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Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4652-4662
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, and
Yasuo Ikeda
From the Division of Hematology, Department of Internal Medicine, and
Blood Center, Keio University, Tokyo, Japan; the Laboratory of Cellular
Biosynthesis, Institute of Molecular and Cellular Biosciences, Tokyo
University, Tokyo, Japan; the Division of Hematology and Medical
Oncology, Oregon Health Sciences University, Portland, OR; the Division
of Hematology, Department of Medicine, Jichi Medical School, Tochigi,
Japan; the Department of Pathophysiology and Therapeutics, Faculty of
Pharmacological Sciences, Hoshi University, Tokyo, Japan; and the
Pharmaceutical Research Laboratory, Kirin Brewery Co, Ltd, Takasaki,
Japan.
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
coimmunoprecipitaion 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
 -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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