Inhibition of Spontaneous Receptor Phosphorylation by Residues in a Putative α-Helix in the KIT Intracellular Juxtamembrane Region

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, Trp-557, Val-559, or Val-560, all sitting along the hydrophobic side of an amphipathic ␣-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 ␣-helical conformation is involved in the inhibition. A synthetic peptide comprising Tyr-553 through Ile-563 showed circular dichroism spectra characteristic of ␣-helix, supporting the structural prediction. Thus, the KIT intracellular juxtamembrane region contains important residues which, in a putative ␣-helical conformation, exert inhibitory control on the kinase activity of ligandunoccupied receptor.

Signal transduction by several KIT juxtamembrane domain mutations

“…More recently, it has been predicted that the residues contained in the KIT sequence between residues 553 and 663 can fold into a putative a helix conformation. This sequence could be involved in the repression of spontaneous receptor phosphorylation by producing an inhibitory effect on the adjacent cytoplasmic domain, and it was suggested that the phenyl ring of Tyr 553 and the hydrophobicity of Trp 557 are critical for the inhibition (Ma et al, 1999a), (Figure 1). Now, multiple deletions or missense mutations that affect this region have been identified in the KIT sequence, several times in GIST (Taniguchi et al, 1999) once in a mastocytoma (Furitsu et al, 1993) as well as in our KD27 mutation and in the FLT3 sequence in AML (Nakao et al, 1996).…”

Mechanism of constitutive activation of FLT3 with internal tandem duplication in the juxtamembrane domain

“…Chemical-cross linking experiments revealed that c-KIT with the former mutations underwent dimerization independent of the ligand, whereas c-KIT with the latter mutations did not (Kitayama et al, 1995). The c-KIT JM domain reportedly exerted inhibitory e ects on the receptor kinase activity (Ma et al, 1999). However, gel-®ltration assay later showed that the missense mutation of the kinase domain also promotes the dimerization, suggesting that dimerization and phosphorylation are closely coupled (Lam et al, 1999).…”

FLT3: ITDoes matter in leukemia

“…96 Activating mutations localized to the juxtamembrane region are also found in other members of the PDGF receptor subfamily, further supporting an autoinhibitory role for this domain. [97][98][99] An activating tandem duplication within the juxtamembrane region of KIT was identified in a canine mastocytoma, and a variety of activating point mutations within this region of the receptor have been found in human gastrointestinal stromal tumors. 81,100 An activating juxtamembrane point mutation was also identified in the PDGF receptor.…”