Gelderloos J A, Anderson S M
Department of Pathology, SUNY at Stony Brook, New York 11794, USA.
Oncogene. 1996 Dec 5;13(11):2367-78.
Interleukin-3 (IL-3) is a hematopoietic growth factor receptor which stimulates the proliferation of multilineage progenitor cells. It is known that IL-3 stimulates tyrosine phosphorylation while transducing a mitogenic signal. The signal transduction pathways activated by the IL-3 receptor, however, are not fully understood. In this study a protein tyrosine phosphatase has been over-expressed in the IL-3 dependent, murine myeloid progenitor cell line, 32D cl3 in order to test whether altering the levels of tyrosine phosphorylation would change IL-3 stimulated proliferation. These cells were transfected with a metal-inducible expression vector containing a rat cDNA encoding PTP1. A low basal level of rat PTP1 message and protein was detected in cells transfected with the PTP1 vector, and zinc treatment resulted in a three- to fourfold increase in the amount of PTP1 message, protein and catalytic activity. Over-expression of PTP1 resulted in a two- to threefold decrease in IL-3 stimulated proliferation. Cells over-expressing PTP1 also exhibited decreased levels of tyrosine phosphorylation; phosphorylation of the IL-3 receptor beta subunit and the Shc protein were both dramatically decreased. Thus, PTP1 over-expression negatively modulated IL-3 signal transduction. To identify potential substrates of PTP1, 32D cl3 cells were transfected with a catalytically inactive PTP1 mutant, PTP1(C/S). Three tyrosine-phosphorylated proteins of MW 140, 79 and 69 k coprecipitated with PTP1(C/S). We believe that the 140 kDa protein represents the beta subunit of the IL-3 receptor. In addition, a GST-fusion protein containing active PTP1 dephosphorylated the beta-subunit in an in vitro assay. By immunofluorescent microscopy over-expressed PTP1(C/S) co-localized largely with calnexin, an endoplasmic reticulum-associated protein. Immunofluorescent microscopy also indicated that PTP1(C/S) and the beta subunit co-localized at discrete sites at the plasma membrane and around a cytoplasmic organelle where most of the beta subunit was located. These observations suggest PTP1 over-expression may down-regulate the growth response to IL-3 through dephosphorylation of the IL-3 receptor, perhaps in an intracellular compartment, thereby inhibiting propagation of the IL-3 mitogenic signal.
白细胞介素-3（IL-3）是一种造血生长因子受体，可刺激多谱系祖细胞的增殖。已知IL-3在转导促有丝分裂信号时会刺激酪氨酸磷酸化。然而，IL-3受体激活的信号转导途径尚未完全明确。在本研究中，一种蛋白酪氨酸磷酸酶在依赖IL-3的小鼠骨髓祖细胞系32D cl3中过表达，以测试改变酪氨酸磷酸化水平是否会改变IL-3刺激的增殖。这些细胞用含有编码PTP1的大鼠cDNA的金属诱导表达载体进行转染。在用PTP1载体转染的细胞中检测到低水平的大鼠PTP1信使核糖核酸和蛋白质，锌处理导致PTP1信使核糖核酸、蛋白质和催化活性增加三到四倍。PTP1的过表达导致IL-3刺激的增殖降低两到三倍。过表达PTP1的细胞也表现出酪氨酸磷酸化水平降低；IL-3受体β亚基和Shc蛋白的磷酸化均显著降低。因此，PTP1的过表达对IL-3信号转导产生负调节作用。为了鉴定PTP1的潜在底物，用催化失活的PTP1突变体PTP1（C/S）转染32D cl3细胞。三种分子量分别为140、79和69kDa的酪氨酸磷酸化蛋白与PTP1（C/S）共沉淀。我们认为140kDa的蛋白代表IL-3受体的β亚基。此外，含有活性PTP1的GST融合蛋白在体外试验中使β亚基去磷酸化。通过免疫荧光显微镜观察，过表达的PTP1（C/S）主要与钙连蛋白共定位，钙连蛋白是一种内质网相关蛋白。免疫荧光显微镜还表明，PTP1（C/S）和β亚基在质膜的离散位点以及β亚基所在的细胞质细胞器周围共定位。这些观察结果表明，PTP1的过表达可能通过使IL-3受体去磷酸化来下调对IL-3的生长反应，可能是在细胞内区室中，从而抑制IL-3促有丝分裂信号的传播。
