Abstract
SLP-76(SH2 domain-containing leukocyte protein of 76 kD) is a hematopoietic adapter protein that is expressed in myeloid and T cells. SLP-76 is a substrate for tyrosine kinase in the src and syk family activation pathway required for T-cell receptor-mediated signaling. Cross-linking of the human FcgammaRIIa1 (CD32) in myeloid cells which contains an immune receptor tyrosine-based activation motif (ITAM) causes phosphorylation of SLP-76. Mice deficient in SLP76 develop fetal hemorrhage along with failure of T cell development and perinatal mortality. We have found that K562 cells express SLP-76. We hypothesized that SLP-76 or associated proteins may be substrates of Bcr-Abl in the K562 cell line and thus promote survival signals.
Materials and Methods: The K-562 cell line was obtained from the American collection of Cell Cultures. SLP deficient (−/−) KO mice were obtained from Dr. James Clements (Roswell Park Cancer Institute). Antibodies used include Sheep polyclonal IgG Anti-Human SLP 76, Peroxidase-conjugated Affinipure Donkey Anti-Sheep IgG at 0.8mg/ml, Mouse monoclonal IgG Anti-Phosphotyrosine, and Polyclonal goat Anti-mouse.
Immunoprecipitation and Immunoblot: Cells were either untreated or stimulated with purvanidate (phosphatase inhibitors). Purvanidate treated cells were used as a positive control. Spleenocytes isolated from a SLP-76 deficient mouse (KO) were used as negative control. Lysates were then subject to standard immunoprecipitation for SLP-76 followed by immunoblotting with a SLP-76 specific antibody. The denatured samples were then resolved by SDS-PAGE.
Results: SLP-76 is expressed in untreated and treated K562 with purvanidate and not detectable from lysates derived from SLP-76 KO spleenocytes. To assess the phosphorylation status of SLP-76 and any co-associated proteins in K562 cells, the SLP-76 blot was stripped and then immunoblotted for total phosphotyrosine content. SLP-76 does not appear to be constitutively phosphorylated in the K562 cells. However, significant tyrosine phosphorylation of SLP-76 was readily detectable in the purvanidate treated K562 cells.
Conclusion: The current studies reveal that although SLP-76 is indeed found in the K562 cell line expressing the Bcr-Abl oncogene. Somewhat surprisingly, despite the constitutive activation of the Bcr-Abl tyrosine kinase in K562 cells, we detected no obvious phosphoproteins co-precipitating with SLP-76 in the absence of purvanidate stimulation. Together, the lack of SLP-76 tyrosine phosphorylation and the lack of co-associated proteins in K562 cells suggest that SLP-76 is not a major player in the signal transduction pathways emanating from Bcr-Abl. However, a more stringent confirmation of this conclusion would require inhibiting SLP-76 expression in K562 cells and than assessing the growth and survival characteristics.
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