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

Dananagoud Hiregowdara,Hava Avraham,Yigong Fu,Roanna London,Shalom Avraham

doi:10.1074/jbc.272.16.10804

Dananagoud Hiregowdara, Hava Avraham + Show 3 more

Open Access

https://doi.org/10.1074/jbc.272.16.10804

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Abstract

We have characterized signaling pathways involving the related adhesion focal tyrosine kinase (RAFTK, also known as PYK2 or CAK-beta) 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.

Highlights

Protein tyrosine kinases regulate various signal transduction pathways, including those controlling cell growth, differentiation, and activation [1,2,3,4,5]
stem cell factor (SCF) Induces the Tyrosine Phosphorylation of RAFTK in CMK Megakaryocytic Cells—To investigate whether RAFTK participated in growth-related signal transduction pathways in megakaryocytes, we studied model CMK megakaryocytic cells and focused on the c-KIT/SCF pathway, which is known to be important in the proliferation of this line [28, 29]
The effect of SCF, phorbol myristate acetate (PMA), and Ca2ϩ on the phosphorylation of RAFTK appeared to be mediated by a PKCdependent pathway

Summary

Introduction

Protein tyrosine kinases regulate various signal transduction pathways, including those controlling cell growth, differentiation, and activation [1,2,3,4,5]. No signal was observed when normal rabbit serum was used to immunoprecipitate lysates from unstimulated or SCF- or PMA-stimulated CMK cells (data not shown), indicating that the R-4250 antibody reacted with RAFTK. SCF treatment of CMK cells induced (within 1 min) the rapid phosphorylation of RAFTK, which was inhibited by the PKC inhibitors calphostin C (Fig. 1C) or staurosporine (data not shown).

Results

Conclusion