FIG. 3.

In vivo phosphorylation of p21^Cip1. (A) Detection of phosphorylated p21^Cip1 by immunoblot analysis of HUVEC extracts using a phospho-specific antibody against the Akt phosphorylation consensus motif. Right lane, effect of Ly294002 (10 μM) for 1 h before lysis. The lower panel shows total endogenous p21^Cip1 for comparison. (B) In vivo phosphorylation of p21^Cip1 by a serum-induced, PI3K-sensitive mechanism. HUVEC were labeled with ³²P and starved for 1 h in FCS-free medium before the addition of 10% phosphate-free FCS and Ly294002 (10 μM) for 30 min as indicated. Endogenous p21^Cip1 was immunoprecipitated (IP) with anti-p21^Cip1 antibodies. (C). In vivo phosphorylation of p21^Cip1 by Akt. COS-7 cells overexpressing myc-tagged p21^Cip1 constructs and vector (pcDNA3.1) or Akt constructs were labeled with ³²P for 3 h, and p21^Cip1 was immunoprecipitated with anti-myc antibodies. In panels B and C, representative autoradiographs are shown: lower panels, expression of p21^Cip1 as a loading control. (D) Phospho-peptide analysis. (Top) Elution of the synthetic peptide QTSMTDFYHSK (where T is the phospho-acceptor amino acid) corresponding to amino acids 144 to 154 of p21 from the reverse phase column. This peptide contains the putative Akt phosphorylation site (¹⁴⁵Thr) in p21 and would result from tryptic digestion of p21. (Middle) Endogenous p21 was immunoprecipitated from COS cells, and tryptic p21 peptides were separated by reverse-phase chromatography. Note the peak corresponding to the peptide QTSMTDFYHSK. (Bottom) Following serum starvation, COS cells were serum treated in the presence of ³²P with or without Ly294002 (10 μM). Tryptic p21 peptides were seperated as above, and fractions surrounding peptide QTSMTDFYHSK were collected for determination of radioactivity. A representative result is shown. WB, Western blotting.