In vitro PKA phosphorylation-mediated human PDE4A4 activation

The PDE4 catalytic machinery comprises, in part, two divalent cations in a binuclear motif. Here we report that PDE4A4 expressed in Sf9 cells exhibits a biphasic Mg 2+ dose–response (EC 50 of ∼0.15 and >10 mM) in catalyzing cAMP hydrolysis. In vitro phosphorylation of PDE4A4 by the PKA-catalytic subunit increases the enzyme’s sensitivity to Mg 2+, leading to 4-fold increased cAMP hydrolysis without affecting its K m. The phosphorylation also increases the potencies of ( R)- and ( S)-rolipram without affecting CDP-840 and SB-207499. The results support that modulating the cofactor binding affinity of PDE4 represents a mechanism for regulating its activity.