Schuster Björn, Hendry Lisa, Byers Helen, Lynham Steven F, Ward Malcolm A, John Susan
Division of Infection, Immunity and Inflammatory Diseases, Guy's Campus, King's College London, London SE1 9RT, UK.
Biochem J. 2007 May 15;404(1):81-7. doi: 10.1042/BJ20061877.
STAT (signal transducer and activator of transcription) proteins are critical regulators of cytokine-induced cell proliferation, differentiation and survival. STAT functional activity can be variably regulated by post-translational modifications, including phosphorylation, acetylation, methylation and sumoylation. Additionally, limited proteolytic digestion of full-length STAT proteins (STATalpha) generates C-terminally truncated forms (STATgamma) in different cell lineages, which have significantly reduced transcriptional activity due to the lack of the transactivation domain. Previously, it has been shown that STAT5gamma, generated by an unidentified nuclear serine protease, plays an important role in myeloid cell differentiation and is aberrantly expressed in acute myeloid leukaemia. To better understand this regulatory mechanism for STAT5 function, we have purified the STAT5 protease from the immature myeloid cell line 32D and identified it by MS analysis as the granule-derived serine protease, CatG (cathepsin G). We show that purified CatG can specifically cleave full-length STAT5 to generate STAT5gamma, and this activity can be inhibited by AEBSF [4-(2-aminoethyl)benzenesulfonyl fluoride] in an in vitro protease assay. Importantly, preparation of nuclear and cytoplasmic extracts from immature myeloid cell lines, 32D and FDC-P1, in the presence of a specific inhibitor for CatG results in the identification of STAT5alpha only. These studies indicate that nuclear STAT5gamma does not naturally exist in immature myeloid cells and is artificially generated from STAT5alpha during the preparation of extracts due to the abundance of CatG in these cells. Therefore in contrast with earlier studies, our data suggest that STAT5alpha, rather than STAT5gamma is the active form in immature myeloid cells.
信号转导与转录激活因子（STAT）蛋白是细胞因子诱导的细胞增殖、分化和存活的关键调节因子。STAT的功能活性可通过翻译后修饰进行多种调控，包括磷酸化、乙酰化、甲基化和类泛素化。此外，全长STAT蛋白（STATα）的有限蛋白水解消化在不同细胞谱系中产生C末端截短形式（STATγ），由于缺乏反式激活结构域，其转录活性显著降低。此前已表明，由一种未鉴定的核丝氨酸蛋白酶产生的STAT5γ在髓系细胞分化中起重要作用，且在急性髓系白血病中异常表达。为了更好地理解STAT5功能的这种调节机制，我们从未成熟髓系细胞系32D中纯化了STAT5蛋白酶，并通过质谱分析鉴定其为颗粒源性丝氨酸蛋白酶组织蛋白酶G（CatG）。我们发现纯化的CatG可特异性切割全长STAT5以产生STAT5γ，并且在体外蛋白酶测定中，这种活性可被4-（2-氨基乙基）苯磺酰氟（AEBSF）抑制。重要的是，在存在CatG特异性抑制剂的情况下，从未成熟髓系细胞系32D和FDC-P1制备核提取物和细胞质提取物，结果仅鉴定出STAT5α。这些研究表明，核STAT5γ在未成熟髓系细胞中并非天然存在，而是由于这些细胞中CatG含量丰富，在提取物制备过程中由STAT5α人工产生。因此，与早期研究相反，我们的数据表明在未成熟髓系细胞中，STAT5α而非STAT5γ是活性形式。
