Figure 2.

PKA phosphorylates Ser258. In each panel, the amount of P450c17 is indicated by staining with Coomassie brilliant blue (CBB), and phosphorylation is assessed by autoradiography of incorporated ³²P. A, S258A is not phosphorylated. Wild-type (wt) and S258A human P450c17 (3 μg each) were treated without (−) and with (+) 0.5 U PKA catalytic subunit (one time) or with 1.0 U PKA catalytic subunit (two times) and 20 μm ATP (1μCi γ-³²P-ATP) for 30 min at 30 C. B, S256R increases phosphorylation. S256R and wild-type (wt) P450c17 (5 μg each) were incubated with 0.5U PKA catalytic subunit for 10 min at 30 C. The percentages of ³²P incorporated (picomoles ³²P per picomole P450c17) of each sample are indicated below the figure. C, S256R does not promote phosphorylation of S258A. S256R mutant (1 μg) or S256R/S258A double mutant (2 μg) was treated with 0.5 U PKA catalytic subunit for 30 min at 30 C. D, D257A promotes phosphorylation. Wild-type human P450c17, S256R mutant, and S256R/D257A double mutant (0.75 μg each) were treated with 0.5 U PKA catalytic subunit for 10 min at 30 C. The percentages of ³²P incorporation are indicated below the figure, as in B.