INTRAMOLECULAR REGULATION OF PROTEIN-TYROSINE-PHOSPHATASE SH-PTP1 - A NEW FUNCTION FOR SRC HOMOLOGY-2 DOMAINS
INTRAMOLECULAR REGULATION OF PROTEIN-TYROSINE-PHOSPHATASE SH-PTP1 - A NEW FUNCTION FOR SRC HOMOLOGY-2 DOMAINS
复制标题
DOI：
10.1021/bi00255a030
复制
发表时间：
1994-12-27
期刊：
影响因子：
2.9
通讯作者：
WALSH, CT
中科院分区：
文献类型：
--
作者：
PEI, DH;LORENZ, U;WALSH, CT
以对硝基磷酸苯酯、磷酸酪氨酰（pY）肽、还原型溶菌酶、羧酰胺甲基化溶菌酶、马来酰化溶菌酶和酪氨酰磷酸化溶菌酶为底物，研究了含Src同源2（SH 2）结构域的蛋白酪氨酸磷酸酶（PTP 1）的稳态动力学性质，并与3种截短突变体的稳态动力学性质进行了比较。在生理pH（7.4）下，相对于野生型酶，两个N-末端SH 2结构域[SH-PTP 1（Delta SH 2）]或C-末端的最后35个氨基酸[SH-PTP 1（Delta C35）]的截短分别激活磷酸酶活性30倍和20-34倍。最后60个氨基酸的截短导致具有野生型活性的突变体[SH-PTP 1（Delta C60）]。SH-PTP 1和SH-PTP 1（Δ C60）仅在酸性pH（pH小于或等于5.4）下显示出对pNPP的表观饱和动力学;当pH升高至5.5以上时，其表观K-M值急剧增加。相比之下，SH-PTP 1（Delta SH 2）在所有测试的pH值（pH 5.1-7.4）下均符合正常的米氏动力学，K-M恒定（10-14 mM）。此外，两个合成的pY肽对应于促红细胞生成素上已知的和潜在的磷酸化位点，（EPOR pY 429）和白细胞介素-3（IL-3R pY 628）受体与SH-PTP 1的N-末端SH 2结构域特异性结合（K-D = 1.8-10 μ M），并以浓度依赖性方式激活SH-PTP 1和SH-PTP 1（Delta C60）的催化活性，但不激活SH-PTP 1（Delta SH 2）。在70 μ M EPOR pY 429下实现SH-PTP 1的最大活化（25-30倍），并且最大活化的酶接近SH-PTP 1的活性（Δ SH 2）。加入40 μ M的EPOR pY 429肽（其对应于最近鉴定的SH-PTP 1的体内结合位点）也完全恢复了SH-PTP 1（pH 7.4）对pNPP的饱和动力学行为，其催化参数（K-M = 12.8 mM，k（cat）= 3.2 s（-1））与SH-PTP 1（Δ SH 2）的催化参数相似。这些数据表明，SH-PTP 1的SH 2结构域起到自抑制PTP 1结构域的磷酸酶活性的作用。提出了一种模型，其中SH 2结构域与PTP 1结构域以不依赖于pY的方式相互作用，并将PTP 1结构域驱动成非活性构象。
The steady-state kinetic properties of SH-PTP1 (PTP1C, SHP, HCP), a Src homology 2 (SH2) domain-containing protein tyrosine phosphatase (PTPase), were assessed and compared with those of three truncation mutants, using p-nitrophenyl phosphate, phosphotyrosyl (pY) peptides, and reduced, carboxyamido-methylated, maleylated, and tyrosyl-phosphorylated lysozyme as substrates. At physiological pH (7.4), truncation of the two N-terminal SH2 domains [SH-PTP1(Delta SH2)] or the last 35 amino acids of the C-terminus [SH-PTP1(Delta C35)] activated the phosphatase 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 [SH-PTP1(Delta C60)] with wild-type activity. SH-PTP1 and SH-PTP1(Delta C60) displayed apparent saturation kinetics toward pNPP only at acidic pH (pH less than or equal to 5.4); as pH increased above 5.5, their apparent K-M values increased dramatically. In contrast, SH-PTP1(Delta SH2) obeyed normal Michaelis-Menten kinetics at all pH values tested (pH 5.1-7.4) with a constant K-M (10-14 mM). Furthermore, two synthetic pY peptides corresponding to known and potential phosphorylation sites on the erythropoietin (EPOR pY429) and interleukin-3 (IL-3R pY628) receptors bound specifically to the N-terminal SH2 domain of SH-PTP1 (K-D = 1.8-10 mu M) and activated the catalytic activity of SH-PTP1 and SH-PTP1(Delta C60) but not SH-PTP1(Delta SH2), in a concentration-dependent manner. Maximal activation (25-30-fold) of SH-PTP1 was achieved at 70 mu M EPOR pY429, and the maximally activated enzyme approached the activity of SH-PTP1(Delta SH2). Addition of EPOR pY429 peptide, which corresponds to the recently identified in vivo binding site for SH-PTP1, at 40 mu M also completely restored the saturation kinetic behavior of SH-PTP1 (at pH 7.4) toward pNPP, with catalytic parameters (K-M = 12.8 mM, k(cat) = 3.2 s(-1)) similar to those of SH-PTP1(Delta SH2). These data suggest that the SH2 domains of SH-PTP1 serve to autoinhibit the phosphatase activity of the PTPase domain, A model is proposed in which the SH2 domains interact with the PTPase domain in a pY-independent fashion and drive the PTPase domain into an inactive conformation.