title: Dephosphorylation of endogenous GABA(B) receptor R2 subunit and AMPK α subunits which were measured by in vitro method using transfer membrane.
abstract: Protein phosphorylation can be regulated by changes in kinase activity, phosphatase activity, or both. GABA(B) receptor R2 subunit (GABA(B)R2) is phosphorylated at S783 by 5'-AMP-activated-protein kinase (AMPK), and this phosphorylation modulates GABA(B) receptor desensitization. Since the GABA(B) receptor is an integral membrane protein, solubilizing GABA(B)R2 is difficult. To circumvent this problem and to identify specific phosphatases that dephosphorylate S783, we employed an in vitro assay based on dephosphorylation of proteins on PVDF membranes by purified phosphatases. Our method allowed us to demonstrate that S783 in GABA(B)R2 is directly dephosphorylated by PP2A (but not by PP1, PP2B nor PP2C) in a dose-dependent and okadaic acid-sensitive manner. We also show that the level of phosphorylation of the catalytic subunit of AMPK at T172 is reduced by PP1, PP2A and PP2C. Our data indicate that PP2A dephosphorylates GABA(B)R2(S783) less efficiently than AMPK(T172), and that additional phosphatases might be involved in S783 dephosphorylation.
authors.affiliation: Department of Pharmacology, Setsunan University, Hirakata, Osaka 573-0101, Japan. kuramoto@pharm.setsunan.ac.jp
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Kuramoto
authors.forename: Machiko
authors.initials: M
authors.lastname: Ito
authors.forename: Yukari
authors.initials: Y
authors.lastname: Saito
authors.forename: Hiroki
authors.initials: H
authors.lastname: Niihara
authors.forename: Natsuki
authors.initials: N
authors.lastname: Tanaka
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Yamada
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Yamamura
authors.forename: Kaname
authors.initials: K
authors.lastname: Iwasaki
authors.forename: Yuki
authors.initials: Y
authors.lastname: Onishi
authors.forename: Kiyokazu
authors.initials: K
authors.lastname: Ogita
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000818:Animals; D020134:Catalytic Domain; D002478:Cells, Cultured; D008567:Membranes, Artificial; D051379:Mice; D010766:Phosphorylation; D054648:Protein Phosphatase 2 / Q000378:metabolism; D018080:Receptors, GABA-B / Q000378:metabolism*
chemical_list: C116566:Gabbr2 protein, rat; D008567:Membranes, Artificial; D018080:Receptors, GABA-B; C548179:Prkaa1 protein, mouse; D055372:AMP-Activated Protein Kinases; D054648:Protein Phosphatase 2