title: Differential regulation of phosphoglucose isomerase/autocrine motility factor activities by protein kinase CK2 phosphorylation.
abstract: Phosphoglucose isomerase (PGI; EC 5.3.1.9) is a cytosolic housekeeping enzyme of the sugar metabolism pathways that plays a key role in both glycolysis and gluconeogenesis. PGI is a multifunctional dimeric protein that extracellularly acts as a cytokine with properties that include autocrine motility factor (AMF)-eliciting mitogenic, motogenic, and differentiation functions, and PGI has been implicated in tumor progression and metastasis. Little is known of the biochemical regulation of PGI/AMF activities, although it is known that human PGI/AMF is phosphorylated at Ser(185) by protein kinase CK2 (CK2); however, the physiological significance of this phosphorylation is unknown. Thus, by site-directed mutagenesis, we substituted Ser(185) with aspartic acid (S185D) or glutamic acid (S185E), which introduces a negative charge and conformational changes that mimic phosphorylation. A Ser-to-Ala mutant protein (S185A) was generated to abolish phosphorylation. Biochemical analyses revealed that the phosphorylation mutant proteins of PGI exhibited decreased enzymatic activity, whereas the S185A mutant PGI protein retained full enzymatic activity. PGI phosphorylation by CK2 also led to down-regulation of enzymatic activity. Furthermore, CK2 knockdown by RNA interference was associated with up-regulation of cellular PGI enzymatic activity. The three recombinant mutant proteins exhibited indistinguishable cytokine activity and receptor-binding affinities compared with the wild-type protein. In both in vitro and in vivo assays, the wild-type and S185A mutant proteins underwent active species dimerization, whereas both the S185D and S185E mutant proteins also formed tetramers. These results demonstrate that phosphorylation affects the allosteric kinetic properties of the enzyme, resulting in a less active form of PGI, whereas non-phosphorylated protein species retain cytokine activity. The process by which phosphorylation modulates the enzymatic activity of PGI thus has an important implication for the understanding of the biological regulation of this key glucose metabolism-regulating enzyme.
authors.affiliation: Tumor Progression and Metastasis Program, Karmanos Cancer Institute, 110 E. Warren Ave., Detroit, Michigan 48201, USA.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yanagawa
authors.forename: Tatsuyoshi
authors.initials: T
authors.lastname: Funasaka
authors.forename: Soichi
authors.initials: S
authors.lastname: Tsutsumi
authors.forename: Tirza
authors.initials: T
authors.lastname: Raz
authors.forename: Nobutada
authors.initials: N
authors.lastname: Tanaka
authors.forename: Avraham
authors.initials: A
authors.lastname: Raz
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D047390:Casein Kinase II / Q000378:metabolism*; D045744:Cell Line, Tumor; D003432:Cross-Linking Reagents / Q000494:pharmacology; D016207:Cytokines / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D019281:Dimerization; D018450:Disease Progression; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D015971:Gene Expression Regulation, Enzymologic; D005947:Glucose / Q000378:metabolism; D005956:Glucose-6-Phosphate Isomerase / Q000096:biosynthesis*; D006019:Glycolysis; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009362:Neoplasm Metastasis; D010766:Phosphorylation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation
chemical_list: D003432:Cross-Linking Reagents; D016207:Cytokines; D008024:Ligands; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D012694:Serine; D047390:Casein Kinase II; D005956:Glucose-6-Phosphate Isomerase; D005947:Glucose