Waterman W H, Sha'afi R I
Department of Physiology, University of Connecticut Health Center, Farmington 06030, USA.
Biochem J. 1995 Apr 1;307 ( Pt 1)(Pt 1):39-45. doi: 10.1042/bj3070039.
The present study was undertaken to determine the identities and characteristics of proteins with molecular masses between 40 and 44 kDa whose tyrosine phosphorylation increases in human neutrophils following stimulation of these cells with tumour necrosis factor alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Immunoblotting results demonstrate that addition of GM-CSF to human neutrophils increases the tyrosine phosphorylation of two proteins with molecular masses of 42 and 44 kDa. However, the addition of TNF-alpha to neutrophils induces a time- and dose-dependent increase in tyrosine phosphorylation of a 40 kDa protein. Immunoprecipitation using specific mitogen-activated protein kinase (MAPK) isoform antibodies and an antibody which recognizes phosphotyrosine-containing proteins demonstrated that the 42 and 44 kDa proteins are isoforms of MAPKs. Utilizing an in situ gel kinase activity assay, GM-CSF increases the kinase activity of the 42 and 44 kDa proteins. Moreover, using immunoprecipitated p42 and p44 MAPK isoforms in this gel assay revealed activity associated with the p42 and p44 MAPK isoforms. Using the same in situ assay, TNF-alpha induces an increase in kinase activity of a 40-42 kDa protein. However, the 40 kDa protein whose phosphorylation on tyrosine residues increased in human neutrophils following stimulation with TNF-alpha is not a member of the known MAPK family, demonstrating the divergences in pathways utilized by GM-CSF and TNF-alpha. This 40 kDa protein may be related to the recently identified protein that becomes phosphorylated on tyrosine residues upon stimulation of the human epidermal carcinoma cell line KB by interleukin-1. In these cells the p40 protein is part of a protein kinase cascade which results in the phosphorylation of the small heat shock protein, hsp27.
本研究旨在确定分子量在40至44 kDa之间的蛋白质的身份和特征，这些蛋白质在肿瘤坏死因子α（TNF-α）和粒细胞-巨噬细胞集落刺激因子（GM-CSF）刺激人中性粒细胞后酪氨酸磷酸化增加。免疫印迹结果表明，向人中性粒细胞中添加GM-CSF会增加两种分子量分别为42和44 kDa的蛋白质的酪氨酸磷酸化。然而，向中性粒细胞中添加TNF-α会诱导一种40 kDa蛋白质的酪氨酸磷酸化呈时间和剂量依赖性增加。使用特异性丝裂原活化蛋白激酶（MAPK）同工型抗体和识别含磷酸酪氨酸蛋白质的抗体进行免疫沉淀表明，42和44 kDa的蛋白质是MAPK的同工型。利用原位凝胶激酶活性测定法，GM-CSF增加了42和44 kDa蛋白质的激酶活性。此外，在该凝胶测定中使用免疫沉淀的p42和p44 MAPK同工型揭示了与p42和p44 MAPK同工型相关的活性。使用相同的原位测定法，TNF-α诱导一种40 - 42 kDa蛋白质的激酶活性增加。然而，在用TNF-α刺激后人中性粒细胞中酪氨酸残基磷酸化增加的40 kDa蛋白质不是已知MAPK家族的成员，这表明GM-CSF和TNF-α利用的信号通路存在差异。这种40 kDa蛋白质可能与最近鉴定的一种蛋白质有关，该蛋白质在白细胞介素-1刺激人表皮癌细胞系KB后酪氨酸残基发生磷酸化。在这些细胞中，p40蛋白质是蛋白激酶级联反应的一部分，该级联反应导致小热休克蛋白hsp27的磷酸化。
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