Proteolytic conversion of STAT3α to STAT3γ in human neutrophils -: Role of granule-derived serine proteases
Proteolytic conversion of STAT3α to STAT3γ in human neutrophils -: Role of granule-derived serine proteases
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DOI：
10.1074/jbc.m400637200
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发表时间：
2004-07-23
影响因子：
4.8
通讯作者：
Kitagawa, S
中科院分区：
文献类型：
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作者：
Kato, T;Sakamoto, E;Kitagawa, S
四种亚型（α, β， γ和δ）已被确定为信号换能器和转录激活子3 （STAT3）。据报道，STAT3gamma是STAT3alpha在粒细胞分化过程中通过有限的蛋白水解而产生的，是人中性粒细胞中表达的一个主要的STAT3亚型。我们证实，在用常规裂解缓冲液制备的人中性粒细胞裂解物中检测到STAT3gamma是一个主要的STAT3亚型。能够在体外将STAT3alpha转化为STAT3gamma的酶定位于中性粒细胞颗粒部分，并在离子霉素刺激下释放到培养基中。该酶活性被苯基甲基磺酰氟、CuSO4和ONO-5046（一种中性粒细胞弹性酶特异性抑制剂）强烈抑制，但不被抑蛋白蛋白、白细胞蛋白酶、苯甲脒和EDTA抑制。通过与人中性粒细胞弹性酶或蛋白酶3（而非组织蛋白酶g）进行有限的蛋白水解，可以在体外有效地从STAT3alpha生成STAT3gamma。中性粒细胞裂解物的转化活性因弹性酶而非蛋白酶3的免疫缺失而降低。出乎意料的是，在中性粒细胞来源的细胞质（缺乏颗粒）的裂解物和氮空化制备的细胞质部分中未检测到STAT3gamma。在这些制剂中检测到的STAT3亚型主要是STAT3 α。在pmsf预处理的中性粒细胞的裂解物中也未检测到STAT3gamma，而在离子霉素预处理的中性粒细胞的裂解物中则明显减少。这些发现表明，与之前的报道相反，STAT3alpha，而不是STAT3gamma，主要在人中性粒细胞中表达，并且在用常规裂解缓冲液制备中性粒细胞裂解物时，通过颗粒衍生的丝氨酸蛋白酶的有限蛋白水解，STAT3alpha可以迅速产生STAT3gamma。
Four isoforms (alpha, beta, gamma, and delta) have been identified for signal transducer and activator of transcription 3 (STAT3). It has been reported that STAT3gamma, which is derived from STAT3alpha by limited proteolysis during granulocytic differentiation, is a major STAT3 isoform expressed in human neutrophils. We confirmed that STAT3gamma was a major STAT3 isoform detected in human neutrophil lysates prepared with the conventional lysis buffer. The enzymes capable of converting STAT3alpha to STAT3gamma in vitro were localized in neutrophil granule fraction and were released into the medium upon ionomycin stimulation. The enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride, CuSO4, and ONO-5046 ( a specific inhibitor of neutrophil elastase), but not by aprotinin, leupeptin, benzamidine, and EDTA. STAT3gamma was effectively generated in vitro from STAT3alpha by limited proteolysis with human neutrophil elastase or proteinase 3 but not cathepsin G. The converting activity in neutrophil lysates was reduced by immunodepletion of elastase but not proteinase 3. Unexpectedly, STAT3gamma was undetected in the lysates of neutrophil-derived cytoplasts, which lack granules, and the cytosol fraction prepared by nitrogen cavitation. The STAT3 isoform detected in these preparations was primarily STAT3alpha. STAT3gamma was also undetected in the lysates of PMSF-pretreated neutrophils and was markedly decreased in the lysates of ionomycin-pretreated neutrophils. These findings indicate that, in contrast to the previous reports, STAT3alpha, but not STAT3gamma, is primarily expressed in human neutrophils, and STAT3gamma is rapidly generated from STAT3alpha by limited proteolysis with granule-derived serine proteases during preparation of neutrophil lysates with the conventional lysis buffer.