Figure 4.

CaMKII phosphorylates Stat1 S727 in vitro. (A Upper) Kinase assays were performed with purified rat brain CaMKIIα/β and a CaMKIIγ/δ-containing fraction purified from U3A nuclear extracts by using GST-Stat1 TAD affinity columns (EPGSTS1C) or eluates from a GST column (EPGST) as control. The incorporation of ³²P in the CaMKII substrate, autocamtide-3, was measured by a scintillation counter. (Lower) Western blot analyses of 10 μl of eluates from a GST column (lane 1), from a GST-Stat1 TAD affinity column (lane 2), and 250 ng of pCaMKIIα/β (lane 3). S1C, Stat1 TAD; pCaMKII, purified CaMKIIα/β, Ca, calcium; CaM, calmodulin; EP, eluted protein. (B) GST-fusion proteins containing WT or S727A mutant Stat1 TAD were used as substrates for the indicated CaMKIIs. Approximately 250 ng of CaMKIIα/β, 200 ng of CaMKIIγ/δ, and 2.5 μg of various GST-fusion proteins were used in the kinase assays. The incorporation of ³²P in the Stat1 TAD was visualized by autoradiography after SDS/PAGE. SA, S727A. (C) Purified histone H3 (1 μg) was used as a substrate for the indicated CaMKIIs, and the incorporation of ³²P was visualized by autoradiography after SDS/PAGE. pCK, purified CaMKIIα/β.