Involvement of the Lyn Interactome in the Regulation of Erythropoiesis.

Src family kinases, such as Lyn, are involved in signalling pathways regulating a myriad of cellular processes. We have shown previously that Lyn is involved in differentiation signals emanating from activated erythropoietin (Epo) receptors. We have highlighted the importance of Lyn to red cell maturation in vivo with Lyn^{−/ −} mice, which develop anaemia and display extramedullary stress erythropoiesis. Here, we show that ex vivo cultures of primary Lyn^{−/ −} erythroblasts display an imbalance between proliferation and differentiation. In addition we have identified several new Lyn pathways. One centres on Cbp (Csk binding protein), an adaptor protein that recruits negative regulators Csk/Ctk. Our data show that Lyn phosphorylates Cbp, which then recruits Csk/Ctk to suppress Lyn kinase activity. Intriguingly, through the use of a phosphotyrosine-specific yeast two-hybrid assay that we developed, phosphorylated Cbp also binds SOCS1, another well-characterised negative regulator of cell signalling - this results in elevated ubiquitination and degradation of Lyn in Epo-stimulated erythroid cells. Altering Cbp expression in primary erythroblasts significantly affected their ability to transmit Epo-receptor signals and differentiate correctly. Another Lyn pathway we have identified centres on a novel molecule we termed LACM, which transmits Lyn signals to Vav2 and Nckβ via phospho-tyrosine-specific interactions. These novel associations regulate the cytoskeleton/cell shape, which lead to changes in cell attachment and migration. Significantly, primary erythroblasts from Lyn^{−/ −} mice display altered subcellular localization of LACM. Another novel Lyn interactor (LIAR) regulates nuclear import/export of signalling molecules - impairing LIAR’s normal function inhibits primary erythroblast development. Taken together, these results illustrate that Lyn is intimately involved in multiple signalling complexes, which play crucial roles in regulating numerous aspects of red blood cell biology.