Olli Silvennoinen

JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin.

Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid cells through interaction with its receptor (EPOR). Although EPOR is a member of the cytokine receptor superfamily and lacks a kinase domain, EPO induces tyrosine phosphorylation, which is correlated with gene transcription and mitogenesis. Here we demonstrate that EPO induces tyrosine phosphorylation of JAK2 kinase and activates its in vitro autophosphorylation. Using EPOR mutants, phosphorylation and activation of kinase activity correlate with the induction of mitogenesis. Furthermore, JAK2 physically associates with a membrane-proximal region of the EPOR cytoplasmic domain that is required for biological activity. The results support the hypothesis that JAK2 is the kinase that couples EPO binding to tyrosine phosphorylation and mitogenesis.

Identification of JAK2 as a growth hormone receptor-associated tyrosine kinase

Growth hormone receptor (GHR) forms a complex with a tyrosine kinase, suggesting involvement of a ligand-activated tyrosine kinase in intracellular signaling by growth hormone (GH). Here we identify JAK2, a nonreceptor tyrosine kinase, as a GHR-associated tyrosine kinase. Immunological approaches were used to establish GH-dependent complex formation between JAK2 and GHR, activation of JAK2 tyrosine kinase activity, and tyrosyl phosphorylation of both JAK2 and GHR. The JAK2-GHR and JAK2-erythropoietin receptor interactions described here and in the accompanying paper provide a molecular basis for involvement of tyrosyl phosphorylation in physiological responses to these ligands and suggest a shared signaling mechanism among members of the cytokine/hematopoietin receptor family.

Signaling by the cytokine receptor superfamily: JAKs and STATs.

A variety of cytokines, lymphokines and growth factors function by interacting with receptors that are members of the cytokine receptor superfamily. These receptors share extracellular motifs and have limited similarity in their cytoplasmic domains. Although lacking catalytic domains, this family of receptors couples ligand binding with the induction of tyrosine phosphorylation. Recent studies have shown that this is mediated by members of the Janus kinase (JAK) family of cytoplasmic protein tyrosine kinases. JAKs physically associate with the membrane-proximal region of the ligand-bound receptor, leading to their tyrosine phosphorylation and activation. The activated JAKs phosphorylate the receptors as well as cytoplasmic proteins belonging to a family of transcription factors called the signal transducers and activators of transcription (STATs), providing a novel signaling pathway that is shared by all members of the cytokine receptor superfamily.

Stat4, a novel gamma interferon activation site-binding protein expressed in early myeloid differentiation

Interferon regulation of gene expression is dependent on the tyrosine phosphorylation and activation of the DNA-binding activity of two related proteins of 91 kDa (STAT1) and/or 113 kDa (STAT2). Recent studies have suggested that these proteins are substrates of Janus kinases and that proteins related in STAT1 are involved in a number of signalling pathways, including those activated in myeloid cells by erythropoietin and interleukin-3 (IL-3). To clone STAT-related proteins from myeloid cells, degenerate oligonucleotides were used in PCRs to identify novel family members expressed in myeloid cells. This approach allowed the identification and cloning of the Stat4 gene, which is 52% identical to STAT1. Unlike STAT1, Stat4 expression is restricted but includes myeloid cells and spermatogonia. In the erythroid lineage, Stat4 expression is differentially regulated during differentiation. Functionally, Stat4 has the properties of other STAT family genes. In particular, cotransfection of expression constructs for Stat4 and Jak1 and Jak2 results in the tyrosine phosphorylation of Stat4 and the acquisition of the ability to bind to the gamma interferon (IFN-gamma)-activated sequence of the interferon regulatory factor 1 (IRF-1) gene. Stat4 is located on mouse chromosome 1 and is tightly linked to the Stat1 gene, suggesting that the genes arose by gene duplication. Unlike Stat1, neither IFN-alpha nor IFN-gamma activates Stat4. Nor is Stat4 activated in myeloid cells by a number of cytokines, including erythropoietin, IL-3, granulocyte colony-stimulating factor, stem cell factor, colon-stimulating factor 1, hepatocyte growth factor, IL-2, IL-4, and IL-6.

Identification of a carbonic anhydrase-like domain in the extracellular region of RPTP gamma defines a new subfamily of receptor tyrosine phosphatases.

The tyrosine phosphatase RPTP gamma is a candidate tumor suppressor gene since it is located on human chromosome 3p14.2-p21 in a region frequently deleted in certain types of renal and lung carcinomas. In order to evaluate its oncogenic potential and to explore its normal in vivo functions, we have isolated cDNAs and deduced the complete sequences of both human and murine RPTP gamma. The murine RPTP gamma gene has been localized to chromosome 14 to a region syntenic to the location of the human gene. Northern (RNA) blot analysis reveals the presence of two major transcripts of 5.5 and 8.5 kb in a variety of murine tissues. In situ hybridization analysis reveals that RPTP gamma mRNA is expressed in specific regions of the brain and that the localization of RPTP gamma changes during brain development. RPTP gamma is composed of a putative extracellular domain, a single transmembrane domain, and a cytoplasmic portion with two tandem catalytic tyrosine phosphatase domains. The extracellular domain contains a stretch of 266 amino acids with striking homology to the zinc-containing enzyme carbonic anhydrase (CAH), indicating that RPTP gamma and RPTP beta (HPTP zeta) represent a subfamily of receptor tyrosine phosphatases. We have constructed a model for the CAH-like domain of RPTP gamma based upon the crystal structure of CAH. It appears that 11 of the 19 residues that form the active site of CAH are conserved in RPTP gamma. Yet only one of the three His residues that ligate the zinc atom and are required for catalytic activity is conserved. On the basis of this model we propose that the CAH-like domain of RPTP gamma may have a function other than catalysis of hydration of metabolic CO2.

Interaction between the components of the interferon gamma receptor complex

Interferon γ (IFN-γ) signals through a multimeric receptor complex consisting of two different chains: the IFN-γ receptor binding subunit (IFN-γR, IFN-γR1), and a transmembrane accessory factor (AF-1, IFN-γR2) necessary for signal transduction. Using cell lines expressing different cloned components of the IFN-γ receptor complex, we examined the function of the receptor components in signal transduction upon IFN-γ treatment. A specific IFN-γR2:IFN-γ cross-linked complex was observed in cells expressing both IFN-γR1 and IFN-γR2 indicating that IFN-γR2 (AF-1) interacts with IFN-γ and is closely associated with IFN-γR1. We show that the intracellular domain of IFN-γR2 is necessary for signaling. Cells coexpressing IFN-γR1 and truncated IFN-γR2, lacking the COOH-terminal 51 amino acids (residues 286-337), or cells expressing IFN-γR1 alone were unresponsive to IFN-γ treatment as measured by MHC class I antigen induction. Jak1, Jak2, and Stat1α were activated, and IFN-γR1 was phosphorylated only in cells expressing both IFN-γR1 and IFN-γR2. Jak2 kinase was shown to associate with the intracellular domain of the IFN-γR2.

The expression of a novel receptor-type tyrosine phosphatase suggests a role in morphogenesis and plasticity of the nervous system

Analysis of the localization of receptor-type protein tyrosine phosphatase-beta (RPTP-beta) by in situ hybridization and immunocytochemistry indicates that it is predominantly expressed in the developing central nervous system (CNS). RPTP-beta is highly expressed in radial glia and other forms of glial cells that play an important role during development. The immunoreactivity localizes to the radial processes of these cells, which act as guides during neuronal migration and axonal elongation. The pattern of RPTP-beta expression changes with the progression of glial cell differentiation. In the adult, high levels of RPTP-beta are seen in regions of the brain where there is continued neurogenesis and neurite outgrowth. The spatial and temporal patterns of RPTP-beta expression suggest that this receptor phosphatase plays a role in morphogenesis and plasticity of the nervous system.

The catalytic activity of the CD45 membrane-proximal phosphatase domain is required for TCR signaling and regulation

Cell surface expression of CD45, a receptor‐like protein tyrosine phosphatase (PTPase), is required for T cell antigen receptor (TCR)‐mediated signal transduction. Like the majority of transmembrane PTPases, CD45 contains two cytoplasmic phosphatase domains, whose relative in vivo function is not known. Site‐directed mutagenesis of the individual catalytic residues of the two CD45 phosphatase domains indicates that the catalytic activity of the membrane‐proximal domain is both necessary and sufficient for restoration of TCR signal transduction in a CD45‐deficient cell. The putative catalytic activity of the distal phosphatase domain is not required for proximal TCR‐mediated signaling events. Moreover, in the context of a chimeric PTPase receptor, the putative catalytic activity of the distal phosphatase domain is not required for ligand‐induced negative regulation of PTPase function. We also demonstrate that the phosphorylation of the C‐terminal tyrosine of Lck, a site of negative regulation, is reduced only when CD45 mutants with demonstrable in vitro phosphatase activity are introduced into the CD45‐deficient cells. These results demonstrate that the phosphatase activity of CD45 is critical for TCR signaling, and for regulating the levels of C‐terminal phosphorylated Lck molecules.