摘要：

The steady-state kinetic properties of (, , ), a homology 2 () domain-containing protein tyrosine (), were assessed and compared with those of three truncation mutants, using p-nitrophenyl phosphate, phosphotyrosyl (pY) , and reduced, carboxyamido-methylated, maleylated, and -phosphorylated as substrates. At physiological pH (7.4), truncation of the two N-terminal domains [(delta )] or the last 35 amino acids of the C-terminus [(delta C35)] activated the activity by 30-fold and 20-34-fold relative to the wild-type enzyme, respectively. Truncation of the last 60 amino acids resulted in a mutant [(delta )] with wild-type activity. and (delta ) displayed apparent saturation kinetics toward pNPP only at acidic pH (pH < or = 5.4); as pH increased above 5.5, their apparent KM values increased dramatically. In contrast, (delta ) obeyed normal Michaelis-Menten kinetics at all pH values tested (pH 5.1-7.4) with a constant KM (10-14 mM). Furthermore, two synthetic pY corresponding to known and potential sites on the erythropoietin (pY429) and interleukin-3 (pY628) receptors bound specifically to the N-terminal domain of (KD = 1.8-10 microM) and activated the of and (delta ) but not (delta ), in a concentration-dependent manner. Maximal activation (25-30-fold) of was achieved at 70 microM pY429, and the maximally activated enzyme approached the activity of (delta ). Addition of pY429 , which corresponds to the recently identified in vivo site for , at 40 microM also completely restored the saturation kinetic of (at pH 7.4) toward pNPP, with catalytic parameters (KM = 12.8 mM, kcat = 3.2 s-1) similar to those of (delta ). These data suggest that the domains of serve to autoinhibit the activity of the domain. A model is proposed in which the domains interact with the domain in a pY-independent fashion and drive the domain into an inactive conformation.

展开