Inhibition of spontaneous receptor phosphorylation by residues in a putative alpha-helix in the KIT intracellular juxtamembrane region

KIT receptor kinase activity is repressed, prior to stem cell factor bindin g, by unknown structural constraints. Using site-directed mutagenesis, we e xamined the role of KIT intracellular juxtamembrane residues Met-552 throug h Ile-563 in controlling receptor autophosphorylation, Alanine substitution for Tyr-553, Trp-557, Val-559, or Val-560, all sitting along the hydrophob ic side of an amphipathic alpha-helix (Tyr-553-Ile-563) predicted by the Ch ou-Fasman algorithm, resulted in substantially increased spontaneous recept or phosphorylation, revealing inhibitory roles for these residues Alanine s ubstitution for other residues, most of which are on the hydrophilic side o f the helix, caused no or slightly increased basal receptor phosphorylation , Converting Tyr-553 or Trp-557 to phenylalanine generated slight or no ele vation, respectively, in basal KIT phosphorylation, indicating that the phe nyl ring of Tyr-553 and the hydrophobicity of Trp-557 are critical for the inhibition. Although alanine substitution for Lys-558 had no effect on rece ptor phosphorylation, its substitution with proline produced high spontaneo us receptor phosphorylation, suggesting that the predicted alpha-helical co nformation is involved in the inhibition. A synthetic peptide comprising Ty r-553 through Ile-563 showed circular dichroism spectra characteristic of a lpha-helix, supporting the structural prediction. Thus, the KIT intracellul ar juxtamembrane region contains important residues which, in a putative al pha-helical conformation, exert inhibitory control on the kinase activity o f ligand-unoccupied receptor.