FIGURE 3.

Protein kinase A activity in Sf9 membranes allows regulatory phosphorylation in Ccc2 and its mutants to be detected. A, membranes from Sf9 cells expressing D627A, Ccc2 wt, S258A, or transfected with an empty bacmid (MOCK) were electrophoretically separated and subsequently immunodetected with the PKA-specific polyclonal antibody, as described under “Experimental Procedures.” The extreme right lane was loaded with five units of purified PKA α-catalytic subunit as a positive control. B, densitometric representation of the phosphorylation of histone 2B (1.5 mg/ml) by membrane-anchored protein kinases of Sf9 cells and sensitivity to the inhibitor PKAi_5–24. Bars represent mean ± S.E. of >3 determinations. Phosphorylation was carried out as described under “Experimental Procedures,” with the use of membranes expressing Ccc2 wt or from Sf9 cells transfected with empty plasmid (MOCK) in the absence or presence of 100 nm PKAi_5–24, as indicated on the abscissa. *: different from histone 2B phosphorylation with no PKA inhibitor. The data were corrected for the phosphorylation measured without histone (usually <10% of the total phosphorylation).