Immunoprecipitation of PDE2 phosphorylated and inactivated by an associated protein kinase.
A PDE2A2-associated protein kinase phosphorylates PDE2A2 in vivo and in vitro to inhibit its catalytic activity. Rat brain PDE2A2 may be solubilized using nona (ethylene glycol) mono dodecyl ether (Lubrol 12A9). PDE2A2 exists in a complex with a protein kinase regulating its activity in an adenosine triphosphate-dependent manner. When native or recombinant PDE2 is immunoprecipitated from PC12 cells using an antibody to the amino terminus in a buffer containing Lubrol 12A9, protease inhibitors, and phosphatase inhibitors, a coimmunoprecipitating nerve growth factor-stimulated protein kinase acts to phosphorylate it. PDE2A2 phosphoryla-tion occurs optimally at pH 6.5 in a sodium 2-(4-morpholino)-ethane sulfonate buffer with 5 mM MgCl2 and 1 mM Na3VO4. I describe protocols for producing an antibody to an amino-terminal bacterial fusion protein encoding amino acids 1-251 of PDE2A2 as well as the use of this antibody in immunoprecipitating a PDE2: tyrosine protein-kinase complex from rat brain or PC12 cells.
Department of Pediatrics and Pulmonary Medicine, The University of Michigan, Ann Arbor, USA.
J Kelley
JK
Bentley
D000818:Animals; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2; D015870:Gene Expression; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D016716:PC12 Cells; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry* / Q000276:immunology; D051381:Rats; D014162:Transfection
D009097:Multienzyme Complexes; D011494:Protein Kinases; D010727:Phosphoric Diester Hydrolases; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2; C517275:PDE2A protein, human; C517278:Pde2a protein, rat