RESUMO
The mammalian JNK/p38 MAP kinase kinase kinase MEKK4 and the Saccharomyces cerevisiae Ssk2p are highly homologous. MEKK4 can replace all of the known functions of Ssk2p in yeast, including functioning in the high osmolarity glycerol (HOG) MAPK pathway and the recently described actin recovery pathway. MEKK4 and Ssk2p share a number of conserved domains and appear to be activated by a similar mechanism. Binding of an activating protein to the N-terminal region alleviates auto-inhibition and causes the kinase to auto-phosphorylate, resulting in activation. In this review we will examine the role of the MAP kinase kinase kinase isoform Ssk2p/MEKK4 in the adaptation of both yeast and mammalian systems to specific external stimuli. Recent work has provided a wealth of information about the activation, regulation, and functions of these MEKK kinases to extra-cellular signals. We will also highlight evidence supporting a role for MEKK4 in mediating actin recovery following osmotic shock in mammalian cells.
Assuntos
MAP Quinase Quinase Quinase 4/metabolismo , Pressão Osmótica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Actinas/metabolismo , Adaptação Fisiológica , Processamento Alternativo , Sequência de Aminoácidos , Animais , Ativação Enzimática , Previsões , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , MAP Quinase Quinase Quinase 4/química , MAP Quinase Quinase Quinases , Mamíferos , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Osmotic stress induces activation of an adaptive mitogen-activated protein kinase pathway in concert with disassembly of the actin cytoskeleton by a mechanism that is not understood. We have previously shown that the conserved actin-interacting MAP kinase kinase kinase Ssk2p/MEKK4, a member of the high-osmolarity glycerol (HOG) MAPK pathway of Saccharomyces cerevisiae, mediates recovery of the actin cytoskeleton following osmotic stress. In this study, we have employed in vitro kinase assays to show that Ssk2p kinase activity is activated for the actin recovery pathway via a noncanonical, Ssk1p-independent mechanism. Our work also shows that Ssk2p requires the polarisome proteins Bud6p and Pea2p to promote efficient, polarized actin reassembly but that this requirement can be bypassed by overexpression of Ssk2p. Formin (BNI1 or BNR1) and tropomyosin functions are also required for actin recovery but, unlike for Bud6p and Pea2p, these requirements cannot be bypassed by overexpression of Ssk2p. These results suggest that Ssk2p acts downstream of Bud6p and Pea2p and upstream of tropomyosin to drive actin recovery, possibly by upregulating the actin nucleation activity of the formins.
Assuntos
Actinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Primers do DNA/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Genes Fúngicos , MAP Quinase Quinase Quinases , Sistema de Sinalização das MAP Quinases , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Pressão Osmótica , Plasmídeos/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Tropomiosina/metabolismoRESUMO
Synthetic genetic analysis was improved by eliminating leaky expression of the HIS3 reporter and gene conversion between the HIS3 reporter and his3Delta1. Leaky expression was eliminated using 3-aminotriazole and gene conversion was eliminated by using the Schizosaccharomyces pombe his5+ gene, resulting in a 5- to 10-fold improvement in the efficiency of SGA.
Assuntos
Amitrol (Herbicida)/metabolismo , Proteínas Fúngicas/genética , Conversão Gênica , Histidina/genética , Análise em Microsséries , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Cromossomos Fúngicos , Genoma FúngicoRESUMO
Many proteins are built from structurally and functionally distinct domains. A major goal is to understand how conformational change transmits information between domains in order to achieve biological activity. A two-domain, bi-functional fusion protein has been designed so that the mechanical stress imposed by the folded structure of one subunit causes the other subunit to unfold, and vice versa. The construct consists of ubiquitin inserted into a surface loop of barnase. The distance between the amino and carboxyl ends of ubiquitin is much greater than the distance between the termini of the barnase loop. This topological constraint causes the two domains to engage in a thermodynamic tug-of-war in which only one can exist in its folded state at any given time. This conformational equilibrium, which is cooperative, reversible, and controllable by ligand binding, serves as a model for the coupled binding and folding mechanism widely used to mediate protein-protein interactions and cellular signaling processes. The position of the equilibrium can be adjusted by temperature or ligand binding and is monitored in vivo by cell death. This design forms the basis for a new class of cytotoxic proteins that can be activated by cell-specific effector molecules, and can thus target particular cell types for destruction.
