title: Type II cGMP-dependent protein kinase negatively regulates fibroblast growth factor signaling by phosphorylating Raf-1 at serine 43 in rat chondrosarcoma cells.
abstract: Although type II cGMP-dependent protein kinase (PKGII) is a major downstream effector of cGMP in chondrocytes and attenuates the FGF receptor 3/ERK signaling pathway, its direct target proteins have not been fully explored. In the present study, we attempted to identify PKGII-targeted proteins, which are associated with the inhibition of FGF-induced MAPK activation. Although FGF2 stimulation induced the phosphorylation of ERK1/2, MEK1/2, and Raf-1 at Ser-338 in rat chondrosarcoma cells, pretreatment with a cell-permeable cGMP analog strongly inhibited their phosphorylation. On the other hand, Ser-43 of Raf-1 was phosphorylated by cGMP in a dose-dependent manner. Therefore, we examined the direct phosphorylation of Raf-1 by PKGII. Wild-type PKGII phosphorylated Raf-1 at Ser-43 in a cGMP-dependent manner, but a PKGII D412A/R415A mutant, which has a low affinity for cGMP, did not. Finally, we found that a phospho-mimic mutant, Raf-1 S43D, suppressed FGF2-induced MAPK pathway. These results suggest that PKGII counters FGF-induced MEK/ERK activation through the phosphorylation of Raf-1 at Ser-43 in chondrocytes.
authors.affiliation: Department of Bioscience and Bioindustry, Tokushima University Graduate School, Minamijosanjima, Tokushima, Japan.
authors.forename: Norio
authors.initials: N
authors.lastname: Kamemura
authors.affiliation: Department of Biological Science and Technology, Tokushima University Graduate School, Minamijosanjima, Tokushima, Japan.
authors.forename: Sara
authors.initials: S
authors.lastname: Murakami
authors.affiliation: Department of Biological Science and Technology, Tokushima University Graduate School, Minamijosanjima, Tokushima, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Komatsu
authors.affiliation: Department of Biological Science and Technology, Tokushima University Graduate School, Minamijosanjima, Tokushima, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sawanoi
authors.affiliation: Department of Biological Science and Technology, Tokushima University Graduate School, Minamijosanjima, Tokushima, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Miyamoto
authors.affiliation: Division of Molecular Biology, Institute for Health Sciences, Tokushima Bunri University, Tokushima, Japan.
authors.forename: Kazumi
authors.initials: K
authors.lastname: Ishidoh
authors.affiliation: Department of Bioscience and Bioindustry, Tokushima University Graduate School, Minamijosanjima, Tokushima, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Kishimoto
authors.affiliation: Department of Bioscience and Bioindustry, Tokushima University Graduate School, Minamijosanjima, Tokushima, Japan; Department of Biological Science and Technology, Tokushima University Graduate School, Minamijosanjima, Tokushima, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Tsuji
authors.affiliation: Department of Bioscience and Bioindustry, Tokushima University Graduate School, Minamijosanjima, Tokushima, Japan; Department of Biological Science and Technology, Tokushima University Graduate School, Minamijosanjima, Tokushima, Japan. Electronic address: kyuasa@tokushima-u.ac.jp.
authors.forename: Keizo
authors.initials: K
authors.lastname: Yuasa
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D019902:Chondrocytes / Q000378:metabolism; D002813:Chondrosarcoma / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism; D062967:Cyclic GMP-Dependent Protein Kinase Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
chemical_list: D016222:Fibroblast Growth Factor 2; D012694:Serine; D019908:Proto-Oncogene Proteins c-raf; C546782:Raf1 protein, rat; D062967:Cyclic GMP-Dependent Protein Kinase Type II; C569732:Prkg2 protein, rat; D006152:Cyclic GMP
keywords: Chondrocytes; FGF; PKGII; Raf-1