RESUMO
Pro-hormone or pro-protein convertases are a conserved family of eukaryotic serine proteases found in the secretory pathway. These endoproteases mature precursors for peptides and proteins that perform a wide range of physiologically important and clinically relevant functions. The first member of this family to be identified was Kex2 in the yeast Saccharomyces cerevisiae. One mammalian member of this family - furin - is responsible for processing substrates that include insulin pro-receptor, human immunodeficiency virus gp160 glycoprotein, Ebola virus glycoprotein, and anthrax protective antigen. Recent determination of the crystal structures for the catalytic core domains of both Kex2 and furin - the first for any members of this family - provide remarkable insights and a new level of understanding of substrate specificity and catalysis by the pro-protein convertases.
Assuntos
Cristalografia por Raios X , Furina/metabolismo , Pró-Proteína Convertases/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Animais , Sítios de Ligação , Cálcio/metabolismo , Catálise , Endopeptidase K/química , Previsões , Furina/química , Humanos , Modelos Moleculares , Pró-Proteína Convertases/química , Conformação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Especificidade por Substrato , Subtilisinas/química , Terminologia como AssuntoAssuntos
Proteínas de Ciclo Celular , Mitose/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiologia , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Regulação Fúngica da Expressão Gênica , Morfogênese , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genéticaRESUMO
The MCK1 gene of Saccharomyces cerevisiae encodes a protein kinase homologous to metazoan glycogen synthase kinase-3. Previous studies implicated Mck1p in negative regulation of pyruvate kinase. In this study we find that purified Mck1p does not phosphorylate pyruvate kinase, suggesting that the link is indirect. We find that purified Tpk1p, a cAMP-dependent protein kinase catalytic subunit, phosphorylates purified pyruvate kinase in vitro, and that loss of the cAMP-dependent protein kinase regulatory subunit, Bcy1p, increases pyruvate kinase activity in vivo. We find that purified Mck1p inhibits purified Tpk1p in vitro, in the presence or absence of Bcy1p. Mck1p must be catalytically active to inhibit Tpk1p, but Mck1p does not phosphorylate this target. We find that abolition of Mck1p autophosphorylation on tyrosine prevents the kinase from efficiently phosphorylating exogenous substrates, but does not block its ability to inhibit Tpk1p in vitro. We find that this mutant form of Mck1p appears to retain the ability to negatively regulate cAMP-dependent protein kinase in vivo. We propose that Mck1p, in addition to phosphorylating some target proteins, also acts by a separate, novel mechanism: autophosphorylated Mck1p binds to and directly inhibits, but does not phosphorylate, the catalytic subunits of cAMP-dependent protein kinase.
