RESUMO
Mitotic cell division is controlled by cyclin-dependent kinases (Cdks), which phosphorylate hundreds of protein substrates responsible for executing the division program. Cdk inactivation and reversal of Cdk-catalyzed phosphorylation are universal requirements for completing and exiting mitosis and resetting the cell cycle machinery. Mechanisms that define the timing and order of Cdk substrate dephosphorylation remain poorly understood. Cdc14 phosphatases have been implicated in Cdk inactivation and are thought to be generally specific for Cdk-type phosphorylation sites. We show that budding yeast Cdc14 possesses a strong and unusual preference for phosphoserine over phosphothreonine at Pro-directed sites in vitro. Using serine to threonine substitutions in the Cdk consensus sites of the Cdc14 substrate Acm1, we demonstrate that phosphoserine specificity exists in vivo. Furthermore, it appears to be a conserved property of all Cdc14 family phosphatases. An invariant active site residue was identified that sterically restricts phosphothreonine binding and is largely responsible for phosphoserine selectivity. Optimal Cdc14 substrates also possessed a basic residue at the +3 position relative to the phosphoserine, whereas substrates lacking this basic residue were not effectively hydrolyzed. The intrinsic selectivity of Cdc14 may help establish the order of Cdk substrate dephosphorylation during mitotic exit and contribute to roles in other cellular processes.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Fosfatases de Especificidade Dupla/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Substituição de Aminoácidos , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/genética , Fosfatases de Especificidade Dupla/química , Fosfatases de Especificidade Dupla/genética , Humanos , Mutação de Sentido Incorreto , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/genética , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Fosforilação/fisiologia , Fosfosserina/química , Fosfosserina/metabolismo , Proteínas Tirosina Fosfatases , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Especificidade por Substrato/fisiologiaRESUMO
The majority of eukaryotic proteins are phosphorylated in vivo, and phosphorylation may be the most common regulatory posttranslational modification. Many proteins are phosphorylated at numerous sites, often by multiple kinases, which may have different functional consequences. Understanding biological functions of phosphorylation events requires methods to detect and quantify individual sites within a substrate. Here we outline a general strategy that addresses this need and relies on the high sensitivity and specificity of selected reaction monitoring (SRM) mass spectrometry, making it potentially useful for studying in vivo phosphorylation without the need to isolate target proteins. Our approach uses label-free quantification for simplicity and general applicability, although it is equally compatible with stable isotope quantification methods. We demonstrate that label-free SRM-based quantification is comparable to conventional assays for measuring the kinetics of phosphatase and kinase reactions in vitro. We also demonstrate the capability of this method to simultaneously measure relative rates of phosphorylation and dephosphorylation of substrate mixtures, including individual sites on intact protein substrates in the context of a whole cell extract. This strategy should be particularly useful for characterizing the physiological substrate specificity of kinases and phosphatases and can be applied to studies of other protein modifications as well.
Assuntos
Proteínas de Bactérias/análise , Proteínas Fúngicas/análise , Espectrometria de Massas/métodos , Proteômica/métodos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Fosfoproteínas/análise , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Quinases/análise , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/metabolismo , Especificidade por SubstratoRESUMO
A generalizable method for detecting protease activity via gelation is described. A recognition sequence is used to target the protease of interest while a second protease is used to remove the residual residues from the gelator scaffold. Using this approach, selective assays for both MMP-9 and PSA are demonstrated.
Assuntos
Ensaios Enzimáticos/métodos , Metaloproteinase 9 da Matriz/metabolismo , Antígeno Prostático Específico/metabolismo , Sítios de Ligação , Técnicas Biossensoriais , Antígenos CD13/metabolismo , GéisRESUMO
A modular system for detecting protease activity via enzyme-triggered gel formation is described. Protease-specific recognition sequences are utilized to achieve enzyme specificity. Artificial blood clotting is demonstrated by activating endogenous thrombin to trigger gelation in fibrinogen-deficient blood plasma.
Assuntos
Biomimética/métodos , Dipeptídeos/química , Trombina/análise , Ácido 4-Aminobenzoico/química , Aminoácidos/química , Coagulação Sanguínea , Géis/química , Humanos , Concentração de Íons de Hidrogênio , Transição de Fase , Proteólise , Especificidade por Substrato , Trombina/químicaRESUMO
The anaphase-promoting complex (APC) regulates the eukaryotic cell cycle by targeting specific proteins for proteasomal degradation. Its activity must be strictly controlled to ensure proper cell cycle progression. The co-activator proteins Cdc20 and Cdh1 are required for APC activity and are important regulatory targets. Recently, budding yeast Acm1 was identified as a Cdh1 binding partner and APC(Cdh1) inhibitor. Acm1 disappears in late mitosis when APC(Cdh1) becomes active and contains conserved degron-like sequences common to APC substrates, suggesting it could be both an inhibitor and substrate. Surprisingly, we found that Acm1 proteolysis is independent of APC. A major determinant of Acm1 stability is phosphorylation at consensus cyclin-dependent kinase sites. Acm1 is a substrate of Cdc28 cyclin-dependent kinase and Cdc14 phosphatase both in vivo and in vitro. Mutation of Cdc28 phosphorylation sites or conditional inactivation of Cdc28 destabilizes Acm1. In contrast, inactivation of Cdc14 prevents Acm1 dephosphorylation and proteolysis. Cdc28 stabilizes Acm1 in part by promoting binding of the 14-3-3 proteins Bmh1 and Bmh2. We conclude that the opposing actions of Cdc28 and Cdc14 are primary factors limiting Acm1 to the interval from G(1)/S to late mitosis and are capable of establishing APC-independent expression patterns similar to APC substrates.