Kit PY567- Directed Signals Are Important for Efficient Erythroid Progenitor Cell Proliferation and Survival, and Are Attenuated by Kit PY569.

Anemia incurred by mice with naturally occurring Kit mutations (eg, W41) clearly illustrates important contributions of SCF/Kit signaling to erythropoiesis. Specific cytoplasmic features of Kit and linked signaling events that support such contributions, however, are not well understood. To address this issue, the proliferative and survival signaling activities of a panel of Kit cytoplasmic PY mutants first were studied in SCF- and Epo- dependent G1E2 cells, and also in multi-potential SCF-dependent EML cells. Kit forms studied included PY567, PY569, PY567-plus-569, PY702, PY719, PY728 and PY745 phosphotyrosine add-back forms as well as a biologically inactive baseline mutant, Kit-F6 in which these six PY sites were mutated to phenylalanine. To by-pass endogenous Kit, cytoplasmic PY mutants were expressed from a MIEG3 vector as MCSF-R/Kit “MK” chimeras, and cells expressing matched and physiological levels of M/K chimeras were isolated by FACS. PY567 per se (ie, M/K PY567) selectively supported near wild-type proliferative and anti-apoptotic activities in both G1E2 and EML cells. By direct comparison, PY569 per se has diminished activity in G1E2 cells, and only baseline activity in EML cells. Moreover, the combined restoration of both PY567 and 569 uniformly attenuated the otherwise high proliferative and survival activities of PY567. These findings therefore indicate that Kit PY567 and PY569 do not necessarily exert overlapping roles, but that PY569 instead may preferentially link to negative effects and also act to attenuate key positive signals relayed via Kit PY567 (possibly in a lineage- restricted fashion). In keeping with recent studies in 32D cells, little if any bioactivities were conferred by PY702, 719, 728, or 745 per se. Mechanistically, PY567 also was observed to link with high efficiency to sustained Bcl-xl expression, and to be selectively efficient in Epo receptor co-signaling. While these findings predict that the mutation of Kit Y567 should perturb red cell development, erythropoiesis in Kit-F567 knock-in mice appears near-normal. However, primary erythroid progenitor cells isolated from the spleen of Kit F567 mice at 50hr post- phenylhydrazine injection proved to be markedly deficient in SCF responsiveness. In addition, a >5-fold decrease in SCF and Epo co-signaling was reproducibly observed for Kit-F567 progenitor cell preparations. These novel findings therefore lend new evidence to the case that PY567, in fact, contributes in important ways to Kit’s erythropoietic biofunction.