Abstract
Csk-binding Protein Mediates Sequential Enzymatic Down-regulation and Degradation of Lyn in Erythropoietin-stimulated Cells
Highlights
Receptor dimerizes and Janus kinase 2 (JAK2) becomes activated by transphosphorylation [5, 6]
C-terminal Src kinase-binding protein (Cbp) contains a number of tyrosine residues, which may become phosphorylated and associate with the Lyn SH2 domain
Because our investigations focused primarily on the effects of Lyn/Cbp in erythroid cells, we examined whether Csk-like protein-tyrosine kinase (Ctk) [46], the closely related hemopoietic homologue of C-terminal Src kinase (Csk), could associate with Cbp
Summary
Plasmid Construction—The Myc-tagged Cbp expression constructs pCMV-Cbp and pCMV-CbpY314F [31] were generously provided by Dr M. Subcloning the yeast ADE2 gene from pASZ11 [42] into pMET416-Lyn generated the yeast Lyn kinase domain expression plasmid pRSADE-MET-Lyn, using adenine rather than uracil selection. J2E subclones expressing Lyn[Y397F], Cbp, Cbp[Y314F], or SOCS1, were generated by infection with amphitropic retroviruses produced by PA317 packaging cells transfected with the pMSCV constructs; multiple unique clonal lines were isolated using methylcellulose cultures as previously described [36]. Kinase Assays—Lyn exokinase and autokinase activity were determined by immunoprecipitation with anti-Lyn antibodies (sc-15, Santa Cruz Biotechnology) and subsequent incubation with [␥-32P]ATP in the presence or absence of the substrates (acid-denatured enolase or purified GST, GST-Cbp, GSTCbp[Y314F], GST-Cbp[Y381F/Y409F]), according to the protocol described previously [21]. Statistical Analysis—Quantitated data were analyzed for statistical significance by two-way ANOVA using Excel (Microsoft). p values less than 0.05 were taken as significant
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