Thrombopoietin, c‐Mpl ligand, induces tyrosine phosphorylation of Tyk2, JAK2, and STAT3, and enhances agonists‐induced aggregation in platelets in vitro

Ezumi, Yasuharu; Takayama, Hiromitsu; Okuma, Minoru

doi:10.1016/0014-5793(95)01072-m

Cited by 140 publications

(101 citation statements)

References 30 publications

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“…Mpl is a member of the hematopoietic growth factor receptor superfamily and like the other hematopoietic growth factor receptors, binding of its cognate ligand causes Mpl oligomerization and activation of members of the Janus family of tyrosine kinases, in particular JAK2 and Tyk2. Activation of these kinases is associated with tyrosine phosphorylation of a number of proteins including members of the STAT family (in platelets, STAT3, and STATS, specifically) and Shc [14][15][16][17][18]291. To our surprise, exposure of PV platelets to TPO failed to activate JAK2 or Tyk2 and as a consequence neither STAT5 nor Shc were tyrosine phosphorylated even though these kinases and their substrates were expressed normally in PV platelets and thrombin-induced protein tyrosine phosphorylation was intact.…”

Section: Discussionmentioning

confidence: 99%

A novel thrombopoietin signaling defect in polycythemia vera platelets

Moliterno¹,

Siebel²,

Sun³

et al. 1998

Stem Cells

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Section: Discussionmentioning

confidence: 99%

A novel thrombopoietin signaling defect in polycythemia vera platelets

Moliterno¹,

Siebel²,

Sun³

et al. 1998

Stem Cells

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“…Platelet-rich plasma was incubated with 1 mM aspirin for 30 min at 37°C. Gel-filtered platelets were then prepared as described previously [8] at a final concentration of 1ϫ10 9 cells/ml in Hepes buffer (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2, 5.6 mM glucose, 1 mg/ml bovine serum albumin, 3.3 mM NaH 2 PO 4 and 20 mM Hepes, pH 7.4), containing 1 U/ ml apyrase and 500 µM RGDS. For 32 Pi-labeling of platelets, platelets were incubated with 0.5 mCi/ml 32 Pi in Hepes buffer without NaH 2 PO 4 for 1 h at 37°C before gel filtration.…”

Section: Reagents Glutathione S-transferase (Gst)-[k97a]mek1mentioning

confidence: 99%

“…Kinase activity of ERK2, MEKs and Raf-1 were determined by in vitro kinase assays with a peptide derived from the epidermal growth factor receptor, kinase-negative GST-MAPK and kinase-negative GST-MEK1 (GST-[K97A]MEK1) as exogenous substrates, respectively. The kinases were isolated from unstimulated or stimulated platelets by immunoprecipitation using specific antibodies for each kinase (1 µg/ml for ERK2 and 5 µg/ml for Raf-1 and MEKs) as described previously [8] with following modified lysis buffers : MAPK-lysis buffer [50 mM Tris/HCl, pH 7.5, 100 mM NaCl, 50 mM NaF, 5 mM EDTA, 1% (mass/vol.) Triton X-100, 40 mM β-glycerophosphate, 40 mM p-nitrophenylphosphate, 1 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin and 20 µg/ml aprotinin] for MEKs and ERK2, or Raf-lysis buffer [20 mM Tris/HCl, pH 8.0, 137 mM NaCl, 1% (mass/vol.)…”

Section: Reagents Glutathione S-transferase (Gst)-[k97a]mek1mentioning

confidence: 99%

“…Unstimulated or stimulated platelets were lysed in the lysis buffers as described above, and whole cell lysates and immunoprecipitates of each protein were prepared as described previously [8]. Samples (whole cell lysates from 6ϫ10 6 cells/lane or immunoprecipitates from 2.6ϫ10 8 cells/ lane) were subjected to 7.5 % (mass/vol.)…”

Section: Reagents Glutathione S-transferase (Gst)-[k97a]mek1mentioning

confidence: 99%

“…TPO does not induce platelet aggregation by itself, but enhances aggregation induced by other agonists [8,9, 11Ϫ14]. Since we found that both the activities of MEKs and ERKs were enhanced by TPO, we investigated whether they were involved in the potentiation effect of TPO on thrombin-induced or PDBu-induced aggregation using a specific MEK inhibitor, PD098059 [26,27].…”

Section: Tyrphostin 47 But Not Wortmannin Inhibits the Priming Effectmentioning

confidence: 99%

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Thrombopoietin potentiates the protein‐kinase‐C‐mediated activation of mitogen‐activated protein kinase/ERK kinases and extracellular signal‐regulated kinases in human platelets

Ezumi

Uchiyama

Takayama

1998

European Journal of Biochemistry

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The thrombopoietin (TPO) receptor is expressed in the megakaryocytic lineage from late progenitors to platelets. We investigated the effect of TPO on the extracellular signal-regulated kinase (ERK) activation pathway in human platelets. TPO by itself did not activate ERK1, ERK2 and protein kinase C (PKC), whereas TPO directly enhanced the PKC-dependent activation of ERKs induced by other agonists including thrombin and phorbol esters, without affecting the PKC activation by those agonists. TPO did not activate the mitogen-activated protein kinase/ERK kinases, MEK1 and MEK2, but activated Raf-1 and directly augmented the PKC-mediated MEK activation, suggesting that TPO primarily potentiates the ERK pathway through regulating MEKs or upstream steps of MEKs including Raf-1. The MEK inhibitor PD098059 failed to affect not only thrombin-induced or phorbol ester-induced aggregation, but also potentiation of aggregation by TPO, denying the primary involvement of ERKs and MEKs in those events. ERKs and MEKs were located mainly in the detergent-soluble/non-cytoskeletal fractions. ERKs but not MEKs were relocated to the cytoskeleton following platelet aggregation and actin polymerization. These data indicate that TPO synergizes with other agonists in the ERK activation pathway of platelets and that this synergy might affect functions of the cytoskeleton possibly regulated by ERKs.Keywords : platelet; thrombopoietin; extracellular signal-regulated kinase ; protein kinase C; signal transduction. Thrombopoietin (TPO) [1Ϫ4], also called the c-Mpl ligand or the megakaryocyte growth and development factor, is the newly identified cytokine that plays a critical role in megakaryocytopoiesis [5]. The TPO receptor, c-Mpl, is expressed in the megakaryocytic lineage from late progenitors to platelets [6]. Others and we have recently reported that TPO induces tyrosine phosphorylation of signaling molecules including Janus tyrosine kinases, JAK2 and Tyk2, Shc, STAT3 and STAT5 in platelets [7Ϫ14], and that TPO does not activate platelets by itself but potentiates other agonists-induced aggregation and release reaction [8, 9, 11Ϫ14].The extracellular signal-regulated kinases (ERKs), ERK1 and ERK2, are the prototypic members of the mitogen-activated protein kinase (MAPK) family and are activated by divergent extracellular stimuli [15Ϫ18]. The ERK signaling pathway through growth factor receptors or cytokine receptors has been well studied. Binding of ligands to receptors leads to activationCorrespondence to H. Takayama, Department of Hematology and Oncology, Clinical Sciences for Pathological Organs, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawaharacho, Sakyo-ku, Kyoto, Japan 606-8507Fax: ϩ81 75 751 3201. E-mail: hiro@kuhp.kyoto-u.ac.jp Abbreviations. TPO, thrombopoietin; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK/ ERK kinase; PKC, protein kinase C; GST, glutathione S-transferase ; STA2, 9, 11-epithio-11, 12-methano-thromboxane A2; PMA, phorbol 12-myristate 13-acetate ; ...

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Thrombopoietin is synergistic with other hematopoietic growth factors and physiologic platelet agonists for platelet activation in vitro

et al. 1997

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Thrombopoietin (TPO) is the primary physiologic regulator of platelet production. The effect of TPO on platelet function, both alone and in combination with other hematopoietic growth factors, adenosine diphosphate (ADP), and epinephrine, was investigated using fluorescent-labeled antibodies to the activation-dependent antigen CD62 (Pselectin) and flow cytometry. TPO stimulated CD62 expression on normal human platelets, and this expression was completely inhibited by the soluble extracellular domain of the TPO receptor, MPL. The growth factors granulocyte colony-stimulating factor (G-CSF) and erythropoietin (EPO), but not interleukin-3 (IL-3) or stem-cell factor (SCF), also stimulated platelet activation. The combination of EPO, SCF, ADP, and epinephrine with TPO were synergistic for platelet CD62 expression. These data further support a role for TPO in modulating platelet function. Am.

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Thrombopoietin, c‐Mpl ligand, induces tyrosine phosphorylation of Tyk2, JAK2, and STAT3, and enhances agonists‐induced aggregation in platelets in vitro

Cited by 140 publications

References 30 publications

A novel thrombopoietin signaling defect in polycythemia vera platelets

A novel thrombopoietin signaling defect in polycythemia vera platelets

Thrombopoietin potentiates the protein‐kinase‐C‐mediated activation of mitogen‐activated protein kinase/ERK kinases and extracellular signal‐regulated kinases in human platelets

Thrombopoietin is synergistic with other hematopoietic growth factors and physiologic platelet agonists for platelet activation in vitro

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