PPM1D regulates p21 expression via dephoshporylation at serine 123

Abstract

The cyclin-dependent kinase inhibitor p21 plays a critical role in regulating cell cycle and cell proliferation. We previously cloned the dog p21 gene and found that unlike human p21, dog p21 is expressed as 2 isoforms due to the proline-directed phosphorylation at serine 123 (S123). Here, we identified that PPM1D, also called Wip1 and a Mg(2+)-dependent phosphatase, dephosphorylates dog p21 protein at serine 123. Specifically, we showed that the level of S123-phosphorylated dog p21 is increased by a PPM1D inhibitor in a dose-dependent manner. We also showed that over-expression of PPM1D decreases, whereas knockdown of PPM1D increases, the level of S123-phosphorylated dog p21 regardless of p53. Additionally, in vitro phosphatase assay was performed and showed that phosphorylated S123 in dog p21 is dephosphorylated by recombinant rPPM1D, which contains the catalytic domain of human PPM1D (residue 1-420), but not by the phosphatase dead rPPM1D (D314A). Furthermore, dephosphorylation of S123 by rPPM1D can be abrogated by PPM1D inhibitor or by withdrawal of Mg(2+). Finally, we showed that upon PPM1D inhibition, the level of S123-phosphorylated dog p21 was increased, concomitantly with decreased expression of cyclin A, cyclin B, Rb, and PCNA. Together, our results indicate that PPM1D functions as a phosphatase of dog p21 at serine 123 and plays a role in cell cycle control via p21.

Keywords: ATM, ataxia telangiectasia mutated; CDKs, cyclin-dependent kinases; Cip1, Cdk interacting protein 1; PCNA, proliferating cell nuclear antigen; PP2C, type 2C protein phosphatases; PPM1D; PPM1D, Protein phosphatase Mg2+/Mn2+ dependent 1D; WAF1, wild-type p53 activated factor 1; Wip1; Wip1, wild-type p53 induced phosphatase 1; p21; p53; phosphatase.

Figure 1.

The level of S123-phosphorylated dog p21 is increased by PPM1D inhibitor independent of p53. (A-B) MDCK (A) and MDCK-p53KD (B) cells were mock-treated or treated with various amounts of PPM1D inhibitor for 12 h, and the level of p21 was determined by Western blot analysis with antibodies against p21 and actin. The relative level of underphosphorylated p21 (p21) and S123-phohosphorylated p21 (p-p21) was measured by densitometry, and the ratio of p-p21 versus p21 is shown below. (C) The experiment was performed as in (A) except that Cf2Th cells were induced to express dog p21 for 12 h prior to the treatment.

Figure 2.

Knockdown of PPM1D increases the level of S123-phosphorylated dog p21. (A) MDCK cells were transiently transfected with a control or PPM1D SiRNA for 60 h, followed by RT-PCR analysis to measure the level of dog PPM1D and actin transcript. (B-C) MDCK cells were treated as described in (A) and the level of actin and total dog p21 (B) or S123-phosphorylated dog p21 (C) was determined by Western blot analysis with actin, total p21, and p-dog p21 antibodies. (D) The experiment was performed as in (A) except MDCK-p53KD cells were used. (E-F) The level of actin, total dog p21 (E), and S123-phosphorylated dog p21 (F) was determined by Western blot analysis using MDCK-p53KD cells treated as in (D). (G) The experiment was performed as in (A) except that Cf2Th cells were induced to express dog p21 for 12 h prior to SiRNA transfection. (H-I) The level of actin, total dog p21 (H), and S123-phosphorylated dog p21 (I) was determined by Western blot analysis using dog p21-expressing Cf2Th cells treated as in (G).

Figure 3.

Over-expression of dog PPM1D decreases S123-phosphorylated dog p21. (A) Cf2Th cells were induced to express dog p21 for 12 h, and then transiently transfected with a control or dog PPM1D expression vector. The level of dog p21, PPM1D, and actin was determined by Western blot analysis. (B) Melanoma-36 cells were transiently transfected with 1 μg of dog p21 expression vector together with various amounts (0–1 μg) of dog PPM1D expression vector. Twenty-four h post transfection, the level of p21, PPM1D, and actin was determined by Western blot analysis. (C) Cf2Th cells were induced to express dog p21 for 12 h, and then transiently transfected with a control or human rPPM1D expression vector. The level of p-p21, rPPM1D, and actin was determined by Western blot analysis. (D) Melanoma-36 cells were transiently transfected with 1 μg of dog p21 expression vector together with a control or rPPM1D expression vector. Twenty-four h post transfection, the level of p-p21, PPM1D, and actin was determined by Western blot analysis. (E) The experiment was performed as in (B) except that dog p21(S137A) expression vector was used.

Figure 4.

PPM1D dephosphorylates dog p21 at serine 123. (A) Cf2Th cells were induced to express dog p21 for 24 h, followed by immunoprecipatation with p21 antibody. The immunocomplexes were then incubated with recombinant GST or GST-tagged rPPM1D or rPPM1D (D314A) in the presence or absence of Mg²⁺ or PPM1D inhibitor for 4 h at 30°C, followed by Western blot analysis. (B) MDCK cells were treated with various amounts of PPM1D inhibitor for 24 h and the level of p-p21, cyclin A, cyclin B, Rb, and PCNA was determined by Western blot analysis.