Sequential Enzymatic Down-Regulation and Degradation of the Lyn Tyrosine Kinase in Erythropoietin-Stimulated Cells.

Erythropoietin (Epo) is a key molecule in the development of red blood cells. We have shown previously that the tyrosine kinase Lyn is involved in differentiation signals emanating from an activated Epo-receptor. We have recently identified Cbp (Csk binding protein, a negative regulator of Src) as a novel interactor with Lyn. The proline-rich region of Cbp associates with the SH3 domain of Lyn, which then phosphorylates Cbp on multiple tyrosine residues forming additional binding sites for the Lyn SH2 domain. Phosphorylated Y314 of Cbp, in turn, recruits the negative regulators Csk/Ctk, which phosphorylate Lyn and down-regulate its kinase activity. A phosphotyrosine-specific yeast two-hybrid system we developed, confirmed that the SH2 domain of Csk associated with Y314 of Cbp. Significantly, another well characterized negative regulator, SOCS1, also bound this phosphorylated tyrosine residue, resulting in elevated ubiquitination and degradation of Lyn. Thus, Cbp co-ordinates a two step down-regulation of Lyn. In Epo-responsive J2E cells, Cbp is rapidly phosphorylated by Lyn and the enzymatic activity of Lyn is suppressed by Csk/Ctk after 10 minutes of Epo stimulation. Subsequently, SOCS1 is involved in the turn over of Lyn protein two hours post-Epo induction. These data demonstrate for the first time a two phase process regulated by Cbp for inactivating and degrading Lyn after Epo stimulation.