amano et al, 2009).…”

Protein Phosphatase-1 Modulates the Function of Pax-6, a Transcription Factor Controlling Brain and Eye Development

“…To prepare active p38 kinase and parallel control, 10 g of mouse anti-p38 (pT180/pY182) or 10 g of mouse anti-␤-actin antibody (control) was added into 1 mg of HLE total proteins, and after vortexing for 10 s the mixture was incubated on ice for 1 h. Then 100 l of protein A/G plusagarose beads were added into the protein solution, and the protein solution was subjected to rotation at 4°C for overnight. The pellet was collected by centrifugation and washed four times with washing buffer as previously described (11). After washing, the pellet was left in 50 l of 1ϫ kinase buffer (50 mM Tris, pH 7.5, 10 mM MgCl 2 , 1 mM EGTA, 2 mM dithiothreitol, 0.01% Brij 35, 0.02% bovine serum albumin) and used for dephosphorylation substrate.…”

“…In Vivo Dephosphorylation Assays-The in vivo dephosphorylation assays were conducted as described before (11,41). Briefly, HLECs transfected with pCI-neo-hPax-6 were labeled with [ 32 P]orthophosphate (200 Ci/ml) in phosphatase-free Dulbecco's modified Eagle's minimal essential medium.…”

“…In Vitro Dephosphorylation Assays-The in vitro dephosphorylation assays were conducted as previously described (11).…”

“…Part of the mechanism by which PP-1 promotes survival is derived from its negative regulation of p53, a master regulator of apoptosis. PP-1 can directly dephosphorylate p53 at Ser-15 and Ser-37 to attenuate its transcriptional and apoptotic activities (11). Second, PP-1 regulates gene expression through modulation of various transcription factors.…”