Isao Oishi

Identification of a Member of the MAPKKK Family as a Potential Mediator of TGF-β Signal Transduction

The mitogen-activated protein kinase (MAPK) pathway is a conserved eukaryotic signaling module that converts receptor signals into various outputs. MAPK is activated through phosphorylation by MAPK kinase (MAPKK), which is first activated by MAPKK kinase (MAPKKK). A genetic selection based on a MAPK pathway in yeast was used to identify a mouse protein kinase (TAK1) distinct from other members of the MAPKKK family. TAK1 was shown to participate in regulation of transcription by transforming growth factor-β (TGF-β). Furthermore, kinase activity of TAK1 was stimulated in response to TGF-β and bone morphogenetic protein. These results suggest that TAK1 functions as a mediator in the signaling pathway of TGF-β superfamily members.

The receptor tyrosine kinase Ror2 is involved in non-canonical Wnt5a/JNK signalling pathway

Background: Ror2 is an orphan receptor, belonging to the Ror family of receptor tyrosine kinases. Although Ror2 has been shown to play crucial roles in developmental morphogenesis, the precise signalling events that Ror2 mediates remain elusive. Since Ror2 possesses an extracellular cysteine‐rich domain (CRD) that resembles the Wnt‐binding sites of the Frizzled (Fz) proteins, it is conceivable that Ror2 interacts with members of the Wnt family.

Mouse Ror2 receptor tyrosine kinase is required for the heart development and limb formation

A mouse receptor tyrosine kinase (RTK), mRor2, which belongs to the Ror‐family of RTKs consisting of at least two structurally related members, is primarily expressed in the heart and nervous system during mouse development. To elucidate the function of mRor2, we generated mice with a mutated mRor2 locus.

Regulation of the antioncogenic Chk2 kinase by the oncogenic Wip1 phosphatase

The antioncogenic Chk2 kinase plays a crucial role in DNA damage-induced cell-cycle checkpoint regulation. Here we show that Chk2 associates with the oncogenic protein Wip1 (wild-type p53-inducible phosphatase 1) (PPM1D), a p53-inducible protein phosphatase. Phosphorylation of Chk2 at threonine68 (Thr68), a critical event for Chk2 activation, which is normally induced by DNA damage or overexpression of Chk2, is inhibited by expression of wild-type (WT), but not a phosphatase-deficient mutant (D314A) of Wip1 in cultured cells. Furthermore, an in vitro phosphatase assay revealed that Wip1 (WT), but not Wip1 (D314A), dephosphorylates Thr68 on phosphorylated Chk2 in vitro, resulting in the inhibition of Chk2 kinase activity toward glutathione S-transferase-Cdc25C. Moreover, inhibition of Wip1 expression by RNA interference results in abnormally sustained Thr68 phosphorylation of Chk2 and increased susceptibility of cells in response to DNA damage, indicating that Wip1 acts as a negative regulator of Chk2 in response to DNA damage.

Expression of the receptor tyrosine kinase genes, Ror1 and Ror2, during mouse development.

In mammals, the Ror-family receptor tyrosine kinases consist of two structurally related proteins, Ror1 and Ror2, characterized by the extracellular Frizzled-like cysteine-rich domain and membrane proximal kringle domains. As an attempt to gain insights into their roles in mouse development, expression patterns of Ror1 and Ror2 during early embryogenesis were examined and compared. Interestingly, at early stages, Ror1 and Ror2 exhibit similar expression patterns in the developing face, including the frontonasal process and pharyngeal arches, which are derived from cephalic neural crest cells. On the other hand, they exhibit different expression patterns in the developing limbs and brain, where the expression of Ror2 was detected broadly compared with that of Ror1. At a later stage, both genes are expressed in a similar fashion in the developing heart and lung, yet in a distinct manner in the brain and eye.

Spatio‐temporally regulated expression of receptor tyrosine kinases, mRor1, mRor2, during mouse development: implications in development and function of the nervous system

Drosophila neurospecific receptor tyrosine kinases (RTKs), Dror and Dnrk, as well as Ror1 and Ror2 RTKs, isolated from human neuroblastoma, have been identified as a structurally related novel family of RTKs (Ror‐family RTKs). Thus far, little is known about the expression and function of mammalian Ror‐family RTKs.

Loss of mRor1 Enhances the Heart and Skeletal Abnormalities in mRor2-Deficient Mice: Redundant and Pleiotropic Functions of mRor1 and mRor2 Receptor Tyrosine Kinases

ABSTRACT The mammalian Ror family of receptor tyrosine kinases consists of two structurally related proteins, Ror1 and Ror2. We have shown that mRor2-deficient mice exhibit widespread skeletal abnormalities, ventricular septal defects in the heart, and respiratory dysfunction, leading to neonatal lethality (S. Takeuchi, K. Takeda, I. Oishi, M. Nomi, M. Ikeya, K. Itoh, S. Tamura, T. Ueda, T. Hatta, H. Otani, T. Terashima, S. Takada, H. Yamamura, S. Akira, and Y. Minami, Genes Cells 5:71–78, 2000). Here we show thatmRor1-deficient mice have no apparent skeletal or cardiac abnormalities, yet they also die soon after birth due to respiratory dysfunction. Interestingly,mRor1/mRor2 double mutant mice show markedly enhanced skeletal abnormalities compared withmRor2 mutant mice. Furthermore, double mutant mice also exhibit defects not observed in mRor2 mutant mice, including a sternal defect, dysplasia of the symphysis of the pubic bone, and complete transposition of the great arteries. These results indicate that mRor1 and mRor2 interact genetically in skeletal and cardiac development.

Modulation of GDF5/BRI-b signalling through interaction with the tyrosine kinase receptor Ror2

The brachydactylies are a group of inherited disorders of the hands characterized by shortened digits. Mutations in the tyrosine kinase receptor Ror2 cause brachydactyly type B (BDB). Mutations in GDF5, a member of the BMP/TGF‐β ligand family, cause brachydactyly type C (BDC) whereas mutations in the receptor for GDF5, BRI‐b, cause brachydactyly type A2 (BDA2). There is considerable degree of phenotypic overlap between the subtypes BDB, BDC and BDA2. Here we demonstrate that all three components are involved in GDF5 induced regulation of chondrogenesis. We show that Ror2 (tyrosine kinase receptor) and BRI‐b (serine/threonine kinase receptor) form a ligand independent heteromeric complex. The frizzled‐like‐CRD domain of Ror2 is required for this complex. Within that complex Ror2 gets transphosphorylated by BRI‐b. We show that Ror2 modulates GDF5 signalling by inhibition of Smad1/5 signalling and by activating a Smad‐independent pathway. Both pathways however, are needed for chondrogenic differentiation as demonstrated in ATDC5 cells. The functional interaction of Ror2 with GDF5 and BRI‐b was genetically confirmed by the presence of epistatic effects in crosses of Ror2, BRI‐b and Gdf5 deficient mice. These results indicate for the first time a direct interaction of Ser/Thr‐ and Tyr‐Kinase receptors and provide evidence for modulation of the Smad‐pathway and GDF5 triggered chondrogenesis.

Expression and Function of the Ror‐Family Receptor Tyrosine Kinases During Development: Lessons from Genetic Analyses of Nematodes, Mice, and Humans

Abstract Receptor tyrosine kinases (RTKs) play crucial roles in various developmental processes. Ror‐family RTKs are characterized by the intracellular tyrosine kinase domains, highly related to those of the Trk‐family RTKs, and by the extracellular Frizzled‐like cysteine‐rich domains (CRDs) and Kringle domains. Rors are evolutionally conserved among Caenorhabditis elegans, Aplysia, Drosophila melanogaster, Xenopus, mice, and humans. In D. melanogaster and mammals, pairs of structurally related Rors are found, while a single Ror protein is identified in C. elegans or Aplysia. In Aplysia and D. melanogaster, Rors are expressed exclusively in developing nervous systems. On the other hand, rather widespread expression of Rors was observed in C. elegans and mammals. Mutations in Ror of C. elegans cause inappropriate axon outgrowth as well as defects in cell migration and asymmetric cell division. It has also been reported that the nematode Ror possesses kinase‐dependent and kinase‐independent functions. Mouse Rors, Ror1, and Ror2, are expressed mainly in migrating neural crest cells and mesenchymal cells, and Ror2‐deficient mice exhibit skeletal abnormalities and ventricular septal defects in the heart. Although Ror1‐deficient mice exhibit no apparent skeletal or cardiac abnormalities, Ror1/Ror2 double mutant mice show markedly enhanced skeletal and cardiac abnormalities compared with Ror2 mutant mice, indicating genetic interaction of Ror1 and Ror2. In humans, mutations within Ror2 have been found in two genetic skeletal disorders, recessive Robinow syndrome and dominant Brachydactyly type B (BDB), further emphasizing critical functions of Ror2 during developmental morphogenesis. In this article, we also discuss the signaling machinery mediated by Ror‐family RTKs with a particular emphasis on our recent structure‐function analyses of Ror‐family RTKs.

A novel Drosophila nuclear protein serine/threonine kinase expressed in the germline during its establishment

Nuclear protein kinases are believed to play important roles in regulating gene expression. We report here the identification and developmental expression of Dmnk (Drosophila maternal nuclear kinase), a Drosophila gene encoding a putative nuclear protein serine/threonine kinase with no apparent homology to previously identified protein kinases and located at 38B on the second chromosome. Dmnk mRNAs are transcribed in nurse cells and are subsequently localized in the anterior of oocytes during oogenesis, in a manner similar to several maternal transcripts regulating oogenesis and early embryogenesis. At early cleavage-stages Dmnk transcripts are transiently present throughout the embryo, but become restricted to the posterior pole and then to the newly-formed primordial germ cells (pole cells) by the blastoderm stage. The transcripts are sustained in the pole cells during gastrulation until they pass through the midgut pocket wall into the body cavity. Immunostaining with specific antibodies revealed that Dmnk proteins are localized to the nuclei in a speckled pattern. Dmnk proteins become detectable in both somatic and germ line cell nuclei upon their arrival at the periplasm of the syncytial embryo, but then disappear from the somatic cell nuclei. Consistent with mRNA expression, Dmnk proteins in pole cell nuclei are sustained during gastrulation. Taken together, Dmnk represents a novel class of nuclear protein kinases and the dynamic expression of Dmnk suggests a role in germ line establishment. The results are discussed in the light of recent findings concerning germ line establishment in Caenorhabditis and Drosophila.