Nerve growth factor inhibits PC12 cell PDE 2 phosphodiesterase activity and increases PDE 2 binding to phosphoproteins.
Nerve growth factor (NGF) has been shown to increase cyclic AMP in PC12 cells and to potentiate the actions of other agents that raise cyclic AMP. In our studies, NGF causes over 50% loss of PDE 2 activity (cyclic GMP-stimulated cyclic nucleotide phosphodiesterase) in PC12 cells within 24 h. After 72 h of NGF treatment, cyclic AMP hydrolysis in PC12 extracts is no longer cyclic GMP-stimulated. NGF deprivation increases the phosphodiesterase activity of treated cells. NGF does not decrease either PDE 2 mRNA or immunoreactivity of PDE 2A2 protein. Incubation of whole cells with micromolar Na(3)VO(4) mimics NGF treatment, reducing PDE 2 activity in PC12 cells by over 50% after 24 h, suggesting a phosphoprotein-mediated regulation of PDE 2 activity. Protein kinase inhibitor effects were difficult to assess due to their direct interaction with the PDE in cell lysates. To study phosphorylation in PDE 2 regulation, PDE 2A2 was epitope-tagged, and stable clonal PC12 cell transfectants were isolated (PC12B cells). When combined with metabolically labeled (32)P-phosphoproteins in vivo or in vitro, phosphoproteins of 108, 90, 64, 43, 33 and 19 kDa coprecipitated with epitope-tagged PDE 2A2 in an NGF sensitive manner. A 23-kDa phosphoprotein containing immunoreactive phosphoserine associated with the complex in an NGF independent manner. Phosphothreonine plus phosphotyrosine immunoreactivity at 23, 24, and 64 kDa as well as the phosphotyrosine immunoreactivity at 108, 90, 64, 43, 33, and 19 kDa required NGF or orthovanadate treatment. These proteins are hypothesized to be part of an NGF-regulated complex controlling PDE 2A2 activity.
Bentley
J K
JK
Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan, USA. kbentley@umich.edu
Juilfs
D M
DM
Uhler
M D
MD
D000818:Animals; D001345:Autoradiography; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism / Q000494:pharmacology; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020932:Nerve Growth Factor / Q000378:metabolism* / Q000494:pharmacology; D016501:Neurites / Q000187:drug effects / Q000378:metabolism; D016716:PC12 Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
D004791:Enzyme Inhibitors; D010726:Phosphodiesterase Inhibitors; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D020932:Nerve Growth Factor; D000242:Cyclic AMP; D017027:Protein Tyrosine Phosphatases; D010727:Phosphoric Diester Hydrolases; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2; C517278:Pde2a protein, rat; D006152:Cyclic GMP