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

The kinetics of the EGF receptor (EGFR) autophosphorylation and of the phosphorylation by EGFR of a fusion protein (Fp(SH2)) derived from PLC-gamma 1 with two SH2 domains were studied employing purified EGFR or membrane-bound preparations of native and truncated EGFR. With varied ATP concentrations both reactions yielded Michaelis-Menten kinetics. KATP for autophosphorylation was 0.35 microM and for Fp(SH2) phosphorylation 1.35 microM. With Fp(SH2) and were followed by drops to zero velocities at about 1.0 microM 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.