Adaptor protein SKAP55R is associated with myeloid differentiation and growth arrest

Objective. Activation of the SRC family of protein tyrosine kinases is an i mportant component of intracellular signaling in hematopoiesis, but their c ritical substrates are less well understood. In this report, we describe th e cloning and functional characterization of murine SKAP55R (mSKAP55R), an SRC family kinase substrate, Materials and Methods. Expression of mSKAP55R was examined by Northern blot . Phosphorylation of mSKAP55R was examined by transient transfection of COS cells. For overexpression studies, mSKAP55R was cloned into a bicistronic murine stem cell virus-based retrovirus. Transduced cells (FDC-P1 cell line and murine bone marrow) were FACS isolated by expression of the selectable marker green fluorescent protein. Results. mSKAP55R showed 90% amino acid identity to the recently published human SKAP55R. mSKAP55R contained a central pleckstrin homology domain, a C -terminal SH3 domain, and a putative SRC kinase consensus substrate DEIY260 . mSKAP55R was expressed in all hematopoietic lineages, with relative mRNA levels greatest in cells of the myeloid and erythroid lineages. Induced mye loid differentiation of M1 and HL-60 cell lines was associated with an eigh t-fold increase in mSKAP55R mRNA. Transient expression of mSKAP55R in COS c ells demonstrated that tyrosine 260 was the predominant site of phosphoryla tion by FYN kinase, Furthermore, this phosphotyrosine was essential for coi mmunoprecipitation of FYN with mSKAP55R. Enforced expression of mSKAP55R in hibited in vitro growth of the myeloid FDC-P1 cell line and primary hematop oietic progenitors, In contrast, a tyrosine 260 mutant mSKAP55R had no effe ct on in vitro growth. Conclusion. These studies implicate mSKAP55R in the processes of myeloid di fferentiation and growth arrest. (C) 2000 International Society for Experim ental Hematology. Published by Elsevier Science Inc.