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

Yongsheng Ma, Matthew E. Cunningham, Xiaomei Wang, Indraneel Ghosh, Lynn Regan, B. Jack Longley

Research output: Contribution to journalArticle

99 Scopus citations

Abstract

KIT receptor kinase activity is repressed, prior to stem cell factor binding, by unknown structural constraints. Using site-directed mutagenesis, we examined the role of KIT intracellular juxtamembrane residues Met-552 through Ile-563 in controlling receptor autophosphorylation. Alanine substitution for Tyr-553, Trp557, Val-559, or Val-560, all sitting along the hydrophobic side of an amphipathic α-helix (Tyr-553-Ile-563) predicted by the Chou-Fasman algorithm, resulted in substantially increased spontaneous receptor phosphorylation, revealing inhibitory roles for these residues. Alanine substitution for other residues, most of which are on the hydrophilic side of the helix, caused no or slightly increased basal receptor phosphorylation. Converting Tyr-553 or Trp-557 to phenylalanine generated slight or no elevation, respectively, in basal KIT phosphorylation, indicating that the phenyl 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 receptor phosphorylation, its substitution with proline produced high spontaneous receptor phosphorylation, suggesting that the predicted α-helical conformation is involved in the inhibition. A synthetic peptide comprising Tyr-553 through Ile-563 showed circular dichroism spectra characteristic of α-helix, supporting the structural prediction. Thus, the KIT intracellular juxtamembrane region contains important residues which, in a putative α-helical conformation, exert inhibitory control on the kinase activity of ligand-unoccupied receptor.

Original languageEnglish (US)
Pages (from-to)13399-13402
Number of pages4
JournalJournal of Biological Chemistry
Volume274
Issue number19
DOIs
StatePublished - May 7 1999
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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