Phosphorylation and activation of phosphodiesterase type 3B (PDE3B) in adipocytes in response to serine/threonine phosphatase inhibitors: deactivation of PDE3B in vitro by protein phosphatase type 2A

Phosphodiesterase type 3B (PDE3B) has been shown to be activated and phosph orylated in response to insulin and hormones that increase cAMP. In order t o study serine/threonine protein phosphatases involved in the regulation of rat adipocyte PDE3B, we investigated the phosphorylation and activation of PDE3B in vivo in response to phosphatase inhibitors and the dephosphorylat ion and deactivation of PDE3B in vitro by phosphatases purified from rat ad ipocyte homogenates, Okadaic acid and calyculin A induced dose- and lime-de pendent activation of PDE3B, Maximal effects were obtained after 30 min usi ng 1 mu M okadaic acid (1.8-fold activation) and 300 nM calyculin A (4-fold activation), respectively. Tautomycin and cyclosporin A did not induce act ivation of PDE3B. Incubation of adipocytes with 300 nM calyculin A inhibite d protein phosphatase (PP) 1 and PP2A completely. Okadaic acid (1 mu M) red uced PP2A activity by approx. 50 % but did not affect PP 1 activity, and 1 mu M tautomycin reduced PP1 activity by approx. 60 % but PP2A activity by o nly 11 %. This indicates an important role for PP2A in the regulation of PD E3B. Furthermore, rat adipocyte PDE3B phosphatase activity co-purified with PP2A but not with PPI during MonoQ chromatography, As compared with insuli n, okadaic acid and calyculin A induced phosphorylation of PDE3B by 2.8- an d 14-fold respectively, whereas tautomycin and cyclosporin A had no effect. Both calyculin A and okadaic acid induced phosphorylation on serine 302, t he site known to be phosphorylated on PDE3B in response to insulin and isop roterenol (isoprenaline), as well as on sites not identified previously. In summary, PP2A seems to be involved in the regulation of PDE3B in vivo and can act as a PDE3B phosphatase in vitro. In comparison with insulin, calycu lin A induced a dramatic activation of PDE3B and both calyculin A and okada ic acid induced phosphorylation on additional sites, which could have a rol e in signalling pathways not yet identified.