Tomoaki Mizuno

The Caenorhabditis elegans MAPK phosphatase VHP-1 mediates a novel JNK-like signaling pathway in stress response

Mitogen‐activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli and to a wide variety of environmental stresses. MAPK cascades can be inactivated at the MAPK activation step by members of the MAPK phosphatase (MKP) family. However, the components that act in MKP‐regulated pathways have not been well characterized in the context of whole organisms. Here we characterize the Caenorhabditis elegans vhp‐1 gene, encoding an MKP that acts preferentially on the c‐Jun N‐terminal kinase (JNK) and p38 MAPKs. We found that animals defective in vhp‐1 are arrested during larval development. This vhp‐1 defect is suppressed by loss‐of‐function mutations in the kgb‐1, mek‐1, and mlk‐1 genes encoding a JNK‐like MAPK, an MKK7‐type MAPKK, and an MLK‐type MAPKKK, respectively. The genetic and biochemical data presented here demonstrate a critical role for VHP‐1 in the KGB‐1 pathway. Loss‐of‐function mutations in each component in the KGB‐1 pathway result in hypersensitivity to heavy metals. These results suggest that VHP‐1 plays a pivotal role in the integration and fine‐tuning of the stress response regulated by the KGB‐1 MAPK pathway.

IDENTIFICATION AND CHARACTERIZATION OF DROSOPHILA HOMOLOG OF RHO-KINASE

The Rho family of small GTPases and their associated regulators and targets are essential mediators of diverse morphogenetic events in development. Mammalian Rho-kinase/ROK alpha, one of the targets of Rho, has been shown to bind to Rho in GTP-bound form and to phosphorylate the myosin light chain (MLC) and the myosin-binding subunit (MBS) of myosin phosphatase, resulting in the activation of myosin. Thus, Rho-kinase/ROK alpha has been suggested to play essential roles in the formation of stress fibers and focal adhesions. We have identified the Drosophila homolog of Rho-kinase/ROK alpha, DRho-kinase, which has conserved the basic structural feature of Rho-kinase/ROK alpha consisting of the N-terminal kinase, central coiled-coil and C-terminal pleckstrin homology (PH) domains. A two-hybrid analysis demonstrated that DRho-kinase interacts with the GTP-bound form of the Drosophila Rho. Drho1, at the conserved Rho-binding site. DRho-kinase can phosphorylate MLC and MBS, preferable substrates for bovine Rho-kinase, in vitro. DRho-kinase is ubiquitously expressed throughout development, in a pattern essentially identical to that of Drho1. These results suggest that DRho-kinase is an effector of Drho1.

Cloning of a sensory-kinase-encoding gene that belongs to the two-component regulatory family from the cyanobacterium Synechococcus sp. PCC7942

A screening method employing Escherichia coli was adopted to clone a sensory-kinase (SK)-encoding gene directly from a phylogenetically distant species, the phototrophic cyanobacterium Synechococcus sp. PCC7942. From the Synechococcus chromosomal DNA, we searched for DNA clones which are able to complement phenotypically not only an E. coli envZ mutant for the expression of ompC, but also an E. coli phoR/creC mutant for the expression of alkaline phosphatase. These E. coli genes are known to encode SK. A 0.75-kb DNA fragment was thus cloned under the control of the E. coli lac promoter carried on an E. coli plasmid vector. A larger DNA fragment encompassing an entire open reading frame was then cloned and its complete nucleotide (nt) sequence determined. The nt sequence corresponds to a gene that encodes a 43,280-Da protein of 387 amino acids with a high degree of homology to the bacterial SK. Thus, we succeeded in cloning a SK-encoding gene, which most likely functions in signal transduction in Synechococcus sp. PCC7942. Hence, the gene was designated sasA (Synechococcus adaptive-response SK A). The purified SasA protein was demonstrated in vitro to undergo autophosphorylation.

Role of the Caenorhabditis elegans Shc Adaptor Protein in the c-Jun N-Terminal Kinase Signaling Pathway

ABSTRACT Mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli and a wide variety of environmental stresses. In Caenorhabditis elegans, the stress response is controlled by a c-Jun N-terminal kinase (JNK)-like mitogen-activated protein kinase (MAPK) signaling pathway, which is regulated by MLK-1 MAPK kinase kinase (MAPKKK), MEK-1 MAPK kinase (MAPKK), and KGB-1 JNK-like MAPK. In this study, we identify the shc-1 gene, which encodes a C. elegans homolog of Shc, as a factor that specifically interacts with MEK-1. The shc-1 loss-of-function mutation is defective in activation of KGB-1, resulting in hypersensitivity to heavy metals. A specific tyrosine residue in the NPXY motif of MLK-1 creates a docking site for SHC-1 with the phosphotyrosine binding (PTB) domain. Introduction of a mutation that perturbs binding to the PTB domain or the NPXY motif abolishes the function of SHC-1 or MLK-1, respectively, thereby abolishing the resistance to heavy metal stress. These results suggest that SHC-1 acts as a scaffold to link MAPKKK to MAPKK activation in the KGB-1 MAPK signal transduction pathway.

The Caenorhabditis elegans Ste20-Related Kinase and Rac-Type Small GTPase Regulate the c-Jun N-Terminal Kinase Signaling Pathway Mediating the Stress Response

ABSTRACT Mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli and a wide variety of environmental stresses. In Caenorhabditis elegans, the stress response is controlled by a c-Jun N-terminal kinase (JNK)-like MAPK signaling pathway, which is regulated by MLK-1 MAPK kinase kinase (MAPKKK), MEK-1 MAPKK, and KGB-1 JNK-like MAPK. In this study, we identify the max-2 gene encoding a C. elegans Ste20-related protein kinase as a component functioning upstream of the MLK-1-MEK-1-KGB-1 pathway. The max-2 loss-of-function mutation is defective in activation of KGB-1, resulting in hypersensitivity to heavy metals. Biochemical analysis reveals that MAX-2 activates MLK-1 through direct phosphorylation of a specific residue in the activation loop of the MLK-1 kinase domain. Our genetic data presented here also show that MIG-2 small GTPase functions upstream of MAX-2 in the KGB-1 pathway. These results suggest that MAX-2 and MIG-2 play a crucial role in mediating the heavy metal stress response regulated by the KGB-1 pathway.