FIGURE 3.

Protein kinase activity regulates the association of Myo5a with Rab3A. Synaptosomal fractions were preincubated with 2 μm staurosporine (Stauro), 2 μm CaMKII inhibitory peptide (CaMKIIi), or 20 nm okadaic acid (OA) and then passed over a GST-Myo5a tail affinity column. Western blots of the total amount of Rab3A or Rabphillin3A (Syn) and bound proteins (BP) eluted from GST-Myo5a tail affinity column are shown. 2 μm staurosporine (Stauro) decreased binding of Rab3A and Rabphilin3A to GST-Myo5a column. Inhibition of CaMKII activity did not affect the binding affinity of Rab3A to Myo5a tail but reduced Rabphilin3A binding. Inhibition of protein phosphatases 1 and 2A increased binding affinity of both Rab3A and Rabphilin3A to Myo5a tail (OA). Signal intensities of Rab3A and Rabphilin3A were individually normalized against the signal obtained with these proteins in synaptosomes. All data are mean ± S.D. of three independent experiments. Statistically significant differences compared with the controls are indicated by *, p < 0.001.