Purification and identification of the STAT5 protease in myeloid cells

Abstract

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.

Targeting Signal Transducer and Activator of Transcription Signaling Pathway in Leukemias

“…7 A recent provocative study claimed cathepsin G as STAT5 protease and argued that COOH-terminally truncated STAT5 was in fact an artifact generated during in vitro sample preparation with no in vivo significance. 8 Further studies are needed to clarify this controversy.…”

STAT5 Is Essential for Akt/p70S6 Kinase Activity during IL-2-Induced Lymphocyte Proliferation

“…Furthermore, in a lymphocyte cell line, caSTAT5 was shown to promote apoptosis by inducing expression of the growth-inhibitory protein JAB (JAK-binding) (38). Finally, naturally occurring isoforms of STAT5 can be produced by alternative splicing or proteolytic cleavage by enzymes such as cathepsin G or calpain (43)(44)(45). Although the exact physiological significance of these isoforms remains to be determined (45,46), overexpression of isoforms lacking the C-terminal trans activation domain can exert a dominant-negative effect on STAT5 signaling and induce apoptosis in certain cell types (43,(47)(48)(49)(50).…”

STAT5 isoforms: controversies and clarifications

“…25 kDa, but whose identity remained unknown. The present study by Schuster et al [15], appearing in this issue of the Biochemical Journal, finally sheds some light on the identity of the protease, and, more importantly, the mechanism by which STAT5 isoforms are generated; however, the answer is not likely to please everyone. The authors purified cathepsin G as the protease that cleaves fulllength STAT5 into the C-terminally truncated forms.…”

“…Other STAT products can be seen on immunoblots, some of which have been argued to be due to proteolysis. A case in point is STAT5, the topic of the study by Schuster et al [15] in this issue of the Biochemical Journal. Work from several groups has pointed to the existence of additional STAT5 isoforms that could not be explained by alternative splicing.…”