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Adaptor protein SKAP55R is associated with myeloid differentiation and growth arrest
D J Curtis1, S M Jane, D J Hilton
1The Walter and Eliza Hall Institute of Medical Research and The Co-operative Research Centre for Cellular Growth Factors, Royal Melbourne Hospital, Victoria, Australia. dcurtis@nhgri.nih.gov
Abstract:
Activation of the SRC family of protein tyrosine kinases is an important component of intracellular signaling in hematopoiesis, but their critical substrates are less well understood. In this report, we describe the cloning and functional characterization of murine SKAP55R (mSKAP55R), an SRC family kinase substrate. 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.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 DEIY(260). mSKAP55R was expressed in all hematopoietic lineages, with relative mRNA levels greatest in cells of the myeloid and erythroid lineages. Induced myeloid differentiation of M1 and HL-60 cell lines was associated with an eight-fold increase in mSKAP55R mRNA. Transient expression of mSKAP55R in COS cells demonstrated that tyrosine 260 was the predominant site of phosphorylation by FYN kinase. Furthermore, this phosphotyrosine was essential for coimmunoprecipitation of FYN with mSKAP55R. Enforced expression of mSKAP55R inhibited in vitro growth of the myeloid FDC-P1 cell line and primary hematopoietic progenitors. In contrast, a tyrosine 260 mutant mSKAP55R had no effect on in vitro growth. These studies implicate mSKAP55R in the processes of myeloid differentiation and growth arrest.
Insights
Murine SKAP55R (mSKAP55R), an SRC family kinase substrate, is crucial for hematopoiesis. Its phosphorylation regulates myeloid differentiation and growth arrest, impacting hematopoietic progenitor cells.
Area of Science:
- Cell Biology
- Molecular Biology
- Hematopoiesis
Background:
- SRC family kinases are vital for intracellular signaling in hematopoiesis.
- Understanding their substrates is key to deciphering these pathways.
Purpose of the Study:
- To clone and functionally characterize murine SKAP55R (mSKAP55R), an SRC family kinase substrate.
- To investigate the role of mSKAP55R in myeloid differentiation and cell growth.
Main Methods:
- Cloning and functional characterization of mSKAP55R.
- Northern blot for expression analysis.
- Transient transfection for phosphorylation studies.
- Retroviral transduction for overexpression in cell lines and bone marrow.
- FACS isolation of transduced cells.
- Coimmunoprecipitation assays.
Main Results:
- mSKAP55R shares high homology with human SKAP55R and possesses key functional domains.
- mSKAP55R is expressed in all hematopoietic lineages, with highest levels in myeloid and erythroid cells.
- Myeloid differentiation increased mSKAP55R mRNA levels.
- Tyrosine 260 is the primary phosphorylation site by FYN kinase and is essential for FYN binding.
- Overexpression of wild-type mSKAP55R inhibited myeloid cell growth, while a Y260 mutant had no effect.
Conclusions:
- mSKAP55R is a significant substrate of SRC family kinases involved in hematopoiesis.
- Phosphorylation at tyrosine 260 is critical for mSKAP55R function.
- mSKAP55R plays a regulatory role in myeloid differentiation and growth arrest.