RESUMO
The mitogen-activated protein kinase (MAPK) Kss1 has a dual role in regulating filamentous (invasive) growth of the yeast Saccharomyces cerevisiae. The stimulatory function of Kss1 requires both its catalytic activity and its activation by the MAPK/ERK kinase (MEK) Ste7; in contrast, the inhibitory function of Kss1 requires neither. This study examines the mechanism by which Kss1 inhibits invasive growth, and how Ste7 action overcomes this inhibition. We found that unphosphorylated Kss1 binds directly to the transcription factor Ste12, that this binding is necessary for Kss1-mediated repression of Ste12, and that Ste7-mediated phosphorylation of Kss1 weakens Kss1-Ste12 interaction and relieves Kss1-mediated repression. Relative to Kss1, the MAPK Fus3 binds less strongly to Ste12 and is correspondingly a weaker inhibitor of invasive growth. Analysis of Kss1 mutants indicated that the activation loop of Kss1 controls binding to Ste12. Potent repression of a transcription factor by its physical interaction with the unactivated isoform of a protein kinase, and relief of this repression by activation of the kinase, is a novel mechanism for signal-dependent regulation of gene expression.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , DNA Recombinante/genética , Ativação Enzimática , Proteínas Fúngicas/genética , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Quinases de Proteína Quinase Ativadas por Mitógeno , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Fosforilação , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Transcrição GênicaRESUMO
Strains of the budding yeast, Saccharomyces cerevisiae, may contain one or more cytoplasmic viruses with double-stranded RNA (dsRNA) genomes. The killer phenomenon in yeast, in which one cell secretes a killer toxin that is lethal to another cell, is dependent upon the presence of the L-A and M1 dsRNA viruses. The L-A viral genome encodes proteins for the viral capsid, and for synthesis and encapsidation of single-stranded RNA replication cycle intermediates. The M1 virus depends upon the L-A-encoded proteins for its capsid and for the replication of its killer-toxin-encoding genome. A full-length cDNA clone of an M genome has been made from a single dsRNA molecule and shown to encode functional killer and killer-immunity functions. The sequence of the clone indicates minor differences from previously published sequences of parts of the M1 genome and of the complete genome of S14 (an internal deletion derivative of M1) but no unreported amino acid variants and no changes in putative secondary structures of the single-stranded RNA. A 118-nucleotide contiguous segment of the M1 genome has not previously been reported; 92 of those nucleotides comprise a segment of A nucleotides in the AU-rich bubble that follows the toxin-encoding reading frame.
