FIGURE 7.

Preferential phosphorylation of the C-terminal region of SAP97-I3I5 by CaMKII. A, the four natural SAP97 U5 splice variants with a FLAG tag were expressed in HEK293 cells. FLAG immune complexes were phosphorylated by exogenous purified CaMKIIα in the presence of [γ-³²P]ATP and analyzed by SDS-PAGE followed by staining of the gel and autoradiography. These data are representative of three experiments. B, wild type or mutated FLAG-SAP97 (S39A, S232A, S39A/S232A) were isolated from lysates of transfected HEK293 cells and then phosphorylated by purified CaMKIIα in the presence of [γ-³²P]ATP (see “Experimental Procedures”). Samples were resolved by SDS-PAGE, and stained/dried gels were autoradiographed. Phosphorylation signals for each mutant were normalized to wild type SAP97. The bar graph shows the mean ± S.E. of data collected from at least six similar experiments. C, GST alone or GST fusion proteins containing the indicated domains from SAP97 splice variants (1 μm each) were phosphorylated by purified CaMKII (0.1 μm) in the presence of [γ-³²P]ATP. Phosphorylated protein bands were detected by autoradiography of SDS-polyacrylamide gels. The bracket indicates ³²P-phosphorylated CaMKII bands that were also observed in the GST blank (data not shown). These data are representative of >5 similar experiments. D, GST-SH3-U5-GK variants were phosphorylated as in panel C, and aliquots of the reactions were spotted on P81-phosphocellulose papers to estimate phosphorylation stoichiometries after subtracting the GST blank. Data are plotted as the mean ± S.E. from >6 experiments.