FIGURE 4.

Identification of MPK38 phosphorylation sites on p53. A, in vitro phosphorylation of p53 at Ser¹⁵ by MPK38. Recombinant p53 proteins (∼3 μg) were mixed with 10 μm ATP, 5 μCi of [γ-³²P]ATP, and 10 mm MgCl₂ in 20 μl of kinase buffer and incubated with precipitated MPK38 from HEK293 cell extracts containing GST-MPK38 for 15 min at 37 °C with frequent gentle mixing. Expression levels of MPK38 and p53 were monitored by immunoblotting with an anti-GST antibody (left panel). Alignment of the MPK38/AMPK consensus motif with putative MPK38 phosphorylation sites on p53 is indicated (right, upper panel). Approximately 3 μg of recombinant p53 or one of its substitution mutants (S15A, T118A, and S260A) were mixed with 10 μm ATP, 5 μCi of [γ-³²P]ATP, and 10 mm MgCl₂ in 20 μl of kinase buffer and incubated with recombinant wild-type MPK38 (∼4 μg) for 15 min at 37 °C (right, lower). Ser¹⁵ phosphorylation of p53 and expression of recombinant p53 and MPK38 were determined by immunoblotting with anti-phospho-p53(S15), anti-GST, and anti-His antibodies, respectively (right lower, 2nd to bottom panels). B, in vivo phosphorylation of p53 at Ser¹⁵ by MPK38. The endogenous level of p53 phosphorylation at Ser¹⁵ in the presence or absence of 5-fluorouracil (or doxorubicin (Dox)) was also analyzed by immunoblotting with an anti-phospho-p53(S15) antibody. The relative level of p53 phosphorylation at Ser¹⁵ was quantitated by densitometric analysis, and the fold increase relative to untreated HEK293 cells was calculated. P-p53 and P-p53(S15) indicate the phosphorylated p53 and p53 at Ser¹⁵, respectively. Φ, hydrophobic residue; β, basic residue; IP, immunoprecipitation; WB, Western blot.