Toshikazu Takeshita

Hrs Is Associated with STAM, a Signal-transducing Adaptor Molecule ITS SUPPRESSIVE EFFECT ON CYTOKINE-INDUCED CELL GROWTH

We previously reported a new type of signal-transducing adaptor molecule, STAM, which was shown to be involved in cytokine-mediated intracellular signal transduction. In this study, we molecularly cloned a 110-kDa phosphotyrosine protein inducible by stimulation with interleukin 2 (IL-2). The 110-kDa molecule was found to be a human counterpart of mouse Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and to be associated with STAM. Tyrosine phosphorylation of Hrs is induced rapidly after stimulation with IL-2 and granulocyte-macrophage colony-stimulating factor as well as hepatocyte growth factor. The mutual association sites of Hrs and STAM include highly conserved coiled-coil sequences, suggesting that their association is mediated by the coiled-coil structures. Exogenous introduction of the wild-type Hrs significantly suppressed DNA synthesis upon stimulation with IL-2 and granulocyte-macrophage colony-stimulating factor, while the Hrs mutant deleted of the STAM-binding site lost such suppressive ability. These results suggest that Hrs counteracts the STAM function which is critical for cell growth signaling mediated by the cytokines.

Hgs (Hrs), a FYVE Domain Protein, Is Involved in Smad Signaling through Cooperation with SARA

ABSTRACT Smad proteins are effector molecules that transmit signals from the receptors for the transforming growth factor β (TGF-β) superfamily to the nucleus; of the Smad proteins, Smad2 and Smad4 are essential components for mouse early embryogenesis. We demonstrated that Hgs, a FYVE domain protein, binds to Smad2 in its C-terminal half and cooperates with another FYVE domain protein, the Smad anchor for receptor activation (SARA), to stimulate activin receptor-mediated signaling through efficient recruitment of Smad2 to the receptor. Furthermore, a LacZ knock-in allele of the C-terminal half-deletion mutant of mouse Hgs was created by gene targeting. The introduced mutation causes an embryonic lethality between embryonic days 8.5 and 10.5. Mutant cells showed significantly decreased responses to stimulation with activin and TGF-β. These findings suggest that the two FYVE domain proteins, Hgs and SARA, are prerequisites for receptor-mediated activation of Smad2.

The Common γ-Chain for Multiple Cytokine Receptors

Publisher Summary The chapter focuses on the common γ chain for multiple cytokine receptors. The signal transducers are composed of both common and specific molecules for the cytokines, resulting in overlapping and pleiotropic functions on various target cells in similar situations to other cytokines that share the common receptor subunits. Elucidation of the modes of signal transduction from IL-2Rγ together with the accompanied subunits will lead to the demonstration of regulatory mechanisms of early T-cell development as well as cellular responses to the ligands. Since no intrinsic effectors function has been seen with cytokine receptors sharing IL-2Rγ, signal transducers are assumed to be associated with the cytoplasmic domains of the receptor subunits. Molecular identification of IL-2Rγ has led to knowledge for understanding the structures and signal-transducing functions of various cytokine receptors, and particularly has made a great contribution toward elucidation of the molecular mechanisms of human XSCID occurrence. IL-BRγ, so-called as the “γc-chain”, is utilized as a common receptor subunit among multiple cytokines such as IL- 2, IL-4, IL-7, IL-9, and IL-15. Mutations of IL-2Rγ in patients with XSCID cause dysfunction of these cytokines, resulting in impairment of early T-cell development, a typical feature of XSCID. These cytokine receptors contain specific subunit(s) for each receptor along with the γc-chain. The γc-chain participates in increasing ligands-binding affinities, except for IL-9, and in intracellular signal transduction for all these cytokines.

STAM, Signal Transducing Adaptor Molecule, Is Associated with Janus Kinases and Involved in Signaling for Cell Growth and c-myc Induction

We previously identified a putative signal transducing adaptor molecule, named STAM, that contains an Src homology 3 (SH3) domain and immunoreceptor tyrosine-based activation motif (ITAM). In this report, we demonstrate the functional significance of STAM in cytokine-mediated signal transduction. STAM is associated with Jak3 and Jak2 tyrosine kinases via its ITAM region and phosphorylated by Jak3 and Jak2 upon stimulation with IL-2 and GM-CSF, respectively. An SH3 deletion mutant of STAM confers a dominant-negative effect on DNA synthesis mediated by IL-2 and GM-CSF. Furthermore, the wild-type STAM, but not STAM mutants deleted of SH3 and ITAM, significantly enhances c-myc induction mediated by IL-2 and GM-CSF. These results strongly implicate STAM in the signaling pathways for cell growth and c-myc induction immediately downstream of the Jaks associated with the cytokine receptors.

Possible involvement of a novel STAM-associated molecule "AMSH" in intracellular signal transduction mediated by cytokines.

STAM containing an SH3 (Src homology 3) domain and an immunoreceptor tyrosine-based activation motif was previously revealed to be implicated in signaling pathways immediately downstream of Jak2 and Jak3 tyrosine kinases associated with cytokine receptors. We molecularly cloned a novel molecule interacting with the SH3 domain of STAM, which was named AMSH (associatedmolecule with the SH3 domain of STAM). AMSH contains a putative bipartite nuclear localization signal and a homologous region of a c-Jun activation domain-binding protein 1 (JAB1) subdomain in addition to a binding site for the SH3 domain of STAM. AMSH mutant deleted of the C-terminal half conferred dominant negative effects on signaling for DNA synthesis and c-myc induction mediated by interleukin 2 and granulocyte macrophage-colony-stimulating factor. These results suggest that AMSH plays a critical role in the cytokine-mediated intracellular signal transduction downstream of the Jak2/Jak3·STAM complex.

Molecular cloning, chromosome mapping and characterization of the mouse CRTH2 gene, a putative member of the leukocyte chemoattractant receptor family.

We have cloned the mouse CRTH2 (chemoattractant receptor-homologous molecule expressed on TH2 cells) gene encoding a putative leukocyte chemoattractant receptor, of which human homologue is expressed selectively in Th2 but not in Th1 clones among T cell clones. The deduced amino-acid sequence of mouse CRTH2 bears 77% identity with its human homologue. Phylogenetic analysis suggested that both mouse and human CRTH2 are closely related to the N-formyl peptide receptor and the C5a receptor among leukocyte chemoattractant receptors. The mouse CRTH2 gene was mapped on chromosome 19c with FISH, where no other genes for leukocyte chemoattractant receptors are mapped. RT-PCR analysis revealed that mouse CRTH2 mRNA is expressed in various cell lineages, including both hematopoietic and non-hematopoietic cell lines. Expression was also observed in liver, lung, kidney, brain, heart, thymus, and spleen. These results suggest that mouse CRTH2 functions in a variety of cells, making the effects of CRTH2 pleiotropic.

STAM2, a new member of the STAM family, binding to the Janus kinases

We here cloned a cDNA encoding STAM2, a new member of the STAM family, which contains an SH3 domain and ITAM. STAM2 like STAM1 is associated with Jak2 and Jak3, and involved in the signaling for DNA synthesis and c‐myc induction mediated by IL‐2 and GM‐CSF. Co‐expression of the SH3 deletion mutants of STAM1 and STAM2 induces an additive effect on suppressing DNA synthesis upon stimulation with IL‐2 and GM‐CSF, suggesting that STAM1 and STAM2 exhibit compensatory effects on the signaling pathways downstream of Jak2 and Jak3 upon stimulation with GM‐SCF and IL‐2, respectively.

Interleukin 2-induced activation of JAK3: Possible involvement in signal transduction for c-myc induction and cell proliferation

We have investigated the role of JAK3 in interleukin 2 (IL‐2)‐induced signal transduction with a human T cell line, ED40515(−), lacking expression of the IL‐2 receptor γ chain and its sublines transfected with wild‐type or mutant cDNAs of the IL‐2 receptor γ chain. Our results demonstrated that the membrane‐proximal cytoplasmic region, encompassing the src homology region 2 (SH2)‐like subdomain, of the γ chain is essential for association and activation of JAK3. Furthermore, IL‐2‐induced activation of JAK3 paralleled induction of the c‐myc gene and DNA synthesis but not induction of the c‐fos and c‐jun genes. These results support the hypothesis that JAK3 plays a pivotal role in the IL‐2 receptor‐mediated signals for cell growth.

Loss of Hippocampal CA3 Pyramidal Neurons in Mice Lacking STAM1

ABSTRACT STAM1, a member of the STAM (signal transducing adapter molecule) family, has a unique structure containing a Src homology 3 domain and ITAM (immunoreceptor tyrosine-based activation motif). STAM1 was previously shown to be associated with the Jak2 and Jak3 tyrosine kinases and to be involved in the regulation of intracellular signal transduction mediated by interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. Here we generated mice lacking STAM1 by using homologous recombination with embryonic stem cells. STAM1−/− mice were morphologically indistinguishable from their littermates at birth. However, growth retardation in the third week after birth was observed for the STAM1−/− mice. Unexpectedly, despite the absence of STAM1, hematopoietic cells, including T- and B-lymphocyte and other hematopoietic cell populations, developed normally and responded well to several cytokines, including IL-2 and GM-CSF. However, histological analyses revealed the disappearance of hippocampal CA3 pyramidal neurons in STAM1−/− mice. Furthermore, we observed that primary hippocampal neurons derived from STAM1−/− mice are vulnerable to cell death induced by excitotoxic amino acids or an NO donor. These data suggest that STAM1 is dispensable for cytokine-mediated signaling in lymphocytes but may be involved in the survival of hippocampal CA3 pyramidal neurons.

IL-2-dependent in vivo and in vitro tyrosine phosphorylation of IL-2 receptor γ chain

We previously reported a molecule, p64, which was tentatively named the γ chain, coprecipitable with the β chain of human interleukin‐2 receptor (IL‐2R). The present study demonstrated that the γ chain, as well as the β chain expressed on IL‐2‐responsive cells, is phosphorylated on tyrosine residues in an IL‐2‐dependent manner in vivo and in vitro. The in vivo tyrosine phosphorylation of both chains was similarly induced within 1 min after IL‐2 stimulation, and their in vitro tyrosine phosphorylation with the anti‐IL‐2Rβ antibody‐directed immunocomplex was also increased by treatment of cells with IL‐2. These results suggest that a tyrosine kinase is associated with the βγ subunit complex, of which activation by IL‐2 may result in transduction of intracellular signals.