Figure 5. Kinase activity of P-TEFb and P-TEFb-Tat complexes with AFF4.
(A) Autoradiogram showing phosphorylation of GST-CTD (500 ng) by P-TEFb and P-TEFb-Tat with and without excess (0.28 μM) AFF42-73 in the presence of low (50 μM) ATP. (B) Phosphorylation of 500 ng GST-CTD by P-TEFb and P-TEFb-Tat with and without excess (0.28 μM) AFF42–73 in the presence of saturating (500 μM) ATP. AFF4 reduces the activity of P-TEFb twofold and has little influence on the kinase activity of Tat-P-TEFb. Tat-P-TEFb is, however, sevenfold to tenfold more active than P-TEFb. Lane 3 in panels (A and B) is a control without GST-CTD. (C) Quantitation of the radioactive GST-CTD in (A and B).
Figure 5—figure supplement 1. SDS polyacrylamide gel of P-TEFb and P-TEFb-Tat at the same ratio as they were used in the kinase assay.
Figure 5—figure supplement 2. Western blots of kinase reaction products from panel B. Phosphorylated CTD was detected with anti-phoshoSer2 and anti-phoshoSer5 antibodies.
The GST-CTD was phosphorylated on Ser2 and Ser5. However, the Ser2 phosphorylation was detected only on the full-length CTD, while Ser5 phosphorylation was detected disproportionately on proteolytic fragments of the CTD compared to the full-length CTD domain.