title: Catalytic activity of cGMP-dependent protein kinase type I in intact cells is independent of N-terminal autophosphorylation.
abstract: Although cGMP-dependent protein kinase type I (cGKI) is an important mediator of cGMP signaling and upcoming drug target, its in vivo-biochemistry is not well understood. Many studies showed that purified cGKI autophosphorylates multiple sites at its N-terminus. Autophosphorylation might be involved in kinase activation, but it is unclear whether this happens also in intact cells. To study cGKI autophosphorylation in vitro and in vivo, we have generated phospho-specific antisera against major in vitro-autophosphorylation sites of the cGKI isoforms, cGKIα and cGKIβ. These antisera detected specifically and with high sensitivity phospho-cGKIα (Thr58), phospho-cGKIα (Thr84), or phospho-cGKIβ (Thr56/Ser63/Ser79). Using these antisera, we show that ATP-induced autophosphorylation of cGKI in purified preparations and cell extracts did neither require nor induce an enzyme conformation capable of substrate heterophosphorylation; it was even inhibited by pre-incubation with cGMP. Interestingly, phospho-cGKI species were not detectable in intact murine cells and tissues, both under basal conditions and after induction of cGKI catalytic activity. We conclude that N-terminal phosphorylation, although readily induced in vitro, is not required for the catalytic activity of cGKIα and cGKIβ in vivo. These results will also inform screening strategies to identify novel cGKI modulators.
authors.affiliation: Interfakultäres Institut für Biochemie, University of Tübingen, Tübingen, Germany; German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany; Graduate School of Cellular & Molecular Neuroscience, University of Tübingen, Tübingen, Germany.
authors.forename: Raghavan
authors.initials: R
authors.lastname: Vallur
authors.affiliation: Interfakultäres Institut für Biochemie, University of Tübingen, Tübingen, Germany.
authors.forename: Hubert
authors.initials: H
authors.lastname: Kalbacher
authors.affiliation: Interfakultäres Institut für Biochemie, University of Tübingen, Tübingen, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Feil
mesh_terms: D000818:Animals; D002384:Catalysis; D002417:Cattle; D062965:Cyclic GMP-Dependent Protein Kinase Type I / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D007106:Immune Sera / Q000276:immunology; D051379:Mice; D010766:Phosphorylation; D054730:Protein Interaction Domains and Motifs; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D007106:Immune Sera; D020033:Protein Isoforms; D011994:Recombinant Proteins; D062965:Cyclic GMP-Dependent Protein Kinase Type I