Structure Function Analysis of Hematopoietic Specific Src Family Kinases Lyn, Hck and Fgr Reveal a Critical Role for Lyn’s Unique Domain in Regulating Growth of Myeloid Cells That Is Distinct from the Role of Hck and Fgr’s Unique Domain.
Authors: Chen Jinbiao , Vemula Sasidhar , Kapur Reuben 
Published: April 2020

Abstract
A flurry of recent publications have implicated hematopoietic specific Src family kinases (SFKs) in contributing to the pathogenesis of lymphoid and myeloid leukemia. However, little is known about the mechanism(s) by which these kinases regulate the growth of myeloid cells. SFKs share significant structural and amino acid sequence homology, particularly in the catalytic domain and in the SH2 and SH3 domains of the regulatory region. Therefore, it is generally believed that SFKs play a redundant role in regulating growth and actin based functions. Although, there are no apparent differences in the known effector domains of SFKs, significant differences exist between the amino terminus unique regions of Hck, Fgr and Lyn. Here, we show that the deficiency of Hck or Fgr or both in myeloid cells results in reduced cytokine (stem cell factor [SCF] & IL-3) induced proliferation. In contrast, deficiency of Lyn SFK results in enhanced growth and survival of myeloid cells. SCF induced hypersensitivity due to Lyn deficiency is observed in spite of the presence of Hck and Fgr in myeloid cells, suggesting that Lyn functions with specificity in negatively regulating cytokine signaling. To determine whether SCF induced hypersensitivity due to Lyn deficiency is contributed via its N-terminal unique domain, we cloned and retrovirally expressed a mutant version of Lyn that lacks its unique domain. As expected, reconstituting Lyn−/− myeloid cells with a cDNA encoding the wildtype version of Lyn completely restored cytokine induced hypersensitivity to wildtype levels. In contrast, expression of a Lyn mutant lacking its N-terminal unique domain, but consistent of all the other domains, including the myristolation and the palmitoylation acetylation sites behaved in a fashion similar to Lyn deficient myeloid cells. Furthermore, neither overexpression of Hck, nor Fgr in Lyn−/− cells affected cytokine induced hypersensitivity. To further characterize the unique nature of Lyn’s N-terminal (unique) domain in cytokine induced hypersensitivity, we cloned and expressed several chimeric proteins in which the unique domain of Lyn was replaced with the unique domain of Hck or Fgr. When expressed in Lyn−/− cells, these chimeric proteins were unable to correct cytokine induced hypersensitivity due to Lyn deficiency. In contrast, swapping Hck’s or Fgr’s unique domain with Lyn’s unique domain completely rescued cytokine induced hypersensitivity in Lyn−/− cells. These results suggest that Lyn’s unique domain plays a critical role in negatively regulating cytokine induced growth in myeloid cells. Although Lyn contains a palmitoylation and an acetylation site, the specific role of these sites in membrane targeting and in cytokine-induced growth is not known. To assess the role of these sites, we generated three additional mutants of Lyn and expressed them in Lyn−/− cells. Mutant of Lyn lacking both the acetylation and the palmitoylation site demonstrated cytokine induced hypersensitivity similar to that seen in Lyn−/− cells. In contrast, mutating the acetylation site alone in Lyn did not affect cytokine induced hyperproliferation, although mutating the palmitoylation site did result in cytokine induced hypersensitivity. Taken together, our results provide compeling evidence for a novel role for Lyn’s unique domain in negatively regulating cytokine signaling, which is distinct from the role of Hck or Fgr’s unique domain.