title: Characterizing PKA-Mediated Phosphorylation of Plexin Using Purified Proteins.
abstract: Protein phosphorylation is one of the widely used posttranslational modifications that alter protein function in vivo. We recently showed phosphorylation of Drosophila Plexin A by cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and subsequent inhibition of plexin-mediated repulsive guidance. This phosphorylation occurs in the active site of the plexin GTPase-activating protein (GAP) domain, which in turn inhibits endogenous GAP activity toward Ras/Rap family small GTP-binding proteins by recruiting the phospho-serine/threonine-binding protein 14-3-3ε. Here we describe how phosphorylation of Plexin A can be detected and quantitated using an in vitro kinase assay and radioactive [γ-P32] adenosine 5'-triphosphate (ATP).
authors: Departments of Neuroscience and Pharmacology and Neuroscience Graduate Program, The University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.|Department of Anatomy, University of California San Francisco, San Francisco, CA, 94158, USA.
authors: Taehong
authors: T
authors: Yang
authors: Departments of Neuroscience and Pharmacology, The University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA. jonathan.terman@utsouthwestern.edu.
authors: Jonathan R
authors: JR
authors: Terman
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D029721:Drosophila Proteins / Q000378:metabolism*; D004331:Drosophila melanogaster; D004591:Electrophoresis, Polyacrylamide Gel; D020558:GTP Phosphohydrolases / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011956:Receptors, Cell Surface / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D029721:Drosophila Proteins; D009419:Nerve Tissue Proteins; D011956:Receptors, Cell Surface; C116483:plexA protein, Drosophila; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D020558:GTP Phosphohydrolases
keywords: Kinase assay; PKA; Phosphorylation; Plexin