A KIT juxtamembrane PY567 -directed pathway provides nonredundant signals for erythroid progenitor cell development and stress erythropoiesis

Abstract

KITL/KIT can elicit diverse sets of signals within lymphoid, myeloid, mast, and erythroid lineages, and exert distinct effects on growth, survival, migration, adhesion, and secretory responses. Presently, we have applied a PY-mutant allele knockin approach to specifically assess possible roles for KIT-PY567 and KIT-PY719 sites, and coupled pathways, during erythropoiesis. Mouse models used to investigate this problem include those harboring knocked-in KIT(Y567F/Y567F), KIT(Y569F/Y569F), KIT(Y719F,Y719F), and KIT(Y567F/Y567F:Y569F/Y569F) alleles. The erythron was stressed by myelosuppression using 5-fluorouracil, and by phenylhydrazine-induced hemolysis. In addition, optimized systems for ex vivo analyses of bone marrow and splenic erythropoiesis were employed to more directly analyze possible stage-specific effects on erythroid cell growth, survival, development and KIT signaling events. In Kit(Y567F/Y567F) mice, steady-state erythropoiesis was unperturbed while recovery from anemia due to 5-fluorouracil or phenylhydrazine was markedly impaired. Deficiencies in erythroid progenitor expansion occurred both in the bone marrow and the spleen. Responses to chronic erythropoietin dosing were also compromised. Ex vivo, Kit(Y567F/Y567F) (pro)erythroblast development was skewed from a Kit(pos)CD71(high) stage toward a subsequent Kit(neg)CD71(high) compartment. Proliferation and, to an extent, survival capacities were also compromised. Similar stage-specific defects existed for erythroid progenitors from Kit(Y567F/Y567F:Y569F/Y569F) but not KIT(Y719F/Y719F) mice. Kit(Y567F/Y567F) erythroblasts were used further to analyze KIT-PY567-dependent signals. MEK-1,2/ERK-1,2 signaling was unaffected while AKT, p70S6K, and especially JNK2/p54 pathways were selectively attenuated. Nonredundant KIT-PY567-directed erythroblast-intrinsic signals are selectively critical for stress erythropoiesis. Investigations also add to an understanding of how KIT directs distinct outcomes among diverse progenitors and lineages.