Purification and identification of the STAT5 protease in myeloid cells

Björn Schuster , Lisa Hendry , Helen Byers , Steven F Lynham , Malcolm A Ward , Susan John (1)

1 Immunobiology

Signal transducer and activator of transcription (STAT) 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 (STATα) generates C-terminally truncated forms (STATγ) in different cell lineages, which have significantly reduced transcriptional activity due to the lack of the transactivation domain. Previously, it was shown that STAT5γ, 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 mass spectrometric analysis as the granule-derived serine protease, Cathepsin G (CatG). We show that purified CatG can specifically cleave full-length STAT5 to generate STAT5γ, and this activity can be inhibited by AEBSF 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 only STAT5α. These studies indicate that nuclear STAT5γ does not naturally exist in immature myeloid cells and is artificially generated from STAT5α during the preparation of extracts due to the abundance of CatG in these cells. Therefore, in contrast to earlier studies, our data suggest that STAT5α, rather than STAT5γ is the active form in immature myeloid cells.

Mots clés: Life Sciences

Björn Schuster, Lisa Hendry, Helen Byers, Steven F Lynham, Malcolm A Ward, et al.. Purification and identification of the STAT5 protease in myeloid cells. Biochemical Journal, 2007, 404 (1), pp.81-87. ⟨10.1042/BJ20061877⟩. ⟨hal-00478721⟩