KIT associated intracellular tyrosines play an essential role in EpoR co-signaling

Abstract

KIT and erythropoietin receptor (EpoR) mediated co-signaling is essential for normal erythroid cell expansion, however the intracellular signals that contribute to cooperative signaling are poorly understood. Here, we examined the role of intracellular tyrosine residues in KIT and EpoR cooperation by co-expressing tyrosine (Y) to phenylalanine (F) and deletion mutants of KIT and EpoR in 32D cells. Of the four EpoR mutants examined, only EpoR-Y343 induced proliferation to near wildtype EpoR levels. A modest increase in the growth was also observed in 32D cells expressing the EpoR-Y343F; however neither EpoR-W282R nor EpoR-F8 showed any increase in growth over baseline. Biochemical analysis revealed that EpoR-Y343 induced the activation of Stat5, PI-3Kinase/ Akt and MAP kinase Erk1/2 to near wildtype EpoR levels, while the remaining mutants failed to activate any of these signals. Interestingly, none of the EpoR mutants cooperated with WT KIT, although EpoR-Y343 showed a modest increase in co-signaling. Loss of seven tyrosine residues in KIT (KIT-F7) completely abrogated EpoR induced co-signaling. Restoring the Src kinase binding sites in KIT-F7 alone or together with the PI3Kinase binding site restored KIT induced signals as well as co-signals with WT EpoR, although restoring the Src kinase binding sites along with the PLC-γ binding site repressed both KIT induced signaling as well as co-signaling with WT EpoR. Taken together, these results suggest that KIT and EpoR mediated co-signaling requires intracellular tyrosine residues and tyrosine residues that bind Src kinases in the KIT receptor appear to be sufficient for restoring both KIT signaling as well as co-signaling with EpoR. In contrast, restoration of the PLC-γ binding site in the context of Src binding sites appears to antagonize the positive signals induced via the Src kinase binding sites in the KIT receptor.

Stem Cell Factor Receptor/c-Kit: From Basic Science to Clinical Implications

“…In some cases, but not all, these types of heterotypic receptor interactions have been shown to correlate with cross-phosphorylation, e.g., SCF stimulation may promote tyrosine phosphorylation of the Epo or IL-7 receptor (123,300). In a study from Kapur's laboratory, it was demonstrated that mutation of seven tyrosine residues in the intracellular region of c-Kit abolished the ability of c-Kit to cooperate with Epo receptor, but interestingly adding back only the Src family kinase binding site in c-Kit restored cooperativity (111). Moreover, adding back the Src family binding site in combination with the PI3=-kinase binding site also allowed cooperatively between cKit and Epo receptor; however, this was inhibited by restoring both Src and PLC-␥ binding (111).…”

“…In a study from Kapur's laboratory, it was demonstrated that mutation of seven tyrosine residues in the intracellular region of c-Kit abolished the ability of c-Kit to cooperate with Epo receptor, but interestingly adding back only the Src family kinase binding site in c-Kit restored cooperativity (111). Moreover, adding back the Src family binding site in combination with the PI3=-kinase binding site also allowed cooperatively between cKit and Epo receptor; however, this was inhibited by restoring both Src and PLC-␥ binding (111). Also, the synergistic interaction between c-Kit and GM-CSF receptor may involve transphosphorylation; GM-CSF by itself did not promote c-Kit phosphorylation, but concurrent treatment with both SCF and GM-CSF did result in a stronger c-Kit phosphorylation compared with SCF by itself (127), although other studies have failed to demonstrate this (162).…”

Kit transduced signals counteract erythroid maturation by MAPK-dependent modulation of erythropoietin signaling and apoptosis induction in mouse fetal liver

“…55 However, studies using EpoR tyrosine-mutants demonstrated that PI3K/Akt activation is also indirectly mediated via Y343, involving the adaptor Gab2. 37,55,56 Stat5 and Akt pathways are thus coupled in EpoR signaling and might cooperate in target gene activation. Interestingly, we could show that KIT murine embryonic stem cells as previously described.…”

Kit activates interleukin‐4 receptor and effector signal transducer and activator of transcription 6 independent of its cognate ligand in mouse mast cells

“…In addition, Kit and Epo-R cross-receptor activation is reported to be necessary for normal erythroid cell expansion. [22][23][24][25][26] Previous in vivo analyses using Kit and c C mutant mice revealed synergistic effects of Kit and c C signaling in thymopoiesis. 27,28 In natural killer cells, activation of Kit and IL-2/15 signaling causes synergy in proliferation through enhanced mitogen-activated protein kinase activity.…”