LaMontagne K R, Flint A J, Franza B R, Pandergast A M, Tonks N K
Cold Spring Harbor Laboratory, New York 11724-2208, USA.
Mol Cell Biol. 1998 May;18(5):2965-75. doi: 10.1128/MCB.18.5.2965.
The p210 bcr-abl protein tyrosine kinase (PTK) appears to be directly responsible for the initial manifestations of chronic myelogenous leukemia (CML). In contrast to the extensive characterization of the PTK and its effects on cell function, relatively little is known about the nature of the protein tyrosine phosphatases (PTPs) that may modulate p210 bcr-abl-induced signalling. In this study, we have demonstrated that expression of PTP1B is enhanced specifically in various cells expressing p210 bcr-abl, including a cell line derived from a patient with CML. This effect on expression of PTP1B required the kinase activity of p210 bcr-abl and occurred rapidly, concomitant with maximal activation of a temperature-sensitive mutant of the PTK. The effect is apparently specific for PTP1B since, among several PTPs tested, we detected no change in the levels of TCPTP, the closest relative of PTP1B. We have developed a strategy for identification of physiological substrates of individual PTPs which utilizes substrate-trapping mutant forms of the enzymes that retain the ability to bind to substrate but fail to catalyze efficient dephosphorylation. We have observed association between a substrate-trapping mutant of PTP1B (PTP1B-D181A) and p210 bcr-abl, but not v-Abl, in a cellular context. Consistent with the trapping data, we observed dephosphorylation of p210 bcr-abl, but not v-Abl, by PTP1B in vivo. We have demonstrated that PTP1B inhibited binding of the adapter protein Grb2 to p210 bcr-abl and suppressed p210 bcr-abl-induced transcriptional activation that is dependent on Ras. These results illustrate selectivity in the effects of PTPs in a cellular context and suggest that PTP1B may function as a specific, negative regulator of p210 bcr-abl signalling in vivo.
p210 bcr-abl蛋白酪氨酸激酶（PTK）似乎直接导致了慢性粒细胞白血病（CML）的初始症状。与对PTK及其对细胞功能影响的广泛研究相比，人们对可能调节p210 bcr-abl诱导信号传导的蛋白酪氨酸磷酸酶（PTP）的性质了解相对较少。在本研究中，我们证明了PTP1B的表达在各种表达p210 bcr-abl的细胞中特异性增强，包括源自一名CML患者的细胞系。对PTP1B表达的这种影响需要p210 bcr-abl的激酶活性，并且迅速发生，与PTK的温度敏感突变体的最大激活同时出现。这种影响显然对PTP1B具有特异性，因为在测试的几种PTP中，我们未检测到PTP1B的近亲TCPTP水平的变化。我们开发了一种鉴定单个PTP生理底物的策略，该策略利用酶的底物捕获突变体形式，这些突变体保留了与底物结合的能力，但无法催化有效的去磷酸化。我们观察到在细胞环境中，PTP1B的底物捕获突变体（PTP1B-D181A）与p210 bcr-abl之间存在关联，但与v-Abl没有关联。与捕获数据一致，我们在体内观察到PTP1B对p210 bcr-abl的去磷酸化作用，但对v-Abl没有作用。我们证明了PTP1B抑制衔接蛋白Grb2与p210 bcr-abl的结合，并抑制了依赖于Ras的p210 bcr-abl诱导的转录激活。这些结果说明了PTP在细胞环境中的作用具有选择性，并表明PTP1B可能在体内作为p210 bcr-abl信号传导的特异性负调节因子发挥作用。
