Purification and identification of the STAT5 protease in myeloid cells.

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.

Lack of suppressive CD4+CD25+FOXP3+ T cells in advanced stages of primary cutaneous T-cell lymphoma.

Mycosis fungoides and its leukemic variant, Sezary syndrome, are the most common primary cutaneous T-cell lymphomas (CTCLs). In an ex vivo study, we investigated the percentage, phenotype, and suppressive function of CD4+CD25+ regulatory T cells (Tregs) from peripheral blood of CTCL patients. The percentage of Tregs did not differ significantly between patients and controls. Functional assays demonstrated a dichotomy in Treg function: in four out of 10 patients CD4+CD25+ T cells were incapable of suppressing autologous CD4+CD25- T-cell proliferation, whereas suppressive function was intact in the other six patients. Suppressive activity of Tregs inversely correlated with the peripheral blood tumor burden. T-plastin gene expression, used as a Sezary cell marker, confirmed that Sezary cells were heterogeneous for CD25 expression. Mixed lymphocyte reactions demonstrated that CD4+CD25- T cells from patients who lacked functional Tregs were susceptible to suppression by Tregs from healthy controls, and had not become suppressive themselves. Furthermore, we found reduced expression of Foxp3 in the CD4+CD25+ Tregs of these patients relative to the other six CTCL patients and controls. Our findings thus indicate a dysfunction of peripheral Tregs in certain CTCL patients, which correlates with tumor burden.

Regulation of STAT signalling by proteolytic processing.

Interaction of cytokines with their cognate receptors leads to the activation of latent transcription factors, the signal transducer and activator of transcription (STAT) proteins. Numerous studies have identified the critical roles played by STAT proteins in regulating cell proliferation, differentiation and survival. Consequently, the activity of STAT proteins is negatively regulated by a variety of different mechanisms, which include alternative splicing, covalent modifications, protein-protein interactions with negative regulatory proteins and proteolytic processing by proteases. Cleavage of STAT proteins by proteases results in the generation of C-terminally truncated proteins, called STATgamma, which lack the transactivation domain and behave as functional dominant-negative proteins. Currently, STATgamma isoforms have been identified for Stat3, Stat5a, Stat5b and Stat6 in different cellular contexts and biological processes. Evidence is mounting for the role of as yet unidentified serine proteases in the proteolytic processing of STAT proteins, although at least one cysteine protease, calpain is also known to cleave these STATs in platelets and mast cells. Recently, studies of acute myeloid leukaemia and cutaneous T cell lymphoma patients have revealed important roles for the aberrant expression of Stat3gamma and Stat5gamma proteins in the pathology of these diseases. Together, these findings indicate that proteolytic processing is an important mechanism in the regulation of STAT protein biological activity and provides a fertile area for future studies.

A single residue modulates tyrosine dephosphorylation, oligomerization, and nuclear accumulation of stat transcription factors.

The NH(2) terminus of Stat proteins forms a versatile protein interaction domain that is believed to use discrete surfaces to mediate oligomerization and tyrosine dephosphorylation of Stat dimers. Here we show for Stat1 and Stat5a/b that these interfaces overlap and need to be reassigned to an unrelated region of the N-domain. Unexpectedly, our study showed for Stat1 that defective oligomerization of DNA-bound dimers was associated with prolonged interferon-induced nuclear accumulation. This uncoupling of DNA binding and nuclear retention was explained by the concomitant dephosphorylation deficiency that both Stat1 and Stat5a/b have in common and that for Stat1 was due to defective dephosphorylation by the phosphatase TC45. Furthermore, diminished N-domain-mediated oligomerization affected transcriptional activation by both Stat1 and Stat5a/b in a promoter-specific manner. DNA binding analysis indicated that oligomerization of Stats on DNA may be common, irrespective of the presence of multiple canonical binding sites. Accordingly, also transcription from promoters with only a single discernable gamma-activated sequence site was negatively effected by reduced tetramerization. Thus, these results indicate that defective oligomerization cannot generally be compensated for by enhanced tyrosine phosphorylation and prolonged nuclear accumulation. In addition, these data clarify the role of DNA binding in nuclear retention of Stat1.