Kinetics of phosphorylation of the SH2‐containing domain of phospholipase Cγ1 by the epidermal growth factor receptor

FEBS Letters

Levitzki, Alexander¹  Posner, Israel¹ 

[1] Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

关键词: Receptor;    EGF receptor;    Phospholipase Cγ1;    SH2 domain;    Tyrosine kinase;    EGFR kinetics;    BMN;    benzenemalononitrile;    CD63;    NIH 3T3 cells expressing an active EGFR mutant lacking 63 amino acids including Y1148 and Y1173;    CD126;    NIH3T3 cells expressing a truncated EGFR lacking 126 amino acid including Y1068;    Y1086;    Y1173;    DTT;    dithiothreitol;    DMSO;    dimethylsulfoxide;    EGF;    epidermal growth factor;    EGFR;    EGF receptor;    EGFR-P;    pre-phosphorylated EGF receptor;    Fp(SH2);    GST fusion protein;    HEPES;    4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;    HER14;    native NIH3T3/ HER14 cells;    GST;    glutathione S-transferase;    MES;    2-[Morpholino]-ethanesulfonic acid;    SH2 domain;    Src homology 2 domain;    TCA;    trichloroacetic acid;    PTK;    protein kinase catalytic subunit;    TK;    tyrosine kinase;   

DOI  :  10.1016/0014-5793(94)00989-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The kinetics of the EGF receptor (EGFR) autophosphorylation and of the phosphorylation by EGFR of a fusion protein (Fp(SH2)) derived from PLC-γ₁, with two SH2 domains were studied employing purfied EGFR or membrane-bound preparations of native and truncated EGFR. With varied ATP concentrations both reactions yielded Michaelis—Menten kinetics. K_ATP, for autophosphorylation was 0.35μM and for Fp(SH2) phosphorylation 1.35 μM. With Fp(SH2) as variable, the velocity curves for substrate phosphorylation by the various EGFR preparations were sigmoidal, reached peaks at 0.45μM Fp(SH2) and were followed by drops to zero velocities at about 1.0μM Fp(SH2). We conclude that (a) our data support the concept that receptor autophosphorylation is a prerequisite for the interactions between EGFR and the substrate's SH2-domains and their eventual phosphorylation by the receptor, and (b) the interactions between EGFR and the physiological substrate seem to involve mechanisms which allow the substrate to act as an on-off switch in the subsequent substrate phosphorylation reaction.

【 授权许可】

Unknown