Identification and activation of Src family kinases in primary megakaryocytes

Abstract

Objectives We have recently shown that the Src family of tyrosine kinases (SFKs) are activated by TPO stimulation in both primary megakaryocytic progenitors and a hematopoietic cells line (BaF3) expressing the TPO receptor (Mpl). In this study, we examine which of the eight Src family members are expressed in primary megakaryocytes (MKs) and determine which of these become activated in response to TPO. Materials and methods High-density oligonucleotide microarrays were used to compare the gene expression profiles of Src kinases from undifferentiated hematopoietic progenitors (CD34 +/CD38 lo) and after in vitro megakaryocytic differentiation. Western blot analysis of lysates from purified, mature murine MKs identified which of SFKs are present. Finally, in vitro kinase assays determined which of the SFKs in primary MKs are activated by TPO stimulation. Results Array profiles demonstrate that Fyn, Lyn, Fgr, Hck, Src, and Yes are all expressed in cultured human MKs (Fyn, Lyn > Src, Yes, Fgr, Hck). Similarly, Western blots of murine MKs identified the same six SFKs (Fyn, Fgr, Hck, Lyn, Src, and Yes). Of these, only Fyn and Lyn demonstrate increased kinase activity after TPO stimulation. Interestingly, gene expression analysis indicates that, among the SFKs, Fyn expression is uniquely upregulated during MK development. Conclusion These results provide the first direct evidence that two Src kinases are activated in primary MKs, Fyn and Lyn. The fact that only Fyn expression is significantly upregulated during MK differentiation suggests variable gene regulation. Specificity of the TPO signaling cascade is demonstrated by the selective activation of Fyn and Lyn.