FIGURE 6.

Alternative splicing of SAP97 in the U5 region modulates CaMKIIα binding. A, GST fusion proteins containing the indicated C-terminal SH3-U5-GK domains of the four alternative splice variants were incubated with purified CaMKIIα (nonphosphorylated or prephosphorylated at Thr-286). Protein complexes collected using glutathione-agarose beads were immunoblotted (IB) using a pan-CaMKII antibody. The bar graph indicates the amount of Thr-286-autophosphorylated CaMKIIα bound to each splice variant, normalized to that bound to GST-SH3-I3I5-GK, revealing significantly less binding to all other splice variants (p < 0.001; n = 4). B, CaMKIIα was immunoprecipitated from lysates of HEK293 cells co-expressing mCherry-CaMKIIα(T286D) with either FLAG-SAP97-I3I5 or -I2I5. Protein complexes were immunoblotted with an anti-SAP97 antibody, and mCherry-CaMKIIα was detected by staining nitrocellulose membranes with Ponceau S. The panel is representative of three similar experiments.