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MINIREVIEW Regulation of STAT signalling by proteolytic processing Lisa Hendry and Susan John Peter Gorer Department of Immunobiology, Programme in Infection and Immunity, King’s College London, UK 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, differ- entiation 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 pro- teases. Cleavage of STAT proteins by proteases results in the generation of C-terminally truncated proteins, called STATc, which lack the transactivation domain and behave as functional dominant-negative proteins. Currently, STATc isoforms have been identified for Stat3, Stat5a, Stat5b and Stat6 in different cellular contexts and biolo- gical processes. Evidence is mounting for the role of as yet unidentified serine proteases in the proteolytic p rocessing of S TAT proteins, although at least one cysteine protease, calpain is a lso known to cleave these STATs in platelets and mast cells. Recently, studies of acute myeloid leukae- mia a nd cutaneous T cell lymphoma patients have revealed important roles for the aberrant expression of Stat3c and Stat5c proteins in the pathology of these diseases. To- gether, these findings indicate that proteolytic processing is an important mechanism in the regulation of STAT pro- tein biological activity and provides a fertile area for future studies. Introduction The Janus kinase-signal transducer and activator of tran- scription ( JAK-STAT) s ignalling p athway, fi rst identified for the interferon-a/b and c receptors, is now known to be employed by many cytokine and growth factor receptors and to be evolutionarily conserved [1,2]. STAT proteins have a c ommon overall structure and are organized into distinct functional modular domains (Fig. 1). After a decade of intense investigation into the structure and biological functions of STAT proteins, their essential roles in cell proliferation, differentiation and survival have been firmly established [2]. A number of studies have iden- tified important negative regulatory mechanisms that exist to curtail the activity of STAT proteins (Fig. 2). These include the activities of phosphatases, suppressors of cytokine signalling (SOCS), interaction of inhibitory proteins such as protein i nhib itor of activated STATs ( PIAS), and targeted proteasome-dependent degradation of active STATs [2,3]. In addition to these direct protein–protein interaction methods o f n egative regulation, STATs a re also regulated a t the level of alternative splicing. The STATb forms, gener- ated by alternative splicing, possess an altered carboxy- terminal (C-terminal) lacking the natural transactivation domain and behave as functional dominant-negative pro- teins when overexpressed in cells [4–6]. However, recent evidence from transgenic mice indicates that STATb proteins are not strict dominant-negatives, and actually contribute to transcriptional activation of selective target genes, despite the absence of the natural transactivation domain [7–9]. The mechanism by which STATb isoforms achieve transactivation remains to be elucidated, but probably involves the differential interaction with other transcription factors. Another mechanism by which STAT signalling is regu- lated occurs at the level of limited proteolytic processing in cellular contexts where there is no evidence for alternative splicing [10]. Proteolytic processing of STAT proteins also results in the generation of C-terminally truncated STAT proteins, referred to as STATc, but these proteins lack the transactivation domain, without the addition of any extra amino acid sequences at their C-termini. Thus, multiple functional forms of STAT proteins, generated by distinct mechanisms exist in different cell lineages. Here we review the generation and function of STATc proteins and their role in human diseases. Processing of Stat5 in haematopoietic progenitor cells Stat5 is activated by a wide variety of haematological and nonhaematological cytokines and growth factors including those which regulate the proliferation and differentiation of Correspondence to S. John, Peter G orer Department of Immunobiol- ogy, Programme in Infection and Imm unity, K i ng’s College Lo ndon, 2nd floor New Guy’s House, St. Thomas Street, London SE1 9RT, UK. E-mail: susan.john@kcl.ac.uk Abbreviations: AML, acute myeloid leukaemia; BMMC, bone mar- row-derived mast cells; CTCL, cuta neous T cell l ymphoma; G-CS F, granulocyte colony stimulating factor; GM-CSF, granulocyte- macrophage colony-stimulating factor; IL, interleukin; JAK, Janus kinase; PBMC, peripheral blood mononuclear cell; PIAS, protein inhibitor of activated STATs; PM SF, ph enylmethanesulfonyl fluoride; SOCS, suppressors of cytokine signalling; SS , Sezary s yndrome; STAT, signal transducer and activator of transcription. (Received 17 August 2004, accepted 7 October 2004) Eur. J. Biochem. 271, 4613–4620 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04424.x myeloid [interleukin (IL)-3, IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF) and thrombopoietin], erythroid (erythropoietin) and lymphoid lineages (the gamma-c family of cytokines, IL-2, IL-7 and IL-15) [11,12]. Targeted deletions in mice of genes encoding Stat5 results in defects in myeloid ce ll differentiation through effects o n early haematopoietic progenitor cells [13]. The two Stat5 proteins, Stat5a and Stat5b, are encoded by separate genes and are e xpressed as both full-length (Stat5a) and shorter, C-termina lly truncated proteins [5,14]. Although alternative splicing generates Stat5b in certain cellular contexts, the lack of abundance of the alternatively Fig. 2. Negative r egulation o f S TAT s ignalling. Cyt okine-induced STAT activation can be in hibited by suppressors of c ytokine signalling (SOCS) proteins, whose gene expression is regulated by STAT proteins, thus fulfilling a negative feedback loop. SOCS proteins inhibit STAT activation either by inh ibition of the a ctivating JAKs or by c o mpetition with STATs for receptor bind ing. Activated STAT proteins can b e dephosphorylated by cytoplasmic and/or nuclear phosphatases. C-terminally truncated STAT pro teins, STATb and STATc, behave as dominant-negative proteins to functionally compete with their full-length coun terparts to alter or inhibit gene expre ssion, respectively. Protein inhibitor of activated STATs (PIAS) proteins interact with STAT proteins to inhibit their DNA binding and/or potentially facilitate their covalent modification by sumoylation and subsequent degradation. Ub, ubiquitin; SUMO, small ubiquitin-like modifier. Fig. 1. Modular structure of STAT proteins. All STAT proteins share a common molecular topology and are organized into distinct functional domains. The NH 2 -terminus (N-domain) is involved in protein–protein interactions between adjacent STAT dimers on DNA, facilitating the formation of S TAT tetramers. It is a lso involved in the formation of d imers between nonphosphorylated STAT monomers, which is important for receptor-mediated activation a nd nuclear translocation of certain STAT p roteins. Interactions with STAT cofactors, which positively o r negatively modulate their t ranscriptional activity, occur via the N-domain, the a djacent coiled -coil domain and the carboxy-terminal transactivation domain (TAD). The con served serine residue (p-S), which is ph osphorylated upon cytokine stimulation and is impo rtant for maximal transcriptional activation, is located within the transactivation domain. The conserved tyrosine residue (p-Y), that becomes phospho rylated upon activation is located immediately preceeding the transactivation domain. 4614 L. Hendry and S. John (Eur. J. Biochem. 271) Ó FEBS 2004 spliced message in haematopoetic progenitor cells led investigators to evaluate other mechanisms for the genera- tion of C-terminally truncated Stat5 proteins. It was noted that distinct forms of Stat5 proteins were activated upon IL-3 treatment of specific myeloid cell lineages. Thus, in myeloid progenitor cell lineages s timula- tion with IL-3, GM-CSF or e rythropoietin activates a shorter, C-terminally truncated isoform of Stat5a (77 kDa) and Stat5b (80 kDa), while full-length Stat5a (96 kDa) and Stat5b (94 kDa) are only activated in differentiated mature myeloid cells [10,15]. The S tat5c proteins in myeloid progenitor cells are generated by a putative S tat5 protease, which is primarily located in the nucleus and cleaves Stat5 proteins indepen dently of their tyrosine-phosphorylation states [10,16]. The protease is an endopeptidase and is inhibited by the broad-spectrum serine protease inhibitor, phenylmethanesulfonyl fluoride (PMSF). Cellular fraction- ation a nd chromatography studies indicate that the protease has an approximate molecular mass of 25 kDa and cleaves murine Stat5a between amino acids 719 (tyrosine; Y) and 720 (methionine; M) and Stat5b between Y724 and M725 [17]. M utant Stat5 proteins bearing amino acid substitutions at these positions were resistant to cleavage by the protease. Importantly, the Stat5-proteolytic activity was absent in mature myeloid cells sugg esting that either the expression of protease is down-regulated or a lternatively inactivated upon myeloid cell differentiation [10,16,17]. Consistent with the distinct function of truncated Stat5 proteins in immature myeloid p rogenitors, they fail to activate several known IL-3-induced target genes that are activated by th e full-length proteins in differ entiated mature myeloid cells [10]. The functional significance o f truncated Stat5 p roteins in maintaining an undifferentiated immature phenotype of myeloid cells was convincingly demon strated by studies using stable enforced expression of mutant, noncleavable forms of Stat5 in undifferentiated myeloid cells [18]. The mutant cell lines developed a partially differentiated phenotype and were resistant t o further differentiation by cytokine treatment. Thus, proteolytic cleavage of Stat5 is an important physiological mechanism in regulating myeloid cell differentiation. Proteolytic cleavage of Stat5 in peripheral T cells Despite the clear role of proteases in regulating myeloid cell development, Schindler and colleagues were unable to demonstrate an analogous situation for lymphoid cell development i n m urine t hymic T cells [17]. However, studies of human peripheral blood mononuclear cells (PBMCs) indicate that naı ¨ ve T cells in the peripheral immune system possess a similar mechanism f or regulating Stat5 a s myeloid progenitor cells. Activation of naı ¨ ve T cells by antigen ic or mitogenic stimulation leads to cell proliferation and differ- entiation into effector T cells, m ediated b y the action of immunologically important cytokines, which signal via Type I and Type II cytokine receptors. Stat5 a ctivation, mediated by IL-2 signalling upon T cell activation, is an important regulator of cell proliferation a nd survival [19,20]. Recently, studies on Stat5 expression and activation in normal human PBMC and peripheral T cells revealed that Stat5 is expressed exclusively as a truncated protein in the nucleus of naı ¨ ve PBMC/T cells [21]. Analysis of the truncated Stat5 proteins using N- and C-terminal Stat5 antibodies revealed that the t runcation is at the C-terminus of the Stat5 protein, as previously noted in myeloid cells. The expression of the t runcated protein in the nucleus i s independent of the phosphorylation state of Stat5a and Stat5b. Unlike myeloid progenitor cells, the cytoplasmic fraction expresses both the full-length and the truncated Stat5 protein, although a t present we cannot exclude the possibility t hat the truncated protein is exclusively generated in the nucleus but is present in the cytoplasmic fraction due to protein shuttling, which has been shown to occur in a cytokine-dependent and independent manner for STAT proteins [22,23]. Upon activation of naı ¨ ve T cells by mitogenic stimula- tion, the expression of truncated Stat5a and Stat5b proteins disappears and is replaced by the expression and activation of the f ull-length Stat5 p roteins [21]. S ignificantly, the normal regulation of truncated vs. full-length Stat5 is dysregulated in cutaneous T cell lymphoma (CTCL) patients and will be described in a later section. Ongoing studies indicate th at the truncated Stat5 protein is generated by the activity of a protease, which is down-regulated o r inactivated upon mitogenic stimulation (Fig. 3). Future biochemical characterization and purification of the prote- ase(s) a nd the identification of t he exact c leavage site on Stat5 will be important in enhancing our understanding of the regulation of Stat5 function by proteolytic cleavage in peripheral T cells. Proteolytic regulation of Stat5 and Stat3 in mature human neutrophils Stat3 and Stat5 isoforms have been identified in differen- tiated human peripheral blood monocytes and polymor- Fig. 3. Stat5a protein is cleaved to Stat5c by the activity of a protease present in peripheral blood mononuclear cell (PBMC) extract. The presence of Stat5-proteolytic activity was evaluated by coinc ubation assay. Extracts prepared from either PBMC (lane 2) or PBMC mito- genically stimulated with phytohaemagglutinin ( PHA-Blasts, lane 3), or a buffer control containing no cell extrac t (lane 1), were incubated with FLAG-tagged Stat5a protein at 37 °C for 15 min. Samples were then analyzed by Western blot analysis using an anti-FLAG IgG. Cleavage of the FLAG-Stat5a input protein was obtained specifically with fresh PBMC extrcats and not with extracts made from PHA- Blasts. Ó FEBS 2004 STAT signalling by proteolytic processing (Eur. J. Biochem. 271) 4615 phonuclear neutrophils [24–27]. During t erminal differen- tiation of n eutrophils, induced by granulocyte c olony stimulating factor (G-CSF), the main STAT that is activated is Stat3 and it is predominantly expressed as Stat3c, generated by proteolytic cleavage of Stat3a [25,28]. Unlike the progenitor myeloid Stat5 protease, the Stat3 protease, activated by G-CSF can only cleave the a ctive, phosphorylated form of Stat3a [25]. The exact specificity of the Stat3 protease appears to be less clear, as the proteolytic activity was shown to be inhibited by di- isopropylfluorophosphate and not PMSF in living cells, but neither was effective at inhibiting the protease in v it ro [25]. The relationship between the S tat3 protease from mature neutrophils and the Stat5 protease from immature myeloid cells is also unknown at present, but the activation of these proteases in different developmental contexts may suggest that they are distinct proteases. More recently, investigators have shown that Stat5 is also similarly regulated by proteolytic processing in mature human neutrophils [26]. Stat5 is activated in human neutrophils by the cytokines IL-2 and GM-CSF, which are both potent modulators of neutrophil activity [29]. In a now familiar theme, these cytokines activate nuclear expression of a C-terminally truncated form of Stat5 in neutrophils, w hich results in a failure to induce expression of known Stat5-regulated genes, such as osm and pim-1, consistent with the inability of these cytokines t o induce proliferation of these cells [26]. No evidence was found for alternative splicing of S tat5 in the se cells and i nstead truncated Stat5 proteins were generated by the activity of a nuclear, PMSF-sensitive serine protease. The exact rela- tionship between the various Stat5-serine proteases derived from myeloid progenitors, human PBMC and mature neutrophils awaits identification by future molecular c lo- ning studies. Regulation of Stat6 activity by proteolytic cleavage in mast cells Unlike Stat3 and Stat5, which are activated by a wide variety of cytokines and growth factors, Stat6 is very selectively activated by IL-4 and the related cytokine, IL-13 [30]. Stat6 deficient m ice reveal defects i n such crucial aspects of normal immune function as Th2 cell differenti- ation, B cell isotype switching and the loss of contact hypersensitivity [30]. While IL-4 induced Stat6 signalling is an activating signal in murine B a nd T cells, its role in bone marrow-derived mast cells (BMMC) is less clear [31,32]. Analysis of Stat6 expression in murine BMMC provided a clue to these apparent cellular differences in response to IL-4. Brown and colleagues first observed that, whereas Stat6 is expressed as a 100 kDa full-length protein in B and T c ells, it is expressed as a 65 kDa protein i n murine B MMC [33]. A similarly truncated Stat6 protein has not been identified in human mast cells and it i s possible that this mechanism of regulation of Stat6 has been lost during evolution. Studies revealed that Stat6 is truncated at its C- terminus and is lacking the transactivation domain in murine BMMCs [33]. While no evidence for alternative splicing of Stat6 was obtained in mast cells, several groups have established that truncated Stat6 protein is generated by proteolytic processing in these cells [33–35]. The activity of the murine Stat6 protease is exclusively nuclear and can be inhibited b y t he serine protease inhibitors, PMSF and 4-(2-aminoethyl)-benzenesulfonyl- fluoride [35]. Moreover, the a ctivity of the Stat6 protease is not dependent on the expression of Stat6, a s Stat6-deficient BMMC also contained Stat6-specific proteolytic activity [35,36]. More recently Iwamoto and c olleagues have further characterized the serine protease to be inhibited by an elastase inhibitor ONO-5046, suggesting that this protease may belong to an elastase family [36,37]. The Stat6 p rotease cleaves Stat6 between amino acids 685 (aspartic acid; D) and 686 (methionine; M). The amino acid sequences surrounding the cleavage site are not conserved in the human Stat6 protein, providing an explanation for the lack of observation of truncated Stat6 in human mast cells. While cleavage-resistant point mutants of Stat6 (Stat6 D685A and M686A) have similar transcriptional activity as their wild-type counterpart in cell transfection assays, the stable expression of these Stat6 mutants in cell lines results in prolonged nuclear accumulation of Stat6 and enhanced IL-4-induced ap optosis and growth inhibition of the mutant mast cell lines [35]. Furthermore, enforced coexpression of truncated Stat6 with Stat6 D685A reverses the functional effect of the l atter mutant indicating that the truncated Stat6 protein can potentially function as a dominan t-negative in BMMC [35]. Despite the finding that both the Stat5 protease and the Stat6 protease a re serine proteas es, the s imilarity apparently does not extend any further. The Stat5 protease from myeloid cells does not cleave Stat6 and is not inhibited by ONO-5046, and the Stat6 protease from BMMC does not cleave Stat5 [35–37]. Thus, t he serine proteases that r egulate STAT activity show STAT and cell-type specificity. Processing of Stat3, Stat5 and Stat6 by calpain While the most common mechanism o f proteolytic process- ing of STAT proteins is mediated by the action of serine proteases, at least one other cellular protease is known to specifically cleave certain STAT proteins. The calcium- dependent cysteine protease calpain w as demonstrated to cleave Stat3 and Stat5 in platelets and Stat6 in mast cells to generate C-terminally truncated proteins [37,38]. Activation of intracellular calpain by thrombin treatment of platelets resulted in a significant i ncrease in the levels of C-terminally truncated Stat3 and Stat5 [38]. Similarly, Stat6 was cleaved upon activation of calpain by dibucaine treatment of BMMC [37]. However, the truncated Stat6 protein that is generated as a result of cleavage by calpain is a 70 kDa protein as c ompared to t he 65 kDa protein generated by the Stat6 protease. Furthermore, the generation of the 70 kDa but not the 65 kDa Stat6 protein is inhibited by the calpain inhibitor calpeptin [37]. Thus, multiple different STATc isoforms can be generated by the activation of different cellular proteases in BMMCs. It is unclear whether the calpain cleaved Stat5 in platelets is identical in size and function to the Stat5c proteins generated by proteolytic processing by the Stat5 proteases from myeloid progenitor or mature neutrophil cells. The physiological importance of STATc isoforms generated by calpain is unknown at present but, as calpain is potently a ctivated by increased intracellular calcium concentrations following cellular 4616 L. Hendry and S. John (Eur. J. Biochem. 271) Ó FEBS 2004 activation, it is plausible t hat c alpain mediated processing of STAT proteins may b e an i mportant mechanism f or regulating STAT-dependent gene expression [39]. Dysregulated expression of proteolytically processed STAT proteins in human diseases The constitutive activation of full-length Stat3 and Stat5 is a common f eature of many primary human tumours of haematopoietic and nonhaematopoeitic origins and is extensively reviewed elsewhere [40,41]. Recent studies of acute m yeloid leukaem ia (AML) and CTCL patients indicate that C-terminally truncated STAT proteins also contribute to the pathology of these diseases. Acute myeloid leukaemia AML is characterized by the clonal expansion of myeloid cells that have been arrested in their maturation. Like their normal counterparts, AML b lasts can proliferate in response to haematopoietic cytokines such as GM-CSF, G-CSF, thrombopoietin and IL-3, which signal via the JAK-STAT pathway [42]. H owever, unlike no rmal myeloid cells, which undergo differentiation in response to specific cytokine treatment, the leukaemic cells proliferate but do not differentiate, sugge sting th at crucial s ignalling pathways that regulate cell proliferation and differentiation may be dysregulated in this disease. Analysis of a number of bone marrow samples from pretreatment AML patients revealed that  20–30% of AML b lasts expressed constitutively activated full-length Stat3 and Stat5 proteins but a much higher proportion ( 80%) expressed C-terminally trun- cated Stat3 and Stat5 proteins [43]. Moreover, 94% of patients in relapse expressed truncated STAT proteins compared to 35% of patie nts with constitutively active full- length STAT proteins, suggesting that the expression of truncated Stat3 and Stat5 proteins may contribute adversely to disease p rogression [44]. Nevertheless, the shortest disease-free survival rate and overall survival was seen in patients that had both constitutive activation of full-length Stat3 and concurrent aberrant expression of truncated Stat3 [45]. These studies suggest that the relative ratio o f full- length : truncated STAT protein may influence the out- come of disease progression. Constitutive expression of C-terminally truncated Stat5 proteins have also been described previously in CD4 T cells from HIV patients undergoing antiretroviral monotherapy or IL-2 treatment and was associated with good response to therapy [46]. However, it is not known whether the truncated Stat5 protein in patient cells is generated by proteolytic activity or by alternative splicing. Biochemical characterization of the AML samples that contained truncated STAT proteins, revealed t hat a pro- teolytic activity was e xpressed in these samples, which could selectively cleave Stat3 and Stat5, but not Stat6 [47]. The serine protease inhibito r PMSF w as able to inhibit the activity of the Stat3/5 prote ase from AML blasts, as previously observed for progenitor myeloid cells. However, this serine protease differed from that present in immature myeloid cells in that it was present in both cytoplasmic and nuclear fractions and chromatographic analysis of the protease from AML blasts yielded a protein of approximate molecular mass of 40 kDa. Thus, the active protease in AML b lasts m ay either represent yet another member o f t he STAT-serine protease family or alternatively may be aberrantly post-translationally modified. Given the clearly established dominant-negative functions of C-terminally truncated STAT proteins, the aberrant constitutive expres- sion of truncated Stat3 and Stat5 proteins in AML blasts has important physiological implications for the pathology of the disease. As cleavage-resistant mutant Stat5 proteins induce differentiation and apoptosis of myeloid cells when artificially expressed, it is plausible to speculate that the selective expression of truncated Stat5 and Stat3 proteins may enhance survival of leukaemic blasts cells in AML, while at the same time preventing cellular differentiation [18]. Cutaneous T cell Lymphoma Primary CTCLs are one of the most frequent extranodal lymphomas affecting the skin, and include mycosis fun- goides and its leukaemic variant Sezary syndrome (SS) [48]. Tumour cells are typically CD4 T cells, which display a memory activated phenot ype, and express Th2-like c yto- kines (IL-4, IL-5 and IL-10) [49]. While the J ak3-Stat3 pathway is constitutively activated in SS, Stat5 activation is inducible [21,50]. R ecently, a s tudy of SS patients with advanced stage disease identified a different form of dysregulation of Stat5 [21]. As mentioned earlier, Stat5 is regulated by proteolytic processing in normal PBMC. Analysis of PBMC from SS patients showed that, unlike in healthy controls, there was elevated or exclusive expres- sion of the C-terminally truncated Stat5 protein even in potently activated cells. DNA binding studies revealed that in SS patients, truncated Stat5 proteins are activated upon IL-2 stimulation and preferentially bind to known S tat5 binding sites, even in patients where a mixture of full-length and truncated Stat5 proteins are expressed. Consistent with these findings, there was a loss of I L-2-induced Stat5- dependent gene expression of target genes such a s pim-1, cis , and bcl-2 in patient samples. However, the Stat5-regulated gene CD25 was still inducible by IL-2, consistent with findings from other studies, which indicated that constitu- tively activated Stat3 aberrantly regulates CD25 expression in SS [50]. Thus, it seems likely t hat the regulation of other important target genes, which a re s hared by Stat5 a nd Stat3 may be similarly dysregulated in SS. Future studies investigating t he repertoire of target genes activated by full-length vs. truncated Stat5 proteins in T cells will enable us to better understand the functional differences between the d ifferent forms o f S tat5 and t heir potential d ysregulation in SS. Nevertheless, the preferential DNA binding of truncated Stat5 proteins and the concomitant loss of Stat5-dependent gene expression in SS patients demon- strates that t runcated Stat5 proteins can behave as physio- logical dominant-negatives. Ongoing studies indicate that the dysregulated activity of a Stat5 protease may be responsible for the elevated expression of C-terminally truncated Stat5 proteins in SS (L. Hendry and S. John, unpublished observations). Given the critical role of IL-2 i nduced Stat5 signalling in normal immune homeostasis and the maintenance of peripheral tolerance, the loss of this pathway has important Ó FEBS 2004 STAT signalling by proteolytic processing (Eur. J. Biochem. 271) 4617 implications for the pathogenesis of SS [20,51]. Thus, sustained expression of C-terminally truncated Stat5 pro- teins may be one m echanism adopted by indolent malignant T cells in SS to escape apoptosis. Conclusions and perspectives Evidence is accumulating to suggest that proteolytic processing is a general mechanism fo r the negative regula- tion of STAT protein function (Fig. 4). Truncated forms of Stat3, Stat5a, Stat5b and S tat6, g enerated by t he proteolytic cleavage of t he C-termini, have been identified in progenitor myeloid cells, mature neutrophils, mast cells and peripheral T c ells. STATc proteins have different C -termini than STATb proteins and behave as functional dominant- negative proteins. Of the STAT proteases that have been characterized most are serine proteases, whose activities are regulated by the developmental or activation state of cells depending on the cellular context. However, their expres- sion and functional activities are not dependent on the presence of the targe t S TAT protein itself and it i s likely that other cellular targets exist f or these proteases. The exact identities and mechanism o f action of the individual proteases are currently unknown but they show STAT and cell-type specificity. Future cloning of the proteases from the different cell sources will reveal whe ther they belong to a family of related serine proteases. In addition, the cysteine protease, calpain, has also been shown to process Stat3 and Stat5 in platelets and Stat6 proteins in mast cells, respectively, although the physiological import- ance of these findings are unknown. Truncated Stat3c and Stat5c proteins generated b y proteases have been shown to contribute s ignificantly to the pathology of AML and CTCL. Thus, future identification of the relevant serine proteases a nd their natural inhibitors from myeloid c ells and T cells will enhance our understanding of these diseases and also provide potential targets for therapeutic intervention b y the rational design of drugs based on these proteins. References 1. Darnell, J.E. Jr, Kerr, I.M. & Stark, G.R. (1994) Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264, 1415–1421. 2. Levy, D.E. & Dar nell, J.E. Jr (2002) Stats: transcriptional control and biological impact. Nat. Rev. Mol. Cell Biol. 3, 651–662. 3. Greenhalgh, C.J. & Hilton, D.J. (2001) Negative regulation of cytokine signaling. J. Leukoc Biol. 70, 348–356. 4. Moriggl, R., Gouilleux-Gruart, V., Jahne, R., Berchtold, S., Gartmann, C., Liu, X., Hennighausen, L., Sotiropoulos, A., Groner,B.&Gouilleux,F.(1996)Deletionofthecarboxyl- terminal transactivation d omain of MGF-Stat5 results in sus- tained DNA binding and a dominant negative phenotype. Mol. Cell. Biol. 16, 5691–5700. 5. Wang, D., St ravopodis, D ., T eglund, S ., Kitazawa, J . & Ihle, J .N. (1996) Naturally occurring dominant negative variants of Stat5. Mol. Cell. Biol. 16, 6141–6148. 6. Mui, A.L., Wakao, H., Kinoshita, T. , K itamura, T. & M iyajima, A. (1996) Supp ression of interleukin-3-induc ed gene expression by Fig. 4. Proteolytic processing of STAT proteins. STAT proteins may be cleaved at the C-terminus by the action of nuclear (A1,A2; progenitor myeloid cells, mature neutrophils, murine BMMC) and/or cytoplasmic (B1,B2; AML blasts, human PBMC) proteases. The activities of the proteases are generally not dependent on STAT-ph osphorylation and therefore the protease can cleave activated (A1,B1) or unactivated STAT proteins (A2,B2). Unactivated, fu ll-length STAT and STATc proteins can also s huttle between the cytoplasm and the nucleus in the absence of cytokine stimulation . Th e t ru ncated S TATc protein lacks th e transactivation d o main a nd behaves a s a dominant-negative protein to functionally compete with the full-length protein. Pr, protease; TAD, transactivation domain. 4618 L. Hendry and S. John (Eur. J. Biochem. 271) Ó FEBS 2004 a C-terminal truncated Stat5: role of Stat5 in proliferation. EMBO J. 15, 2425–2433. 7. Yoo, J.Y., Huso, D.L., Nathans, D. & Desiderio, S. (2002) Spe- cific ablation of Stat3beta d istorts the patte rn of Stat3-respo nsive gene expression and impairs recovery from endotoxic s hock. Cell 108, 331–344. 8. 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(200 1) Constitutive STAT3-activation in Sezary syndrome: tyrphost in AG49 0 inhib its STAT 3-activation, interleukin-2 receptor expression and growth of leukemic Sezary cells. Leukemia 15, 787–793. 51. Antov, A., Yang, L., Vig, M., Baltimore, D. & Van Parijs, L. (2003) Essential role for STAT5 signaling in CD25 + CD4 + regu- latory T cell h omeo stasis and the maintenance of self- tolerance. J. Immunol. 171, 3435–3441. 4620 L. Hendry and S. John (Eur. J. Biochem. 271) Ó FEBS 2004 . MINIREVIEW Regulation of STAT signalling by proteolytic processing Lisa Hendry and Susan John Peter Gorer Department of Immunobiology, Programme in Infection and Immunity, King’s College. cells. Proteolytic regulation of Stat5 and Stat3 in mature human neutrophils Stat3 and Stat5 isoforms have been identified in differen- tiated human peripheral blood monocytes and polymor- Fig. 3. Stat5 a. characterization and purification of the prote- ase(s) a nd the identification of t he exact c leavage site on Stat5 will be important in enhancing our understanding of the regulation of Stat5 function by proteolytic

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