Serine 123 phosphorylation modulates p21 protein stability and activity by suppressing ubiquitin-independent proteasomal degradation

Abstract

The cyclin-dependent kinase inhibitor p21(Waf1/Cip1) is a major regulator of the cell cycle and plays an important role in many cellular processes, including differentiation, stress response, apoptosis, and tumorigenesis. We previously cloned the gene encoding dog p21 and found that unlike its human ortholog, dog p21 is expressed as two isoforms, one high molecular mass band of 23 kDa and one low molecular mass band of 19 kDa. In the current study, we found that the high molecular mass band is phosphorylated, whereas the low molecular mass band is hypophosphorylated. Moreover, by generating multiple mutants of dog p21, we found that serine 123 and proline 124, which form a consensus site for proline-directed phosphorylation, are required for expression of the high molecular mass p21 isoform through phosphorylation at serine 123. Most importantly, we showed that serine 123 phosphorylation inhibits ubiquitin-independent proteasomal degradation of p21 protein and subsequently, prolongs p21 protein half-life and enhances the ability of p21 to suppress cell proliferation. Taken together, these data reveal that serine 123 phosphorylation modulates p21 protein stability and activity by suppressing ubiquitin-independent proteasomal degradation.

FIGURE 1.

Phosphorylation is responsible for expression of two p21 isoforms in dog. A, dog p21 is expressed as two isoforms. MCF7, MDCK, and D17 cells were mock-treated or treated with camptothecin or doxorubincin for 12 h, and the level of p21, actin, and GAPDH was determined by Western blot analysis. B, the high molecular mass dog p21 band is diminished upon λ phosphatase treatment. MDCK cells were mock-treated or treated with camptothecin or doxorubicin for 12 h. The cell lysates were then treated with or without λ phosphatase (300 units) for 30 min, followed by Western blot analysis to determine the level of p21, actin, and GAPDH.

FIGURE 2.

Serine 123 phosphorylation is required for expression of the phosphorylated p21 isoform. A, sequence similarity between human and dog p21. Shaded areas indicate the regions for replacing dog p21 with that of human p21 and vice versa. B, schematic representation of various dog and human p21 mutants. C, region, from amino acid 117 to 126, in dog p21, required for expression of two dog p21 isoforms. Three micrograms of pcDNA3 vectors that express HA-tagged wild-type dog p21, dog p21(Hu 38–74), dog p21(Hu 74–83), dog p21(Hu 84–93), dog p21(Hu 107–116), dog p21(Hu 117–126), and dog p21(Hu 127–136) was transfected into Cf2Th cells for 24 h followed by Western blot analysis to determine the levels of p21 proteins, actin, and GAPDH. D, 3 μg of pCDNA3 vectors that express HA-tagged wild-type dog p21, wild-type human p21, human p21(Dog 117–136), human p21(Dog 117–126), human p21(R122H), and human p21(G124P) transfected into 293T cells for 24 h, followed by Western blot analysis to determine the levels of p21 proteins, actin, and GAPDH. E, Cf2Th cells transiently transfected with pcDNA3 vectors expressing human p21(Dog 117–136), human p21(Dog 117–126), human p21(R122H), and human p21(G124P) for 24 h. The cell lysates were treated with or without λ phosphatase (300 units) for 30 min, followed by Western blot analysis to determine the level of p21 proteins, actin, and GAPDH. F, proline at 124 requirement for expression of two dog p21 isoforms. Three micrograms of pcDNA3 vectors that express HA-tagged wild-type dog p21, dog p21(Hu 117–126), dog p21(L120V), dog p21(H122R), dog p21(P124G), and dog p21(R126Q) was transfected into Cf2Th cells for 24 h followed by Western blot analysis to determine the levels of p21 proteins, actin, and GAPDH. G, serine 123 phosphorylation responsibility for expression of two dog p21 isoforms. Three micrograms of pcDNA3 vectors that express HA-tagged wild-type dog p21, dog p21(Hu 117–126), dog p21(S123A), and dog p21(S123D) was transfected into Cf2Th cells for 24 h followed by Western blot analysis to determine the levels of p21 proteins, actin, and GAPDH.

FIGURE 3.

Serine 123 phosphorylation enhances the ability of dog p21 to block S phase entry and subsequently to suppress cell proliferation. A, Cf2Th cells uninduced or induced to express wild-type p21, dog p21(S123A), and dog p21(S123D) for 24 h. The levels of p21, Rb, PCNA, cyclin A, cyclin B, cdk1, and actin were determined by Western blot analysis. B, DNA histogram analysis of Cf2Th cells in the absence or presence of wild-type dog p21, dog p21(S123A), or dog p21(S123D). Cf2Th cells were uninduced or induced to express wild-type dog p21 (top panel), dog p21(S123D) (middle panel), and dog p21(S123A) (bottom panel) for 72 h, and the percentage of cells in each phase of the cell cycle was determined by DNA histogram analysis. DNA content was quantified using the data from at least three independent experiments. C–E, growth rates of Cf2Th cells in the presence or absence of wild-type dog p21 (C), dog p21(S123D) (D), or dog p21(S123A) (E) over a 9-day period. Error bars, S.E. F, upper panel, colony formation assay performed using Cf2Th cells uninduced or induced to express wild-type dog p21, dog p21(S123D), or dog p21(S123A) for 14 days. Lower panel, relative percentage of colony numbers shown in upper panel. Error bars, S.D.

FIGURE 4.

Phosphorylation of serine 123 increases the stability of dog p21 protein. A, Cf2Th cell expressing wild-type dog p21, dog p21(S123A), or dog p21(S123D) were mock-treated or treated with cycloheximide for various times, followed by Western blot analysis to determine the level of p21 proteins and actin. B and C, left panels, MDCK cells were mock-treated or treated with cycloheximide for various times followed by Western blot analysis to determine the level of p21 proteins and actin. Right panels, upon normalization to actin, the relative half-life of underphosphorylated dog p21 (B) and phosphorylated dog p21 (C) was calculated.

FIGURE 5.

Serine 123 phosphorylation prevents dog p21 degradation through ubiquitin-independent pathway. A, Cf2Th cells expressing wild-type dog p21, dog p21(S123A), and dog p21(S123D) were mock-treated or treated with MG132 for 12 h followed by Western blot analysis to determine the level of dog p21 proteins and actin. B, MDCK cells were mock-treated or treated with MG132 for 12 h followed by Western blot analysis to determine the level of p21 and actin. C–E, ts20 cells were transiently transfected with pcDNA3 vectors expressing wild-type dog p21 (C), dog p21(S123D) (D), and dog p21(S123A) (E) at 35 °C for 24 h and then cultured at 35 °C or 39 °C for 12 h. Cell lysates were collected and subjected to Western blot analysis to determine the level of dog p21 proteins, p53, and GAPDH.