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J G Drachman

Publications and source records attributed to J G Drachman.

18 recordsLinked to original sources
PubMed2001

X-linked thrombocytopenia caused by a novel mutation of GATA-1.

A family with recessive X-linked thrombocytopenia affecting 4 males in 2 generations, characterized by macrothrombocytopenia, profound bleeding, and mild dyserythropoiesis, is described. Microsatellite linkage analysis identified a region of the X chromosome including the GATA-1 gene, which encodes a critical transcription factor involved in erythrocyte and megakaryocyte development. By sequencing the entire coding region of GATA-1, a 2-base mutation was detected that results in a single amino acid substitution (glycine 208 to serine) within a highly conserved portion of the N-terminal zinc finger domain. Restriction fragment length polymorphism confirmed that this novel mutation segregated with the affected males and female carrier. Although not required for DNA binding, Gly208 of GATA-1 is involved in direct interaction with Friend of GATA-1 (FOG), a cofactor required for normal megakaryocytic and erythroid development. These results demonstrate that the GATA-1-FOG interaction is partially disrupted by the mutation and that the greatest effect involves contact with the FOG zinc finger 9. These findings help describe a novel mutation of GATA-1 in humans as a cause of X-linked thrombocytopenia, and they confirm the vital role played by this transcription factor during in vivo megakaryocyte development.

Bone Marrow↗
PubMed2001

Membrane localization is not required for Mpl function in normal hematopoietic cells.

Cellular trafficking of growth factor receptors, including cross-talk among receptors at the cell surface, may be important for signal transduction in normal hematopoietic cells. To test this idea, the signaling domain of Mpl (the thrombopoietin receptor) was targeted to the plasma membrane, or to the cytoplasm of murine marrow cells, and the ability of the cells to proliferate and differentiate in response to Mpl dimerized at the plasma membrane or free in the cytoplasm was assessed. Constructs encoding the signaling domain of Mpl linked to an FK506 binding protein domain (to permit dimerization by the membrane-permeable ligand AP20187) with or without a myristylation sequence (to target the receptor to the plasma membrane) and a hemagglutinin epitope tag were generated and introduced into murine marrow cells using a murine stem cell virus (MSCV)-based retroviral vector. Both populations of transduced marrow cells proliferated in Iscoves modified Dulbecco medium-10% FCS-100 nM AP20187 without exogenous growth factors for more than 100 days and achieved greater than a 10(7)-fold expansion of cells by day 50 (n = 4 transductions). Growth was dimerizer dependent, and myeloid, erythroid, and megakaryocytic progenitors were generated. Activation of Mpl either at the plasma membrane or in the cytoplasm allowed for the terminal maturation of transduced progenitor cells. Introduction of membrane-targeted or cytoplasmic Mpl into fetal liver cells from homozygous JAK2 knock-out mice or wild-type littermates demonstrated that both forms of Mpl require JAK2 for signaling. These data show that the activation of Mpl independent of its normal plasma membrane location can support production of the full range of normal hematopoietic progenitor cells in vitro.

Animals↗
PubMed2000

A structure-function analysis of serine/threonine phosphorylation of the thrombopoietin receptor, c-Mpl.

Thrombopoietin (TPO), the critical regulator of platelet production, acts by binding to its cell surface receptor, c-Mpl. Numerous studies have shown that TPO binding leads to JAK2 kinase activation and Tyr phosphorylation of c-Mpl and several intracellular signaling intermediates, events vital for the biological activity of the hormone. In contrast, virtually nothing is known of the role of Ser or Thr phosphorylation of c-Mpl. By using phosphoamino acid analysis we found that Ser residues of c-Mpl were constitutively phosphorylated in receptor-bearing cells, levels that were increased following exposure of cells to TPO. To identify which residues were modified, and to determine the functional consequences of their phosphorylation, we generated a series of Ser to Ala mutations of a truncated c-Mpl receptor (T69) capable of supporting TPO-induced cell growth. Of the eight Ser within T69 we found that at least four are phosphorylated in TPO-stimulated cells. The mutation of each of these residues alone had minimal effects on TPO-induced proliferation, but substitution of all of the phosphoserine residues with Ala reduced the capacity of the receptor to support cell growth by over 50%. Additionally, the Ser at cytoplasmic position 18 is not detectably phosphorylated. However, the mutation of Ser-18 to Ala nearly abrogates TPO-induced proliferation and co-precipitation of JAK2 with Mpl. This study provides the first systematic analysis of the role of Ser residues in c-Mpl signaling.

Amino Acid Sequence↗
PubMed2000

Autosomal dominant thrombocytopenia: incomplete megakaryocyte differentiation and linkage to human chromosome 10.

We studied a large kindred with nonsyndromic autosomal dominant thrombocytopenia to define the phenotype and used genomic linkage analysis to determine the locus of the abnormal gene. Affected family members are characterized by lifelong moderate thrombocytopenia (mean = 42.7 x 10(9)/L) with moderate propensity toward easy bruising and minor bleeding. Megakaryocytes are present in bone marrow with reduced frequency, and there are no apparent abnormalities of myeloid or erythroid cells. This type of inherited thrombocytopenia has no evident association with hematopoietic malignancy or progression to aplastic anemia. In the past, members of this family have failed therapeutic trials of immunosuppression and splenectomy. In our investigation, we found that affected individuals had normal platelet size compared with unaffected family members and modestly increased thrombopoietin levels. Hematopoietic colony assays from bone marrow and peripheral blood demonstrated that megakaryocyte precursors (CFU-Mk) were dramatically increased in both number and size in affected individuals. Bone marrow cells grown in liquid culture with thrombopoietin failed to develop polyploid cells greater than 8N. Also, electron microscopy demonstrated that megakaryocytes from an affected individual had markedly delayed nuclear and cytoplasmic differentiation. Genome-wide linkage analysis established a single locus for the disease gene on the short arm of chromosome 10 with a maximum 2-point lod score of 5.68 (at theta = 0). By recruiting additional family members, the genomic region was narrowed to 17 centimorgans. We conclude that a gene in this locus plays an important role in megakaryocyte endomitosis and terminal maturation.

Bone Marrow Cells↗
PubMed2000

CDP/Cut DNA binding activity is down-modulated in granulocytes, macrophages and erythrocytes but remains elevated in differentiating megakaryocytes.

DNA binding by the CCAAT-displacement protein, the mammalian homologue of the Drosophila melanogaster Cut protein, was previously found to increase sharply in S phase, suggesting a role for CDP/Cut in cell cycle progression. Genetic studies in Drosophila indicated that cut plays an important role in cell-type specification in several tissues. In the present study, we have investigated CDP/Cut expression and activity in a panel of multipotent hematopoietic cell lines that can be induced to differentiate in vitro into distinct cell types. While CDP/Cut DNA binding activity declined in the pathways leading to macrophages, granulocytes and erythrocytes, it remained elevated in megakaryocytes. CDP/Cut was also highly expressed in primary megakaryocytes isolated from mouse, and some DNA binding activity could be detected. Altogether, these results raise the possibility that CDP/Cut may be a determinant of cell type identity downstream of the myelo-erythroid precursor cell. Another possibility, which does not exclude a role in lineage identity, is that CDP/Cut activity in megakaryocytes is linked to endomitosis. Indeed, elevated CDP/Cut activity in differentiating megakaryocytes and during the S phase of the cell cycle suggests that it may be required for DNA replication.

Animals↗
PubMed2000

Role of thrombopoietin in hematopoietic stem cell and progenitor regulation.

Thrombopoietin performs an essential role during hematopoiesis by regulating the expansion and maturation of megakaryocytes. In keeping with this function, megakaryocytes, platelets, and their precursors all express the thrombopoietin receptor, Mpl, on their cell surface. However, Mpl is also expressed on primitive, pluripotent hematopoietic progenitors and plays an important role in the regulation of lineages other than megakaryocytes as well as primitive progenitors. Recently, the ability of thrombopoietin to maintain and expand repopulating stem cells has been demonstrated. Thus, thrombopoietin is unique among the hematopoietic cytokines because it is necessary both for terminal maturation and regulation of lineage-specific megakaryocytes and also for maintenance of the most primitive hematopoietic stem cells. Many new strategies are evolving to exploit the activity of thrombopoietin on primitive progenitors. This may lead to faster hematopoietic recovery from marrow-suppressive therapy, effective methods of ex vivo expansion of hematopoietic stem cells, and retroviral transduction of stem cells to facilitate gene therapy.

Animals↗
PubMed1999

Thrombopoietin-induced activation of the mitogen-activated protein kinase (MAPK) pathway in normal megakaryocytes: role in endomitosis.

Thrombopoietin (TPO) plays a critical role in megakaryocyte proliferation and differentiation. Using various cultured cell lines, several recent studies have implicated the mitogen-activated protein kinase (MAPK) pathway in megakaryocyte differentiation. In the study reported here, we examined the role played by thrombopoietin-induced MAPK activity in a cytokine-dependent cell line (BAF3/Mpl) and in primary murine megakaryocytes. In both systems, extracellular signal-regulated protein kinase (ERK) 1 and 2 MAPK phosphorylation was rapidly induced by TPO stimulation. To identify the Mpl domain responsible for MAPK activation, BAF3 cells expressing truncated forms of the Mpl receptor were studied. Phosphorylation of ERKs did not require elements of the cytoplasmic signaling domain distal to Box 2 and was not dependent on phosphorylation of the adapter protein Shc. ERK activation in murine megakaryocytes was maximal at 10 minutes and was markedly decreased over the subsequent 3 hours. Next, the physiologic consequences of MAPK inhibition were studied. Using the MAPK kinase (MEK) inhibitor, PD 98059, blockade of MAPK activity substantially reduced TPO-dependent proliferation in BAF3/Mpl cells and markedly decreased mean megakaryocyte ploidy in cultures. To exclude an indirect effect of MAPK inhibition on stromal cells in whole bone marrow, CD41(+) cells were selected and then cultured in TPO. The number of polyploid megakaryocytes derived from the CD41-selected cells was also significantly reduced by MEK inhibition, as was their geometric mean ploidy. These studies show an important role for MAPK in TPO-induced endomitosis and underscore the value of primary cells when studying the physiologic effects of signaling pathways.

Animals↗
PubMed1999

Thrombopoietin signal transduction requires functional JAK2, not TYK2.

The Janus family of tyrosine kinases (JAKs) plays a critical role in signal transduction by members of the cytokine receptor superfamily. In response to ligand-receptor interaction, these nonreceptor tyrosine kinases are rapidly phosphorylated and activated, triggering tyrosine phosphorylation and activation of downstream signaling intermediates. Upon binding to its receptor, the product of the proto-oncogene c-mpl, thrombopoietin (TPO) activates both JAK2 and TYK2 in multiple cell lines as well as megakaryocytes and platelets. To study whether one or both of these kinases are essential for TPO signal transduction, we engineered a parental human sarcoma cell line (2C4) as well as sarcoma cell lines that are deficient in JAK2 expression (gamma2A) or TYK2 expression (U1A) to express the wild-type Mpl receptor. The ability of TPO to induce tyrosine phosphorylation of Mpl and multiple intracellular substrates in each cell line was then examined. Our results demonstrate that JAK2-deficient cells (gamma2A-Mpl) are unable to initiate TPO-mediated signaling. In contrast, cells that are TYK2-deficient (U1A-Mpl) are able to induce tyrosine phosphorylation of Mpl, JAK2, STAT3, and Shc as efficiently as parental cells (2C4-Mpl). These data indicate that JAK2 is an essential component of Mpl signaling and that, in the absence of JAK2, TYK2 is incapable of initiating TPO-induced tyrosine phosphorylation.

Base Sequence↗
PubMed1999

Thrombopoietin signal transduction: studies from cell lines and primary cells.

Thrombopoietin (TPO) and its receptor Mpl support all of the developmental step necessary for megakaryocytopoiesis. In the past few years, the signaling pathways utilized by this member of the cytokine receptor family have been extensively studied, especially JAK/STAT, Ras/MAP kinase, Shc, and other adapter molecules. Many if not most of the secondary signaling pathways activated by thrombopoietin have also been identified upon binding of other hematopoietic growth factors to their cognate receptors, making the study of Mpl signaling representative of the field in general. However, identifying unique molecules or combinations of signals that direct megakaryocyte development has been an elusive goal and has led some investigators to conclude that there is little specificity during Mpl signal transduction. In this article we review the data regarding Mpl signaling with particular attention to the methods employed and critical interpretation of the data generated. Future studies will have to focus on primary bone marrow cells and intact animal models rather than transformed cell lines. Furthermore, it is likely that a comprehensive, integrative analysis of the many pathways activated by ligand binding will be necessary to understand the physiology of cytokine signaling.

Animals↗
PubMed1997

A proliferation switch for genetically modified cells.

Receptor dimerization is the key signaling event for many cytokines, including erythropoietin. A system has been recently developed that permits intracellular protein dimerization to be reversibly activated in response to a lipid-soluble dimeric form of the drug FK506, called FK1012. FK1012 is used as a pharmacological mediator of dimerization to bring together FK506 binding domains, taken from the endogenous protein FKBP12. In experiments reported herein, FK1012-induced dimerization of a fusion protein containing the intracellular portion of the erythropoietin receptor allowed cells normally dependent on interleukin 3 to proliferate in its absence. FK506 competitively reversed the proliferative effect of FK1012 but had no influence on the proliferative effect of interleukin 3. Signaling pathways activated by FK1012 mimicked those activated by erythropoietin, because both JAK2 and STAT5 were phosphorylated in response to FK1012. This approach may provide a means to specifically and reversibly stimulate the proliferation of genetically modified cell populations in vitro or in vivo.

Animals↗
PubMed1997

Dissecting the thrombopoietin receptor: functional elements of the Mpl cytoplasmic domain.

Thrombopoietin (TPO) acts through its receptor, Mpl, to stimulate the proliferation and maturation of megakaryocytes and their progenitors. The Mpl cytoplasmic domain controls this process through assembly of an active signaling complex using various receptor docking sites. In this report, eight carboxyl truncations of the 121-aa murine Mpl cytoplasmic domain were tested for the ability to support growth of a cytokine-dependent cell line (Ba/F3) and for their capacity to induce TPO-stimulated tyrosine phosphorylation of specific signaling proteins. Point mutations of the five tyrosine residues in the cytoplasmic domain of the receptor were subsequently used to confirm our conclusions. From these studies we demonstrate that: (i) TPO-induced proliferation is moderately reduced by truncation of as many as 53 C-terminal amino acids of Mpl, including the sites of receptor tyrosine phosphorylation; (ii) truncation/mutation of residues 69-83 of the Mpl cytoplasmic domain enhances proliferative signaling, perhaps mediated by a decrease in receptor-driven cellular differentiation; (iii) Mpl can be phosphorylated at either Y112 or Y117 but not at the three proximal cytoplasmic tyrosine residues (Y8, Y29, and Y78); (iv) Y112 of Mpl is necessary for tyrosine phosphorylation of Shc and Shc-associated p145 (SHIP); and (v) unlike STAT3, STAT5 is partially phosphorylated in the absence of any tyrosine residues in the Mpl cytoplasmic domain. These studies identify subdomains of Mpl necessary for activation of several critical signaling pathways and point to two potentially novel mechanisms of TPO-induced signal transduction, an indirect pathway to STAT5 activation and a differentiation domain that acts by limiting proliferation.

Animals↗
PubMed1997

Thrombopoietin signal transduction in purified murine megakaryocytes.

Thrombopoietin (TPO) is a recently cloned cytokine that binds to its receptor, Mpl, and promotes hematopoietic expansion and maturation, primarily of the megakaryocyte lineage. The signaling pathways responsible for these events are thought to involve the Janus family of nonreceptor tyrosine kinases (JAKs) and the signal transducers and activators of transcription (STATs), which are activated by tyrosine phosphorylation. Previous investigators have studied these molecules in engineered and naturally occurring cell lines. To investigate the molecular basis for TPO signal transduction in a more physiologic target, we determined the pattern of JAK and STAT activation in purified, normal urine megakaryocytes. These results are compared with those of established cell lines that only proliferate (Ba/F3- mMPL and DA-1-TPO) or only differentiate (L8057) in response to TPO. From these findings, a model is proposed to explain the physiologic roles of JAK2, TYK2, STAT3, and STAT5 in TPO signaling. Furthermore, previous studies of the physical interaction between Mpl and the JAKs are extended, showing a difference in the association of JAK2 and TYK2 with the TPO receptor. Finally, we show that, in the cell line Ba/F3-mMPL, the closely related proteins STAT5A and STAT5B are both activated by TPO stimulation and are capable of heterodimerization. Together, these results further our understanding of the early stages of megakaryocyte and platelet development.

Animals↗
PubMed1995

The c-Mpl ligand (thrombopoietin) stimulates tyrosine phosphorylation of Jak2, Shc, and c-Mpl.

c-Mpl is a member of the cytokine receptor superfamily, expressed primarily on hematopoietic cells. Recently, the c-Mpl ligand was cloned and found to have thrombopoietic activity. In this paper we report that ligand binding induced tyrosine phosphorylation in BaF3 cells engineered to express the murine Mpl receptor (BaF3/mMpl). Phosphorylation occurred within 1 min at cytokine concentrations sufficient for proliferation of receptor-bearing cells. Using specific antibodies for immunoprecipitation and Western blotting, several of these phosphorylated proteins were identified. Shc and Jak2, known cytokine signaling molecules, and the c-Mpl receptor were shown to be major substrates for tyrosine phosphorylation. In contrast, phospholipase C-gamma and phosphatidylinositol 3-kinase displayed little and no tyrosine phosphorylation, respectively, after thrombopoietin stimulation. Co-immunoprecipitation studies demonstrated that Jak2 became physically associated with c-Mpl relatively late in the observed time course (20-60 min), significantly later than tyrosine phosphorylation of Jak2 (1-5 min). These results suggest that c-Mpl induces signal transduction pathways similar to those of other known cytokines. Additionally, in light of its late physical association with c-Mpl following ligand binding, Jak2 may not be the initiating tyrosine kinase in the thrombopoietin-induced signaling cascade.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Structure and function of the cytokine receptor superfamily.

The cytokine receptor superfamily is a group of transmembrane proteins, characterized by a common extracellular structure--two barrels composed of seven beta strands each. Over the past year, several new members of the cytokine receptor family have been described, and new insights have been made into how related receptors share common subunits. Also this year, the ligand for the orphan cytokine receptor c-Mpl was identified and shown to stimulate megakaryocyte development and thrombopoiesis. Progress has been made in unraveling the precise atomic basis for ligand-receptor interactions and the role that subunit association plays in receptor activation. Finally, advances have been made in understanding the organization of the intracellular domain and how signaling to the nucleus is achieved. Together, these new results have led to a greater appreciation of the role that cytokine receptors play in the regulation of proliferation and lineage-specific differentiation during hematopoiesis.

Animals↗
PubMed1994

Increased erythrocyte sedimentation rate and a splenic mass.

This discussion was selected from the weekly Grand Rounds in the Department of Medicine, University of Washington School of Medicine, Seattle. Taken from a transcription, it has been edited by Jonathan G. Drachman, MD, Chief Medical Resident; Henry Rosen, MD, Professor and Associate Chair; and Paul G. Ramsey, MD, Professor and Chair of the Department of Medicine.

Adult↗
PubMed1991

The alpha beta monomer of the insulin receptor has hormone-responsive tyrosine kinase activity.

Insulin receptors from turkey erythrocyte membranes exist as monomers and dimers when membranes are solubilized with detergent. We examined the ability of monomers and dimers to act as protein kinases to effect both autophosphorylation of the receptor and phosphorylation of an exogenous substrate. After separation by sucrose-density-gradient centrifugation, only receptor dimers show significant basal and insulin-stimulated kinase activity, whereas material at the position of receptor monomers is not active. Partial reduction of the membrane-bound receptors with dithiothreitol, however, produces a receptor monomer containing an alpha and a beta chain which has protein kinase activity similar to that of the original dimers. With rat adipocyte plasma membranes, which in the absence of reducing agents only contain receptor dimers, reduction with dithiothreitol also produces monomers with receptor kinase activity. Receptor monomer hormone-dependent kinase activity is insensitive to receptor concentration and shows stimulation after immobilization on an affinity support.

Adipose Tissue↗