Hironobu Asao

Cutting edge : the common gamma-chain is an indispensable subunit of the IL-21 receptor complex

The common γ-chain (γc) is an indispensable subunit of the functional receptor complexes for IL-4, IL-7, IL-9, and IL-15 as well as IL-2. Here we show that the γc is also shared with the IL-21R complex. Although IL-21 binds to the IL-21R expressed on γc-deficient ED40515− cells, IL-21 is unable to transduce any intracytoplasmic signals. However, in EDγ-16 cells, a γc-transfected ED40515− cell line, IL-21 binds to the IL-21R and can activate Janus kinase (JAK)1, JAK3, STAT1, and STAT3. The chemical cross-linking study reveals the direct binding of IL-21 to the γc. These data clearly demonstrate that the γc is an indispensable subunit of the functional IL-21R complex.

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.

Promoting bone morphogenetic protein signaling through negative regulation of inhibitory Smads

Inhibitory Smads, i.e. Smad6 and Smad7, are potent antagonists of the BMP–Smad pathway by interacting with activated bone morphogenetic protein (BMP) type I receptors and thereby preventing the activation of receptor‐regulated Smads, or by competing with activated R‐Smads for heteromeric complex formation with Smad4. The molecular mechanisms that underlie the regulation of I‐Smad activity have remained elusive. Here we report the identification of a cytoplasmic protein, previously termed associated molecule with the SH3 domain of STAM (AMSH), as a direct binding partner for Smad6. AMSH interacts with Smad6, but not with R‐ and Co‐Smads, upon BMP receptor activation in cultured cells. Consistent with this finding, stimulation of cells with BMP induces a co‐localization of Smad6 with AMSH in the cytoplasm. Ectopic expression of AMSH prolongs BMP‐induced Smad1 phosphorylation, and potentiates BMP‐induced activation of transcriptional reporter activity, growth arrest and apoptosis. The data strongly suggest that the molecular mechanism by which AMSH exerts its action is by inhibiting the binding of Smad6 to activated type I receptors or activated R‐Smads.

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.

Ligand-independent degradation of epidermal growth factor receptor involves receptor ubiquitylation and hgs, an adaptor whose ubiquitin-interacting motif targets ubiquitylation by Nedd4

Ligand‐dependent endocytosis of the epidermal growth factor receptor (EGFR) involves recruitment of a ubiquitin ligase, and sorting of ubiquitylated receptors to lysosomal degradation. By studying Hgs, a mammalian homolog of a yeast vacuolar‐sorting adaptor, we provide information on the less understood, ligand‐independent pathway of receptor endocytosis and degradation. Constitutive endocytosis involves receptor ubiquitylation and translocation to Hgs‐containing endosomes. Whereas the lipid‐binding motif of Hgs is necessary for receptor endocytosis, the ubiquitin‐interacting motif negatively regulates receptor degradation. We demonstrate that the ubiquitin‐interacting motif is endowed with two functions: it binds ubiquitylated proteins and it targets self‐ubiquitylation by recruiting Nedd4, an ubiquitin ligase previously implicated in endocytosis. Based upon the dual function of the ubiquitin‐interacting motif and its wide occurrence in endocytic adaptors, we propose a ubiquitin‐interacting motif network that relays ubiquitylated membrane receptors to lysosomal degradation through successive budding events.

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.

Elevated oxidative stress in erythrocytes due to a SOD1 deficiency causes anaemia and triggers autoantibody production

Reactive oxygen species are involved in the aging process and diseases. Despite the important role of Cu/Zn SOD (superoxide dismutase) encoded by SOD1, SOD1-/- mice appear to grow normally under conventional breeding conditions. In the present paper we report on a novel finding showing a distinct connection between oxidative stress in erythrocytes and the production of autoantibodies against erythrocytes in SOD1-/- mice. Evidence is presented to show that SOD1 is primarily required for maintaining erythrocyte lifespan by suppressing oxidative stress. A SOD1 deficiency led to an increased erythrocyte vulnerability by the oxidative modification of proteins and lipids, resulting in anaemia and compensatory activation of erythropoiesis. The continuous destruction of oxidized erythrocytes appears to induce the formation of autoantibodies against certain erythrocyte components, e.g. carbonic anhydrase II, and the immune complex is deposited in the glomeruli. The administration of an antioxidant, N-acetylcysteine, suppressed erythrocyte oxidation, ameliorated the anaemia, and inhibited the production of autoantibodies. These data imply that a high level of oxidative stress in erythrocytes increases the production of autoantibodies, possibly leading to an autoimmune response, and that the intake of antioxidants would prevent certain autoimmune responses by maintaining an appropriate redox balance in erythrocytes.

The Role of G-Protein-Coupled Receptor Kinase 5 in Pathogenesis of Sporadic Parkinson's Disease

Sporadic Parkinsons disease (sPD) is a common neurodegenerative disorder, characterized by selective degeneration of dopaminergic neurons in the substantia nigra. Although the pathogenesis of the disease remains undetermined, phosphorylation of α-synuclein and its oligomer formation seem to play a key role. However, the protein kinase(s) involved in the phosphorylation in the pathogenesis of sPD has not been identified. Here, we found that G-protein-coupled receptor kinase 5 (GRK5) accumulated in Lewy bodies and colocalized with α-synuclein in the pathological structures of the brains of sPD patients. In cotransfected cells, GRK5 phosphorylated Ser-129 of α-synuclein at the plasma membrane and induced translocation of phosphorylated α-synuclein to the perikaryal area. GRK5-catalyzed phosphorylation also promoted the formation of soluble oligomers and aggregates of α-synuclein. Genetic association study revealed haplotypic association of the GRK5 gene with susceptibility to sPD. The haplotype contained two functional single-nucleotide polymorphisms, m22.1 and m24, in introns of the GRK5 gene, which bound to YY1 (Yin Yang-1) and CREB-1 (cAMP response element-binding protein 1), respectively, and increased transcriptional activity of the reporter gene. The results suggest that phosphorylation of α-synuclein by GRK5 plays a crucial role in the pathogenesis of sPD.