Characteristics of the Molecular Interaction of Adaptor Proteins in Protein Tyrosine Kinase Cascades

Park, Young Kyu; Choi, Soo Jin Na; Park, Rae Kil; Chung, Sang Young; Kim, Shin Kon

Abstract

Recent evidence suggests that FcgammaR and FcgammaR receptors signal through the low molecular weight GTPase, Ras. Ras is regulated by the nucleotide exchange protein, Sos, via formation of Grb2/Sos complex. Herein, we examine the modulation of adaptor protein interaction(Shc, Grb2, and Cbl) following FcgammaRI mediate signaling leading to activation of Ras. Cross-linking of FcgammaRI induces the conversion of GDP-Ras to GTP-Ras reaching a maximum 5 min after stimulation. Concomitantly with Ras activation Sos undergos an electrophoretic mobility shift and the Sos/Grb2 association is increased(6 fold). Tyrosine phosphorylated Shc, mainly p52 isoform, onloads to Grb2/Sos complex upon FcgammaRI stimulation. The augmented Sos/Grb2/Shc interaction is unique to ITAM1-related FcgammaRI signaling, not observed with growth factor receptors(insulin and EGF). Reciprocal anti-Shc and anti-Grb2 immunoprecipitations demonstrate that tyrosine phosphorylated Shc inducibly associates with Grb2 through Grb2-SH2 domain and coprecipitates with other phosphoproteins, such as p145 and p35. Cross-linking of FcgammaRI induces the tyrosine phosphorylation of Cbl which forms a complex with Grb2 and Shc via Cbl C-terminus; at least part of Shc/Cbl interaction is mediated through Shc SH2 domain. Kinetics experimetns confirm that Cbl/Grb2 is relatively stable, whereas Grb2/Sos, Grb2/Shc, and Cbl/Shc interactions are highly inducible. These results indicate that distinct adaptor complexes containing Cbl/Grb2/Shc or Shc/Grb2/Sos are modulated by FcgammaRI stimulation potentially regulating Ras in myeloid cells.