Thrombopoietin induces association of Crkl with STAT5 but not STAT3 in human platelets

Katsutoshi Ozaki, Atsushi Oda, Hiroshi Wakao, Jennifer Rhodes, Brian J. Druker, Akaru Ishida, Masatoshi Wakui, Shinichiro Okamoto, Kayo Morita, Makoto Handa, Norio Komatsu, Hideya Ohashi, Atsushi Miyajima, Yasuo Ikeda

Research output: Contribution to journalArticle

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Abstract

Crkl, a 39-kD SH2, SH3 domain-containing adapter protein, is constitutively tyrosine phosphorylated in hematopoietic cells from chronic myelogenous leukemia (CML) patients. We recently reported that thrombopoietin induces tyrosine phosphorylation of Crkl in normal platelets. In this study, we demonstrate that thrombopoietin induces association of Crkl with a tyrosine phosphorylated 95- to 100-kD protein in platelets and in UT7/TPO cells, a thrombopoietin-dependent megakaryocytic cell line. With specific antibodies against STAT5, we demonstrate that the 95- to 100-kD protein in Crkl immunoprecipitates is STAT5. This coimmunoprecipitation was specific in that Crkl immunoprecipitates do not contain STAT3, although STAT3 becomes tyrosine phosphorylated in thrombopoietin-stimulated platelets. The coimmunoprecipitation of Crkl with STAT5 was inhibited by the immunizing peptide for Crkl antisera or phenyl phosphate (20 mmol/L). After denaturing of Crkl immunoprecipitates, Crkl was still immunoprecipitated by Crkl antisera. However, coimmunoprecipitation of STAT5 was not observed. Coincident with STAT5 tyrosine phosphorylation, thrombopoietin induces activation of STAT5 DNA-binding activity as demonstrated by electrophoretic mobility shift assays (EMSA). Using a β-casein promoter STAT5 binding site as a probe, we have also demonstrated that Crkl antisera supershift the STAT5-DNA complex, suggesting that Crkl is a component of the complex in the nucleus. Furthermore, interleukin-3 (IL-3), granulocyte-macrophage colony- stimulating factor (GM-CSF), and erythropoietin also induce Crkl-STAT5 complex formation in responding cells in a stimulation-dependent manner. In vitro, glutathione S-transferase (GST)-Crkl bound to STAT5 inducibly through its SH2 domain. These results indicate that thrombopoietin, IL-3, GM-CSF, and erythropoietin commonly induce association of STAT5 and Crkl and that the complex translocates to the nucleus and binds to DNA. Interestingly, such association between STAT5 and Crkl was not observed in cytokine-stimulated murine cells, suggesting an intriguing possibility that components of the human STAT5-DNA complex may be different from those of the murine counterpart.

Original languageEnglish
Pages (from-to)4652-4662
Number of pages11
JournalBlood
Volume92
Issue number12
Publication statusPublished - 1998 Dec 15
Externally publishedYes

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Thrombopoietin
Platelets
Blood Platelets
Tyrosine
Association reactions
src Homology Domains
Immune Sera
Phosphorylation
Interleukin-3
DNA
Granulocyte-Macrophage Colony-Stimulating Factor
Erythropoietin
Electrophoretic mobility
Proteins
Electrophoretic Mobility Shift Assay
Leukemia, Myelogenous, Chronic, BCR-ABL Positive
Caseins
Glutathione Transferase
Assays
Chemical activation

ASJC Scopus subject areas

  • Hematology

Cite this

Ozaki, K., Oda, A., Wakao, H., Rhodes, J., Druker, B. J., Ishida, A., ... Ikeda, Y. (1998). Thrombopoietin induces association of Crkl with STAT5 but not STAT3 in human platelets. Blood, 92(12), 4652-4662.

Thrombopoietin induces association of Crkl with STAT5 but not STAT3 in human platelets. / Ozaki, Katsutoshi; Oda, Atsushi; Wakao, Hiroshi; Rhodes, Jennifer; Druker, Brian J.; Ishida, Akaru; Wakui, Masatoshi; Okamoto, Shinichiro; Morita, Kayo; Handa, Makoto; Komatsu, Norio; Ohashi, Hideya; Miyajima, Atsushi; Ikeda, Yasuo.

In: Blood, Vol. 92, No. 12, 15.12.1998, p. 4652-4662.

Research output: Contribution to journalArticle

Ozaki, K, Oda, A, Wakao, H, Rhodes, J, Druker, BJ, Ishida, A, Wakui, M, Okamoto, S, Morita, K, Handa, M, Komatsu, N, Ohashi, H, Miyajima, A & Ikeda, Y 1998, 'Thrombopoietin induces association of Crkl with STAT5 but not STAT3 in human platelets', Blood, vol. 92, no. 12, pp. 4652-4662.
Ozaki K, Oda A, Wakao H, Rhodes J, Druker BJ, Ishida A et al. Thrombopoietin induces association of Crkl with STAT5 but not STAT3 in human platelets. Blood. 1998 Dec 15;92(12):4652-4662.
Ozaki, Katsutoshi ; Oda, Atsushi ; Wakao, Hiroshi ; Rhodes, Jennifer ; Druker, Brian J. ; Ishida, Akaru ; Wakui, Masatoshi ; Okamoto, Shinichiro ; Morita, Kayo ; Handa, Makoto ; Komatsu, Norio ; Ohashi, Hideya ; Miyajima, Atsushi ; Ikeda, Yasuo. / Thrombopoietin induces association of Crkl with STAT5 but not STAT3 in human platelets. In: Blood. 1998 ; Vol. 92, No. 12. pp. 4652-4662.
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abstract = "Crkl, a 39-kD SH2, SH3 domain-containing adapter protein, is constitutively tyrosine phosphorylated in hematopoietic cells from chronic myelogenous leukemia (CML) patients. We recently reported that thrombopoietin induces tyrosine phosphorylation of Crkl in normal platelets. In this study, we demonstrate that thrombopoietin induces association of Crkl with a tyrosine phosphorylated 95- to 100-kD protein in platelets and in UT7/TPO cells, a thrombopoietin-dependent megakaryocytic cell line. With specific antibodies against STAT5, we demonstrate that the 95- to 100-kD protein in Crkl immunoprecipitates is STAT5. This coimmunoprecipitation was specific in that Crkl immunoprecipitates do not contain STAT3, although STAT3 becomes tyrosine phosphorylated in thrombopoietin-stimulated platelets. The coimmunoprecipitation of Crkl with STAT5 was inhibited by the immunizing peptide for Crkl antisera or phenyl phosphate (20 mmol/L). After denaturing of Crkl immunoprecipitates, Crkl was still immunoprecipitated by Crkl antisera. However, coimmunoprecipitation of STAT5 was not observed. Coincident with STAT5 tyrosine phosphorylation, thrombopoietin induces activation of STAT5 DNA-binding activity as demonstrated by electrophoretic mobility shift assays (EMSA). Using a β-casein promoter STAT5 binding site as a probe, we have also demonstrated that Crkl antisera supershift the STAT5-DNA complex, suggesting that Crkl is a component of the complex in the nucleus. Furthermore, interleukin-3 (IL-3), granulocyte-macrophage colony- stimulating factor (GM-CSF), and erythropoietin also induce Crkl-STAT5 complex formation in responding cells in a stimulation-dependent manner. In vitro, glutathione S-transferase (GST)-Crkl bound to STAT5 inducibly through its SH2 domain. These results indicate that thrombopoietin, IL-3, GM-CSF, and erythropoietin commonly induce association of STAT5 and Crkl and that the complex translocates to the nucleus and binds to DNA. Interestingly, such association between STAT5 and Crkl was not observed in cytokine-stimulated murine cells, suggesting an intriguing possibility that components of the human STAT5-DNA complex may be different from those of the murine counterpart.",
author = "Katsutoshi Ozaki and Atsushi Oda and Hiroshi Wakao and Jennifer Rhodes and Druker, {Brian J.} and Akaru Ishida and Masatoshi Wakui and Shinichiro Okamoto and Kayo Morita and Makoto Handa and Norio Komatsu and Hideya Ohashi and Atsushi Miyajima and Yasuo Ikeda",
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AU - Ozaki, Katsutoshi

AU - Oda, Atsushi

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AU - Druker, Brian J.

AU - Ishida, Akaru

AU - Wakui, Masatoshi

AU - Okamoto, Shinichiro

AU - Morita, Kayo

AU - Handa, Makoto

AU - Komatsu, Norio

AU - Ohashi, Hideya

AU - Miyajima, Atsushi

AU - Ikeda, Yasuo

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N2 - Crkl, a 39-kD SH2, SH3 domain-containing adapter protein, is constitutively tyrosine phosphorylated in hematopoietic cells from chronic myelogenous leukemia (CML) patients. We recently reported that thrombopoietin induces tyrosine phosphorylation of Crkl in normal platelets. In this study, we demonstrate that thrombopoietin induces association of Crkl with a tyrosine phosphorylated 95- to 100-kD protein in platelets and in UT7/TPO cells, a thrombopoietin-dependent megakaryocytic cell line. With specific antibodies against STAT5, we demonstrate that the 95- to 100-kD protein in Crkl immunoprecipitates is STAT5. This coimmunoprecipitation was specific in that Crkl immunoprecipitates do not contain STAT3, although STAT3 becomes tyrosine phosphorylated in thrombopoietin-stimulated platelets. The coimmunoprecipitation of Crkl with STAT5 was inhibited by the immunizing peptide for Crkl antisera or phenyl phosphate (20 mmol/L). After denaturing of Crkl immunoprecipitates, Crkl was still immunoprecipitated by Crkl antisera. However, coimmunoprecipitation of STAT5 was not observed. Coincident with STAT5 tyrosine phosphorylation, thrombopoietin induces activation of STAT5 DNA-binding activity as demonstrated by electrophoretic mobility shift assays (EMSA). Using a β-casein promoter STAT5 binding site as a probe, we have also demonstrated that Crkl antisera supershift the STAT5-DNA complex, suggesting that Crkl is a component of the complex in the nucleus. Furthermore, interleukin-3 (IL-3), granulocyte-macrophage colony- stimulating factor (GM-CSF), and erythropoietin also induce Crkl-STAT5 complex formation in responding cells in a stimulation-dependent manner. In vitro, glutathione S-transferase (GST)-Crkl bound to STAT5 inducibly through its SH2 domain. These results indicate that thrombopoietin, IL-3, GM-CSF, and erythropoietin commonly induce association of STAT5 and Crkl and that the complex translocates to the nucleus and binds to DNA. Interestingly, such association between STAT5 and Crkl was not observed in cytokine-stimulated murine cells, suggesting an intriguing possibility that components of the human STAT5-DNA complex may be different from those of the murine counterpart.

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