Tyrosine Phosphorylation of the Related Adhesion Focal Tyrosine Kinase in Megakaryocytes upon Stem Cell Factor and Phorbol Myristate Acetate Stimulation and Its Association with Paxillin*
- From the Divisions of Experimental Medicine and Hematology/Oncology, Beth Israel Deaconess Medical Center (West Campus), Harvard Medical School, Boston, Massachusetts 02215
Abstract
We have characterized signaling pathways involving the related adhesion focal tyrosine kinase (RAFTK, also known as PYK2 or CAK-β) in CMK human megakaryocytic cells. Stem cell factor, which potentiates the growth of megakaryocytes and their progenitors, and phorbol myristate acetate, which causes differentiation of megakaryocytic cell lines, induced the tyrosine phosphorylation of RAFTK but not of focal adhesion kinase. Stimulation of CMK cells with stem cell factor resulted in an increase in the autophosphorylation and kinase activity of RAFTK. Phosphorylation of RAFTK under these conditions was mediated by a protein kinase C-dependent pathway. Cytochalasin D, which disrupts the cytoskeleton, abolished the phosphorylation of RAFTK upon phorbol myristate acetate and stem cell factor stimulation, indicating that RAFTK association with the actin cytoskeleton appears to be critical for its phosphorylation. In addition, we observed an association of RAFTK with paxillin, a 68-kDa cytoskeleton protein. Using in vitro binding assays, RAFTK and paxillin were shown to bind directly through the C-terminal proline-rich domain. Transient overexpression of a dominant-negative mutant of RAFTK inhibited significantly the tyrosine phosphorylation of paxillin upon phorbol myristate acetate stimulation. These observations indicate that RAFTK might play an important role in the phosphorylation of signaling pathways within the focal adhesions and that RAFTK participates in signaling events that link signals from the cell surface to the cytoskeleton. Furthermore, this study suggests that RAFTK might be involved in megakaryocyte proliferation and differentiation.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grants HL55445 and HL51456 and by Genentech, Inc.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵† This work is dedicated to the memory of Dananagoud Hiregowdara.
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↵‡ To whom correspondence should be addressed: Divisions of Experimental Medicine and Hematology/Oncology, Beth Israel Deaconess Medical Center (West Campus), Harvard Institutes of Medicine, One Deaconess Rd., Boston, MA 02215. Tel.: 617-667-0063; Fax: 617-975-5240.
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↵1 The abbreviations used are: FAK, focal adhesion kinase; BAPTA-AM, [1,2-bis(2-aminophenoxy)ethan-N,N,N′,N′-tetraacetic acid, tetra(acetoxymethyl)ester]; PKC, protein kinase C; PMA, phorbol myristate acetate; RAFTK, related adhesion focal tyrosine kinase; SCF, stem cell factor; GST, glutathioneS-transferase; PAGE, polyacrylamide gel electrophoresis.
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↵2 D. Price and S. Avraham, unpublished data.
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- Received May 13, 1996.
- Revision received February 6, 1997.
