RESUMEN
In budding yeast, cell division is initiated in late G1 phase once the Cdc28 cyclin-dependent kinase is activated by the G1 cyclins Cln1, Cln2, and Cln3. The extreme instability of the Cln proteins couples environmental signals, which regulate Cln synthesis, to cell division. We isolated Cdc53 as a Cln2-associated protein and show that Cdc53 is required for Cln2 instability and ubiquitination in vivo. The Cln2-Cdc53 interaction, Cln2 ubiquitination, and Cln2 instability all depend on phosphorylation of Cln2. Cdc53 also binds the E2 ubiquitin-conjugating enzyme, Cdc34. These findings suggest that Cdc53 is a component of a ubiquitin-protein ligase complex that targets phosphorylated G1 cyclins for degradation by the ubiquitin-proteasome pathway.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cullin , Ciclinas/metabolismo , Fase G1 , Proteínas de Saccharomyces cerevisiae , Complejos de Ubiquitina-Proteína Ligasa , Ubiquitinas/metabolismo , Secuencia de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Línea Celular , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/genética , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Proteínas Fúngicas/metabolismo , Ligasas/genética , Ligasas/metabolismo , Datos de Secuencia Molecular , Mutación , Fosforilación , Plásmidos , Enzimas Ubiquitina-Conjugadoras , Ubiquitina-Proteína LigasasRESUMEN
The upstream region of the rat CYP17 gene shows significant homology to the upstream regions of the bovine and human genes, 53 and 60 percent, respectively. The start site of transcription was determined by primer extension and S1 nuclease protection to be 41 base pairs (bp) upstream of the initiating methionine codon. Expression vectors were constructed by ligation of upstream sequences into promoterless chloramphenicol acetyl transferase (CAT) vectors. Transient transfection studies using primary cultures of rat Leydig cells indicate a strong cAMP-responsive element located within the -26/+65 region. Stimulation by cyclic AMP was abolished when sequences upstream of -264 were included in expression vectors. No significant expression was seen in Leydig cells in the absence of dbcAMP nor was there any expression in the presence or absence of dbcAMP in rat skin fibroblasts or in mouse adrenal (Y-1) cells in which CYP17 is not normally expressed. Three possible regulatory elements were found in the 5' upstream region: a CRE/ATF consensus sequence (GACGTCA) starting at position -57; a GRE consensus sequence (TGTTCT) starting at position -501; and a consensus sequence for AP-1 binding (TTAGTCA) starting at position -659. It was concluded that the CRE/ATF at -57 is not responsible for increased transcription in the presence of cyclic AMP.
Asunto(s)
AMP Cíclico/fisiología , Regulación Enzimológica de la Expresión Génica , Células Intersticiales del Testículo/enzimología , Esteroide 17-alfa-Hidroxilasa/genética , Animales , Secuencia de Bases , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , ADN , Electroforesis en Gel de Poliacrilamida , Masculino , Datos de Secuencia Molecular , Ratas , Homología de Secuencia de Ácido Nucleico , beta-Galactosidasa/metabolismoRESUMEN
We describe the isolation and characterization of a full-length clone for the porcine 17 alpha-hydroxylase/C(17-20) lyase (CYP17) gene. The complete exon and partial intron sequences are presented including approx. 1000 bp of the 5' upstream sequence. In addition we describe the isolation and characterization of the 5' upstream region of the rat CYP17 gene. The sequences of the first exon, part of the first intron, and approx. 3.5 kb of the 5' upstream region are presented.