Biomedical subjects

T C He

Publications and source records attributed to T C He.

23 records · Page 2Linked to original sources

PubMed1994

Dominant negative effects of a carboxy-truncated Jak2 mutant on Epo-induced proliferation and Jak2 activation.

Members of the Janus family of protein tyrosine kinases are emerging as primary, receptor-associated transducing factors among numerous cytokine systems. However, little is understood regarding mechanisms of recruitment of these kinases to receptor complexes and their ligand-dependent activation. To initially address these questions, we have assessed effects of ectopically expressing a carboxy-truncated form of Jak2 (Jak2-829) in Epo-responsive DAER cells. Expression of this truncation mutant at low levels efficiently inhibited both Epo-dependent activation of endogenous Jak2 and Epo-induced mitogenesis (10% to 39% of parental DAER cells). These results suggest that amino-terminal domains of Jak2 may mediate the assembly of Jak2/Epo receptor complexes and that integration of Jak2-829 into receptor complexes may effectively inhibit the activity of oligomeric Jak2/receptor assemblages.

Animals↗

PubMed1994

Inhibition of erythropoietin-induced mitogenesis by a kinase-deficient form of Jak2.

Receptors for a variety of hematopoietins, interferons alpha/beta and gamma, and growth hormone have recently been shown to mediate rapid, ligand-dependent activation of the Janus-type cytosolic protein-tyrosine kinases Jak1, Jak2, and/or tyk-2. This finding extends relatedness among class I and II cytokine receptors to a functional context and provides an initially satisfying mechanistic analogy to protein-tyrosine kinase-encoding receptors of the epidermal growth factor/platelet-derived growth factor/insulin family. Through the construction and expression of a kinase-deficient form of Jak2 (JK2 delta VIII) in interleukin-3 (IL-3)/erythropoietin (Epo)-dependent DAER cells, we have tested whether activation of Jak2 is required for induced mitogenesis via these class I cytokine receptors. Ectopic expression of JK2 delta VIII inhibited Epo- and IL-3-induced activation of endogenous wild-type Jak2, transiently attenuated IL-3-dependent growth, and essentially abrogated Epo-induced proliferation in this model system. These dominant-negative effects provide the first direct experimental evidence for an essential role for Janus kinase activation in mitogenesis and suggest that distinct effectors may mediate IL-3-induced versus Epo-induced pathways.

Amino Acid Sequence↗

PubMed1994

The extended box 2 subdomain of erythropoietin receptor is nonessential for Jak2 activation yet critical for efficient mitogenesis in FDC-ER cells.

The development of erythroid progenitor cells depends upon exposure to the glycoprotein hormone, erythropoietin (EPO). Binding of EPO to its transmembrane receptor leads to the rapid tyrosine phosphorylation of several cellular targets including Shc, Raf-1, Gap120, the cloned EPO receptor (EPOR), pp100/97, and a M(r) 130,000 EPO-activated receptor-associated Janus protein tyrosine kinase, Jak2. A membrane-proximal cytosolic region of the EPOR recently has been shown to be essential for the activation of Jak2 and sufficient for EPO-induced mitogenesis. This cytosolic region includes 8-12 amino acid box 1 and box 2 subdomains, which are conserved in certain class I receptors as well as a more distal 10-40 amino acid subdomain (extended box 2 subdomain, ExBx2), which likewise is implicated in mitogenic signaling. Through the expression of EPOR carboxyl-terminal truncation mutants in FDC-P1 cells, we presently show that an EPOR form truncated within the ExBx2 domain efficiently activates Jak2, yet is deficient in mitogenesis. Efficient expression of this mutant receptor at the cell surface and its ability to activate Jak2 indicate that poor mitogenic activity does not result from aberrant transport or folding. Rather, failure of this mutant to support proliferation above nominal rates underlines an apparent role for the EPOR ExBx2 subdomain in the activation of a distinct primary mitogenic effector.

Amino Acid Sequence↗

PubMed1993

Association of the p85 regulatory subunit of phosphatidylinositol 3-kinase with an essential erythropoietin receptor subdomain.

Using an active, HAI epitope-tagged form of the murine erythropoietin (EPO) receptor and via direct coimmunoprecipitation, the p85 regulatory subunit of phosphatidyl inositol-3 kinase (p85/PI3-K) is shown to associate with the EPO receptor in transfected FDC-P1 cell lines. Coimmunoprecipitation of p85 with epitope-tagged EPO receptors was observed initially in FDC-HER cells labeled metabolically with [32P]orthophosphate, and association of these factors was confirmed by Western analyses of receptor immunoprecipitates using p85 antiserum. Interestingly, this association occurred in the absence of ligand, and exposure of FDC-HER cells to EPO did not detectably affect levels of receptor-associated p85 or overall levels of p85 phosphorylation. However, EPO was observed to stimulated the rapid formation of phosphatidylinositol 32P-phosphate in FDC-HER and FDC-ER cells. Through baculovirus-mediated expression of epitope-tagged EPO receptor forms in SF9 cells, domains for p85 association were mapped. Analyses of receptor forms with cytosolic truncations and deletions delineated a candidate subdomain for p85 binding to an essential extended box-2 region (P329-E374; including a putative motif for SH2 binding, Y343LVL). These findings extend a mechanistic alignment between the EPO receptor and protein tyrosine kinase-encoding receptors that likewise activate PI3-K, and expand the importance of further defining pathways to PI3-K activation.

Amino Acid Sequence↗

PubMed1993

Signal transduction in the erythropoietin receptor system.

Developing erythroid cells require the glycoprotein hormone, erythropoietin (EPO) as an activator of the rapid proliferation of early proerythroblasts (colony forming units-erythroid [CFU-e]), and subsequently as an activator of late erythroid gene expression. Activation of these growth and differentiation events proceeds from the binding of EPO at its transmembrane receptor (Class I cytokine receptor), to the engagement of a complex set of signaling pathways. Studies of reconstituted activities of the cloned EPO receptor in transfected hematopoietic cell lines have served well in identifying receptor domains and downstream mediators involved in proliferative signaling. Extracellular domains have been defined which contribute to ligand binding, receptor processing and transport, and possible dimerization. Cytosolic regions have been delineated which mediate induced mitogenesis, early gene transcription, activated protein tyrosine phosphorylation, down modulation of EPO- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced proliferation, and direct association with PI3- and JAK-2 kinases. These newly defined properties begin to align the EPO receptor mechanistically with growth factor receptors (GFR) which encode, or likewise associate with, regulated protein tyrosine kinases including the Class II cytokine receptors for interferons alpha/beta and gamma. An improved understanding of factors which mediate EPO-induced late erythroid gene activation also is emerging. These factors and pathways may be distinct from those associated with EPO-induced proliferation and may involve induced increases in cellular Ca++, cAMP and arachidonic acid, as well as the modulation of GATA-1, and/or SCL. Attributes of model systems used in studies of the role of EPO in late erythroid differentiation also are considered.

Animals↗