Abstract 445: Site-Specific Phosphorylation of Tau at Serine-214 by PKA Disrupts Microtubule in Endothelium
Intracellular cAMP strengthens endothelial barrier integrity when its signaling is restricted to subplasma membrane domains. While, when cAMP accesses cytosol by dominant negative phosphodiesterase 4D4 (PDE4D4) expression within membrane of pulmonary microvascular endothelial cells (PMVECs), it disrupts microtubule networks via specific phosphorylation of microtubule binding protein tau at serine-214, the putative substrate of the activated PKA in cytosol (FASEB J 20:738.1). Such PKA-mediated microtubule disruption subsequently induces hyperpermeability/inter-endothelial cells gap formation. Increased phosphorylation of tau at serine-214 was co-localized with PKA catalytic and regulatory RI subunits at a bundle-like structure of reorganized microtubule, the depolymerized pool of tubulins in the cytosol. To verify whether the abnormal cAMP/PKA activation in cytosol by dysfunction of plasma membrane associated PDE4D4 is sufficient to induce microtubule reorganization and depolymerization, a dominant negative retroviral construct of human Tau40 with an alanine point mutation at serine-214 (S214A) was co-expressed in PDE4D4 dominant negative PMVECs. Tau-S214A blocks the forskolin-induced microtubule disruption, hyperpermeabilty and gap formation via the decrease in PKA-mediated phosphorylation of tau and the disassociation of tau with the depolymerized pool of tubulins in the cytosol. Results suggest that PDE4D4 acts as cAMP signaling compartmentalization factor necessary to protect barrier functions in pulmonary endothelial cells via the devoid of PKA-mediated specific phosphorylation of Tau-s214 in cytosol.