Protein Kinase A Phosphorylation of Human Phosphodiesterase 3B Promotes 14-3-3 Protein Binding and Inhibits Phosphatase-catalyzed Inactivation

Recent studies confirm that intracellular cAMP concentrations are nonuniform and that localized subcellular cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) is important in maintaining these cAMP compartments. Human phosphodiesterase 3B (HSPDE3B), a member of the PDE3 family of PDEs, represents the dominant particulate cAMP-PDE activity in many cell types, including adipocytes and cells of hematopoietic lineage. Although several previous reports have shown that phosphorylation of HSPDE3B by either protein kinase A (PKA) or protein kinase B (PKB) activates this enzyme, the mechanisms that allow cells to distinguish these two activated forms of HSPDE3B are unknown. Here we report that PKA phosphorylates HSPDE3B at several distinct sites (Ser-73, Ser-296, and Ser-318), and we show that phosphorylation of HSPDE3B at Ser-318 activates this PDE and stimulates its interaction with 14-3-3 proteins. In contrast, although PKB-catalyzed phosphorylation of HSPDE3B activates this enzyme, it does not promote 14-3-3 protein binding. Interestingly, we report that the PKA-phosphorylated, 14-3-3 protein-bound, form of HSPDE3B is protected from phosphatase-dependent dephosphorylation and inactivation. In contrast, PKA-phosphorylated HSPDE3B that is not bound to 14-3-3 proteins is readily dephosphorylated and inactivated. Our data are presented in the context that a selective interaction between PKA-activated HSPDE3B and 14-3-3 proteins represents a mechanism by which cells can protect this enzyme from deactivation. Moreover, we propose that this mechanism may allow cells to distinguish between PKA- and PKB-activated HSPDE3B. Recent studies confirm that intracellular cAMP concentrations are nonuniform and that localized subcellular cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) is important in maintaining these cAMP compartments. Human phosphodiesterase 3B (HSPDE3B), a member of the PDE3 family of PDEs, represents the dominant particulate cAMP-PDE activity in many cell types, including adipocytes and cells of hematopoietic lineage. Although several previous reports have shown that phosphorylation of HSPDE3B by either protein kinase A (PKA) or protein kinase B (PKB) activates this enzyme, the mechanisms that allow cells to distinguish these two activated forms of HSPDE3B are unknown. Here we report that PKA phosphorylates HSPDE3B at several distinct sites (Ser-73, Ser-296, and Ser-318), and we show that phosphorylation of HSPDE3B at Ser-318 activates this PDE and stimulates its interaction with 14-3-3 proteins. In contrast, although PKB-catalyzed phosphorylation of HSPDE3B activates this enzyme, it does not promote 14-3-3 protein binding. Interestingly, we report that the PKA-phosphorylated, 14-3-3 protein-bound, form of HSPDE3B is protected from phosphatase-dependent dephosphorylation and inactivation. In contrast, PKA-phosphorylated HSPDE3B that is not bound to 14-3-3 proteins is readily dephosphorylated and inactivated. Our data are presented in the context that a selective interaction between PKA-activated HSPDE3B and 14-3-3 proteins represents a mechanism by which cells can protect this enzyme from deactivation. Moreover, we propose that this mechanism may allow cells to distinguish between PKA- and PKB-activated HSPDE3B. Cyclic AMP regulates a diverse array of cellular processes, including intermediary metabolism, vascular and visceral smooth muscle relaxation, hormonal secretion, cytoskeletal organization, as well as transcription, migration, proliferation, and apoptosis (reviewed in Refs.1Taylor P. Insel P.A. Pratt W.B. Taylor P. 3rd Ed. Principles of Drug Action: The Basis of Pharmacology. Churchill Livingstone Inc., New York1988: 144-164Google Scholar, 2De Cesare D. Fimia G.M. Sassone-Corsi P. Trends Biochem. 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Haemostasis. 1999; 82: 407-411Crossref PubMed Scopus (96) Google Scholar, 12Bender A.T. Beavo J.A. Pharmacol. Rev. 2006; 58: 488-520Crossref PubMed Scopus (1478) Google Scholar). PDE3A mRNA is enriched in cells of the cardiovascular system and in oocytes, whereas PDE3B mRNA is abundant in adipocytes, hepatocytes, and cells of hematopoietic lineage (13Reinhardt R.R. Chin E. Zhou J. Taira M. Murata T. Manganiello V.C. Bondy C.A. J. Clin. Investig. 1995; 95: 1528-15238Crossref PubMed Scopus (154) Google Scholar). Full-length PDE3A and PDE3B contain two N-terminal hydrophobic regions (NHR1 and NHR2) that target these enzymes to the endoplasmic reticulum and perhaps the plasma membrane (13Reinhardt R.R. Chin E. Zhou J. Taira M. Murata T. Manganiello V.C. Bondy C.A. J. Clin. Investig. 1995; 95: 1528-15238Crossref PubMed Scopus (154) Google Scholar, 14Degerman E. Belfrage P. Managaniello V.C. J. Biol. 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PubMed Scopus Google Scholar) the of C. of a murine in that of proteins as as of HSPDE3B in which of proteins. for HSPDE3B as and was in with and as for for in the of and for the of from that into in and by and of HSPDE3B was by an into A HSPDE3B was by of a of HSPDE3B into the in 14-3-3 14-3-3 or or HSPDE3B as glutathione proteins in E. A human was a from and the and E. was with at for cell by and by in with and protein on reduced glutathione and with in and was of protein and by Cell or cells in with and of HSPDE3B HSPDE3B V. C. of was into the mammalian was used to HSPDE3B. In in which HSPDE3B was with of to of HSPDE3B to not of HSPDE3B. HSPDE3B, of by PDE activity that of for at or HSPDE3B the to the wild or dominant by D. of HSPDE3B in or cells In of or proteins by or HSPDE3B proteins with PKA or activated Inc., in a or with or with for at with kinase with kinase or or with and the of these proteins to of with and Cell of or cells at for in or protein kinase inhibitors, to the the of the cells either or cell with a in a of DTT, and was by and used in the concentrations by the protein of with or between HSPDE3B, or its or with proteins studied by or with or of with GST, or at 4 for and for at 4 with cell as cell for at 4 the or and with cAMP PDE in was of in an Palmer D. D.H. J. Pharmacol. 1997; PubMed Scopus Google Scholar). or was used to PDE3 and PDE4 Palmer D. D.H. J. Pharmacol. 1997; PubMed Scopus Google Scholar). in HSPDE3B, as well as proteins of in these several including a for HSPDE3B a for from S. and V. or selective and as we D.H. J. Biol. 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Biol. 1999; PubMed Scopus Google Scholar), and this was by the PKA in an in kinase In and in of HSPDE3B and PKA of a the HSPDE3B with either or not with with an important regulatory role for HSPDE3B phosphorylation in regulating this in of HSPDE3B with the of PKA and not the these interactions A and In contrast, although in of HSPDE3B with and HSPDE3B activity by it not promote HSPDE3B to proteins these data are with the that HSPDE3B and 14-3-3 proteins interact in and that phosphorylation of HSPDE3B by PKA, not PKB, this The HSPDE3B by was likely of the of of kinase activity in the HSPDE3B. phosphorylation of HSPDE3B also 14-3-3 in we used a cell that HSPDE3B of cells with a of and HSPDE3B to by of HSPDE3B, the of that with HSPDE3B and with a role for PKA in the effects of F/I, of cells with a PKA this of a or a data not not HSPDE3B to of either the proteins activated by insulin data not or of an of not impact HSPDE3B to these data are with the that HSPDE3B with 14-3-3 proteins in a PKA-dependent and that this interaction is of or of proteins activated by PKC, PI3K, or An of HSPDE3B with HSPDE3B amino bound to in a PKA-dependent in cells In contrast, a HSPDE3B amino not data that 14-3-3 proteins with HSPDE3B through the of HSPDE3B. An in N. S. S. J. Mol. Biol. 1999; PubMed Scopus Google Scholar) several PKA phosphorylation consensus sites and of these (Ser-73, Ser-296, and for HSPDE3B Ser-318 was studied PKA phosphorylation of the in murine PDE3B activated this enzyme T. S. M. K. U. W. M. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar). was a PKA consensus sequence in the used in the was a PKA consensus sequence that 14-3-3 proteins to the protein S. R. E. R. V. G. C. A. J. J. 2002; Scopus Google Scholar). PKA in was not also studied the of in HSPDE3B phosphorylation of this in activated the enzyme T. S. M. K. U. W. M. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar) and was to promote its with 14-3-3 in murine adipocytes K. T. F. 2002; PubMed Scopus Google Scholar). of data with the that phosphorylation of HSPDE3B at Ser-318 coordinated 14-3-3 binding. although of cells an form of not promote of this of cells forms or promote their interactions with effects of in the of cells these to HSPDE3B in these cells with of the in cells these cells not Interestingly, of the or in cells with with data from studies in which we show that can with HSPDE3B in R. Raymond and D. Indeed, of in a cell that does not these not in Full-length HSPDE3B an of cells or HSPDE3B with their interactions with In contrast, of cells an HSPDE3B not these data are with the that Ser-318 is the that PKA-dependent interactions between HSPDE3B and In to the HSPDE3B phosphorylation for and enzyme activation by an PKA substrates we that Ser-318 may also represent a of PKA phosphorylation of HSPDE3B in cells with it is this with to phosphorylation consensus a to the of at of these Although in N. S. S. J. Mol. Biol. 1999; PubMed Scopus Google Scholar) PKA consensus sites HSPDE3B, data are with the that only Ser-318 was for in of phosphorylation of HSPDE3B on enzyme significant between and data in significant between and data in significant between and data in significant between and data in in a previous report that an phosphorylation of at and to and Ser-318 in activated this enzyme and its interaction with in murine adipocytes K. T. F. 2002; PubMed Scopus Google Scholar). In to this although data confirm that can HSPDE3B are with the that phosphorylation of HSPDE3B by at or of this enzyme with data are with the that on HSPDE3B the of PKA phosphorylation at Ser-318 to promote Indeed, of wild a and activated or a dominant and in cells not either the of HSPDE3B to in these cells the of to promote this with their of with a that a fraction of the wild or activated and that the PKB, which is was not to it is also that was in the PKA-dependent of HSPDE3B to in these of the PKA and HSPDE3B to to and data that PKA phosphorylation of Ser-318 in HSPDE3B activated this enzyme and its to we that this interaction the of this fraction of HSPDE3B to dephosphorylated and by Our data are with this Indeed, although a of HSPDE3B from cells with calf intestinal alkaline in dephosphorylation and enzyme this of the HSPDE3B not result in dephosphorylation or enzyme phosphorylation of HSPDE3B not promote the of of this enzyme by it is that PKB-activated HSPDE3B protected from dephosphorylation and inactivation. A these is presented in of on PDE PDE in a HSPDE3B to 14-3-3 proteins and the impact of this on PKA- or A is presented that the data in of the interaction of HSPDE3B and 14-3-3 proteins. The how PKA-phosphorylated HSPDE3B is protected from with PKA- or HSPDE3B in proteins in cellular are regulating several distinct events T. Scott J.D. Trends Biochem. Sci. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, Zhou T. Nat. Rev. Mol. Cell Biol. 2006; PubMed Scopus Google Scholar). Although the events that allow coordination of the of proteins are as regulation of protein phosphorylation has emerged as an important of T. Scott J.D. Trends Biochem. Sci. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, Zhou T. Nat. Rev. Mol. Cell Biol. 2006; PubMed Scopus Google Scholar). In this we have that phosphorylation of HSPDE3B regulates this enzyme activity in and we have a mechanism that may allow cells to between the PKA- and the PKB-activated of this in the cells only one HSPDE3B and this enzyme can to the endoplasmic reticulum perhaps to a to the plasma membrane of cells R. Ahmad F. Sward K. Andersson U. Weston M. Manganiello V. Degerman E. Cell. Signal. 2006; 18: 1713-1721Crossref PubMed Scopus (45) Google Scholar, K. G. D. M.D. Degerman E. Manganiello V.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, H.S. D.H. J. 2005; PubMed Scopus Google Scholar). is a of the proteins with which these of HSPDE3B interact and on the impact protein-protein interactions have on HSPDE3B Indeed, whereas PDE3B in or human adipocytes reported to interact with the insulin receptor with and the enzyme was shown to interact with p87PIKAP in the impact of these events on PDE3 activity and on cellular events by the hydrolysis of cAMP by this enzyme are as the rat adipocyte PDE3B was reported to interact with in an and Indeed, in this it was that phosphorylation of or sites to and Ser-318 in HSPDE3B, coordinated this interaction K. T. F. 2002; PubMed Scopus Google Scholar). we presented data that of the 14-3-3 protein family of proteins or interact with HSPDE3B in human and these interactions are by of these cells with Moreover, a of selective PKA we PKA as the cAMP this cAMP-mediated In to that 14-3-3 proteins with HSPDE3B in cells these proteins and that these events coordinated through cAMP-mediated activation of PKA, we also of a of and in studies to the HSPDE3B this Our data two 14-3-3 sites HSPDE3B. on we a the amino of HSPDE3B. Although this HSPDE3B PKA phosphorylation consensus sites and was by PKA in studies that this to in the in 14-3-3 protein in mammalian cells in to on the that this interaction was in in which PKA not been this 14-3-3 protein of HSPDE3B may in of 14-3-3 in is with a of 14-3-3 protein with HSPDE3B in cells that was not to or the activation of in of 14-3-3 interactions that not on protein phosphorylation have been reported S. N. 2000; 95: PubMed Google Scholar, L. Biochem. J. 1999; Scopus Google Scholar). phosphorylation of and in cells a role in cellular HSPDE3B at a not in studies and form the of in studies we a dominant role for one HSPDE3B in the interaction between HSPDE3B and 14-3-3 proteins. Indeed, of the of HSPDE3B was not cells with Our data also a role for phosphorylation of either of the HSPDE3B PKA sites and or the in PKA-dependent HSPDE3B to Indeed, HSPDE3B, in which PKA sites with in a with the of the interaction between the used in studies and was by activation of PKA, this is to have to 14-3-3 binding. of a activated or of a dominant impact on or PKA-dependent protein we that HSPDE3B does not interact with 14-3-3 proteins in a in In we have shown that does not HSPDE3B to 14-3-3 proteins in either the cell or a human cell from a that several S. and D. Our data the of in HSPDE3B to are Indeed, we to a role for in either of HSPDE3B to or in between these proteins in to in cellular In to of to in adipocytes K. T. F. 2002; PubMed Scopus Google Scholar), it may significant that insulin can in several cell types and that this was J. Pharmacol. PubMed Scopus Google Scholar). insulin to in rat adipocytes through an activation of PKA, or a of was which was not in to the of a to that the of concentrations of as was in the K. T. F. 2002; PubMed Scopus Google Scholar), in the of this to that the PKA-activated form of HSPDE3B was protected from inactivation. Indeed, HSPDE3B was to with PKA-activated HSPDE3B or with PKB-activated HSPDE3B. the PKB-activated HSPDE3B was not Indeed, we that this in of the PKB-activated forms of HSPDE3B may represent a mechanism by which cells can activation of HSPDE3B by these two kinases by F. PubMed Scopus Google Scholar, D. 2002; PubMed Scopus Google Scholar) have shown that 14-3-3 protein to not their of studies in are at the impact that 14-3-3 dephosphorylation of the PKA-activated enzyme, not that activated by PKB, may have on intracellular cAMP and the events with HSPDE3B activation in V. C. Manganiello of R. J. J. A. Beavo of V. S. and D. of for of used in these

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