Multisite phosphorylation of adipocyte and hepatocyte phosphodiesterase 3B.
Phosphodiesterase 3B (PDE3B) is an important component of insulin and cAMP-dependent signalling pathways. In order to study phosphorylation of PDE3B, we have used an adenoviral system to express recombinant flag-tagged PDE3B in primary rat adipocytes and H4IIE hepatoma cells. Phosphorylation of PDE3B after treatment of cells with insulin, cAMP-increasing agents, or the phosphatase inhibitor, calyculin A was analyzed by two-dimensional tryptic phosphopeptide mapping and mass spectrometry. We found that PDE3B is multisite phosphorylated in adipocytes and H4IIE hepatoma cells in response to all these stimuli. Several sites were identified; serine (S)273, S296, S421, S424/5, S474 and S536 were phosphorylated in adipocyte as well as H4IIE hepatoma cells whereas S277 and S507 were phosphorylated in hepatoma cells only. Several of the sites were phosphorylated by insulin as well as cAMP-increasing hormones indicating integration of the two signalling pathways upstream of PDE3B, maybe at the level of protein kinase B.
Lindh
Rebecka
R
Department of Experimental Medical Sciences, Division for Diabetes, Metabolism and Endocrinology, Lund University, Lund, Sweden. rebecka.lindh@med.lu.se
Ahmad
Faiyaz
F
Resjö
Svante
S
James
Peter
P
Yang
Jeong S
JS
Fales
Henry M
HM
Manganiello
Vincent
V
Degerman
Eva
E
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017667:Adipocytes / Q000187:drug effects / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D004789:Enzyme Activation / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology*; D007328:Insulin / Q000494:pharmacology; D008066:Lipolysis / Q000187:drug effects; D008114:Liver Neoplasms, Experimental / Q000201:enzymology; D008297:Male; D008387:Marine Toxins; D051379:Mice; D008969:Molecular Sequence Data; D010080:Oxazoles / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology
D007328:Insulin; D008387:Marine Toxins; D010080:Oxazoles; D011994:Recombinant Proteins; C059041:calyculin A; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; C517293:Pde3b protein, mouse; C517294:Pde3b protein, rat