PDE3A (phosphodiesterase 3A) was identified as a phosphoprotein that co-immunoprecipitates with endogenous 14-3-3 proteins from HeLa cell extracts, and binds directly to 14-3-3 proteins in a phosphorylation-dependent manner. Among cellular stimuli tested, PMA promoted maximal binding of PDE3A to 14-3-3 proteins. While p42/p44 MAPK (mitogen-activated protein kinase), SAPK2 (stress-activated protein kinase 2)/p38 and PKC (protein kinase C) were all activated by PMA in HeLa cells, the PMA-induced binding of PDE3A to 14-3-3 proteins was inhibited by the non-specific PKC inhibitors Ro 318220 and H-7, but not by PD 184352, which inhibits MAPK activation, nor by SB 203580 and BIRB0796, which inhibit SAPK2 activation. Binding of PDE3A to 14-3-3 proteins was also blocked by the DNA replication inhibitors aphidicolin and mimosine, but the PDE3A–14-3-3 interaction was not cell-cycle-regulated. PDE3A isolated from cells was able to bind to 14-3-3 proteins after in vitro phosphorylation with PKC isoforms. Using MS/MS of IMAC (immobilized metal ion affinity chromatography)-enriched tryptic phosphopeptides and phosphospecific antibodies, at least five sites on PDE3A were found to be phosphorylated in vivo, of which Ser428 was selectively phosphorylated in response to PMA and dephosphorylated in cells treated with aphidicolin and mimosine. Phosphorylation of Ser428 therefore correlated with 14-3-3 binding to PDE3A. Ser312 of PDE3A was phosphorylated in an H-89-sensitive response to forskolin, indicative of phosphorylation by PKA (cAMP-dependent protein kinase), but phosphorylation at this site did not stimulate 14-3-3 binding. Thus 14-3-3 proteins can discriminate between sites in a region of multisite phosphorylation on PDE3A. An additional observation was that the cytoskeletal cross-linker protein plectin-1 coimmunoprecipitated with PDE3A independently of 14-3-3 binding.
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Research Article|
November 08 2005
Phosphodiesterase 3A binds to 14-3-3 proteins in response to PMA-induced phosphorylation of Ser428
Mercedes Pozuelo Rubio;
Mercedes Pozuelo Rubio
1
1MRC Protein Phosphorylation Unit, Faculty of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
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David G. Campbell;
David G. Campbell
1MRC Protein Phosphorylation Unit, Faculty of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
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Nicholas A. Morrice;
Nicholas A. Morrice
1MRC Protein Phosphorylation Unit, Faculty of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
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Carol Mackintosh
Carol Mackintosh
2
1MRC Protein Phosphorylation Unit, Faculty of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
2To whom correspondence should be addressed (email c.mackintosh@dundee.ac.uk).
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Publisher: Portland Press Ltd
Received:
July 11 2005
Revision Received:
September 09 2005
Accepted:
September 09 2005
Accepted Manuscript online:
September 09 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 392 (1): 163–172.
Article history
Received:
July 11 2005
Revision Received:
September 09 2005
Accepted:
September 09 2005
Accepted Manuscript online:
September 09 2005
Citation
Mercedes Pozuelo Rubio, David G. Campbell, Nicholas A. Morrice, Carol Mackintosh; Phosphodiesterase 3A binds to 14-3-3 proteins in response to PMA-induced phosphorylation of Ser428. Biochem J 15 November 2005; 392 (1): 163–172. doi: https://doi.org/10.1042/BJ20051103
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