Veillette A, Dumont S, Fournel M
McGill Cancer Centre, Montréal, Canada.
J Biol Chem. 1993 Aug 15;268(23):17547-53.
We have evaluated the possibility that conserved cysteine residues are critical for the enzymatic function of p56lck. Through oligonucleotide-directed mutagenesis, 5 Lck residues (cysteines 217, 224, 378, 464, and 475) were individually mutated to alanines, and the effects of these substitutions were tested in various in vitro and in vivo assays. We found that mutation of either of 2 cysteines located in the carboxyl portion of the kinase domain (cysteines 464 and 475) abolished the catalytic function of Lck. In addition, it was noted that alteration of cysteine 475 resulted in a dramatic reduction of the half-life of p56lck. These cysteine residues are highly conserved throughout the tyrosine protein kinase family, suggesting that they may play important functions in catalysis and/or substrate recognition.
我们评估了保守半胱氨酸残基对p56lck酶功能至关重要的可能性。通过寡核苷酸定向诱变，将5个Lck残基（半胱氨酸217、224、378、464和475）分别突变为丙氨酸，并在各种体外和体内试验中测试这些替代的效果。我们发现位于激酶结构域羧基部分的2个半胱氨酸（半胱氨酸464和475）中的任何一个发生突变都会消除Lck的催化功能。此外，还注意到半胱氨酸475的改变导致p56lck半衰期大幅缩短。这些半胱氨酸残基在整个酪氨酸蛋白激酶家族中高度保守，表明它们可能在催化和/或底物识别中发挥重要作用。
