Biochem. J. (1999) 341
(839–845) (Printed in Great Britain)
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
Svante RESJÖ *1, Alina OKNIANSKA†, Stanislaw ZOLNIEROWICZ†, Vincent MANGANIELLO‡ and Eva DEGERMAN*
*Section for Molecular Signalling, Department of Cell and Molecular Biology, Lund University, P. O. Box 94, S-221 00 Lund, Sweden, †Intercollegiate Faculty of Biotechnology UG-MUG, Debinki 1, 80-211 Gdansk, Poland, and ‡Pulmonary/Critical-Care Medicine Branch, NHLBI, National Institutes of Health, Bethseda, MD 20892, U.S.A.
Phosphodiesterase type 3B (PDE3B) has been shown to be activated and phosphorylated in response to insulin and hormones that increase cAMP. In order to 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 dephosphorylation and deactivation of PDE3B in vitro by phosphatases purified from rat adipocyte homogenates. Okadaic acid and calyculin A induced dose- and time-dependent activation of PDE3B. Maximal effects were obtained after 30 min using 1 µM okadaic acid (1.8-fold activation) and 300 nM calyculin A (4-fold activation), respectively. Tautomycin and cyclosporin A did not induce activation of PDE3B. Incubation of adipocytes with 300 nM calyculin A inhibited protein phosphatase (PP) 1 and PP2A completely. Okadaic acid (1 µM) reduced PP2A activity by approx. 50% but did not affect PP1 activity, and 1 µM tautomycin reduced PP1 activity by approx. 60% but PP2A activity by only 11%. This indicates an important role for PP2A in the regulation of PDE3B. Furthermore, rat adipocyte PDE3B phosphatase activity co-purified with PP2A but not with PP1 during MonoQ chromatography. As compared with insulin, okadaic acid and calyculin A induced phosphorylation of PDE3B by 2.8- and 14-fold respectively, whereas tautomycin and cyclosporin A had no effect. Both calyculin A and okadaic acid induced phosphorylation on serine 302, the site known to be phosphorylated on PDE3B in response to insulin and isoproterenol (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, calyculin A induced a dramatic activation of PDE3B and both calyculin A and okadaic acid induced phosphorylation on additional sites, which could have a role in signalling pathways not yet identified.
Abbreviations used: PP, protein phosphatase; PP1c, PP2Ac, catalytic subunits of PP1 and PP2A respectively; IGF-I, insulin-like growth factor I; PDE, phosphodiesterase; C
13E
12, a non-ionic alkyl poly(oxyethylene glycol) detergent; IL-4, interleukin 4.
1 To whom correspondence should be addressed (e-mail svante.resjo@medkem.lu.se).
Key words: calyculin A, insulin signalling, lipolysis, okadaic acid.
Received 24 November 1998/6 April 1999; accepted 25 May 1999
The Biochemical Society, London © 1999
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