Fig. 6. PKA-mediated phosphorylation of tau at Ser214 reorganizes microtubules. (A) Forskolin (1 µM, 30 minutes) increased co-immunoprecipitation of phosphorylated tau-Ser214 with β-tubulin in the cytosol of PDE4D4_1-166-expressing PMVECs, which was not seen in vector controls. Such enhanced co-immunoprecipitation of phosphorylated tau with β-tubulin was not observed using the phosphorylated tau-Ser262 antibody under the same conditions. (B) The forskolin-stimulated increase in tau-Ser214 phosphorylation was prominent in the depolymerized microtubule-enriched fractions, and was not observed in polymerized fractions. (C) H89 pretreatment (10 µM, 10 minutes before forskolin stimulation) blocked the forskolin-induced phosphorylation of tau-Ser214 binding to β-tubulin in cytosol. (D) Phosphorylated tau-Ser214 (red) and microtubules (green) are shown in control and PDE4D4_1-166-expressing PMVECs. Whereas PDE4D4_1-166 did not influence resting tau-Ser214 or microtubule distribution (top panel), the application of forskolin (bottom panel) abruptly reorganized microtubules into bundles. (White boxes denote area enlarged in right panel of each image set.)