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Eur. J. Biochem.

271, 4613–4620 (2004) Ó FEBS 2004



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, 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 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 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 Stat3c and Stat5c 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.

The Janus kinase-signal transducer and activator of transcription (JAK-STAT) signalling pathway, first 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 common 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 identified 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 inhibitor of activated STATs (PIAS), and targeted proteasome-dependent degradation of active STATs [2,3].

Correspondence to S. John, Peter Gorer Department of Immunobiology, Programme in Infection and Immunity, King’s College London, 2nd floor New Guy’s House, St. Thomas Street, London SE1 9RT, UK. E-mail: Abbreviations: AML, acute myeloid leukaemia; BMMC, bone marrow-derived mast cells; CTCL, cutaneous T cell lymphoma; G-CSF, granulocyte colony stimulating factor; GM-CSF, granulocytemacrophage colony-stimulating factor; IL, interleukin; JAK, Janus kinase; PBMC, peripheral blood mononuclear cell; PIAS, protein inhibitor of activated STATs; PMSF, phenylmethanesulfonyl fluoride; SOCS, suppressors of cytokine signalling; SS, Sezary syndrome; STAT, signal transducer and activator of transcription. (Received 17 August 2004, accepted 7 October 2004)

In addition to these direct protein–protein interaction methods of negative regulation, STATs are also regulated at the level of alternative splicing. The STATb forms, generated by alternative splicing, possess an altered carboxyterminal (C-terminal) lacking the natural transactivation domain and behave as functional dominant-negative proteins 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 regulated 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

small ubiquitin-like modifier. Activated STAT proteins can be dephosphorylated by cytoplasmic and/or nuclear phosphatases. Ub. Negative regulation of STAT signalling. behave as dominant-negative proteins to functionally compete with their full-length counterparts to alter or inhibit gene expression.4614 L. the adjacent coiled-coil domain and the carboxy-terminal transactivation domain (TAD). 1. is located within the transactivation domain. IL-5. facilitating the formation of STAT tetramers. Interactions with STAT cofactors. occur via the N-domain. whose gene expression is regulated by STAT proteins. The NH2-terminus (N-domain) is involved in protein–protein interactions between adjacent STAT dimers on DNA. C-terminally truncated STAT proteins. that becomes phosphorylated upon activation is located immediately preceeding the transactivation domain. Although alternative splicing generates Stat5b in certain cellular contexts. SUMO. The two Stat5 proteins. myeloid [interleukin (IL)-3. thus fulfilling a negative feedback loop. Targeted deletions in mice of genes encoding Stat5 results in defects in myeloid cell differentiation through effects on early haematopoietic progenitor cells [13]. 271) Ó FEBS 2004 Fig. STATb and STATc. which is important for receptor-mediated activation and nuclear translocation of certain STAT proteins. Stat5a and Stat5b. John (Eur. The conserved serine residue (p-S). Modular structure of STAT proteins. J. respectively. granulocyte-macrophage colony-stimulating factor (GM-CSF) and thrombopoietin]. 2. Biochem. ubiquitin. It is also involved in the formation of dimers between nonphosphorylated STAT monomers.12]. are encoded by separate genes and are expressed as both full-length (Stat5a) and shorter. erythroid (erythropoietin) and lymphoid lineages (the gamma-c family of cytokines. Cytokine-induced STAT activation can be inhibited by suppressors of cytokine signalling (SOCS) proteins. The conserved tyrosine residue (p-Y). IL-2.14]. the lack of abundance of the alternatively . which positively or negatively modulate their transcriptional activity. which is phosphorylated upon cytokine stimulation and is important for maximal transcriptional activation. 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. IL-7 and IL-15) [11. C-terminally truncated proteins [5. Hendry and S. Fig. SOCS proteins inhibit STAT activation either by inhibition of the activating JAKs or by competition with STATs for receptor binding. All STAT proteins share a common molecular topology and are organized into distinct functional domains.

while full-length Stat5a (96 kDa) and Stat5b (94 kDa) are only activated in differentiated mature myeloid cells [10. Significantly. 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 Fig. J. The expression of the truncated protein in the nucleus is independent of the phosphorylation state of Stat5a and Stat5b.16. Y) and 720 (methionine. full-length Stat5 is dysregulated in cutaneous T cell lymphoma (CTCL) patients and will be described in a later section. which is down-regulated or inactivated upon mitogenic stimulation (Fig. as previously noted in myeloid cells. the cytoplasmic fraction expresses both the full-length and the truncated Stat5 protein. Consistent with the distinct function of truncated Stat5 proteins in immature myeloid progenitors. Upon activation of naı¨ ve T cells by mitogenic stimulation. However. . mediated by IL-2 signalling upon T cell activation. 271) 4615 spliced message in haematopoetic progenitor cells led investigators to evaluate other mechanisms for the generation of C-terminally truncated Stat5 proteins. they fail to activate several known IL-3-induced target genes that are activated by the full-length proteins in differentiated mature myeloid cells [10]. Proteolytic regulation of Stat5 and Stat3 in mature human neutrophils Stat3 and Stat5 isoforms have been identified in differentiated human peripheral blood monocytes and polymor- Proteolytic cleavage of Stat5 in peripheral T cells Despite the clear role of proteases in regulating myeloid cell development. which is primarily located in the nucleus and cleaves Stat5 proteins independently of their tyrosine-phosphorylation states [10. Thus. noncleavable forms of Stat5 in undifferentiated myeloid cells [18]. Thus. or a buffer control containing no cell extract (lane 1). Stat5 activation.23]. Biochem. The protease is an endopeptidase and is inhibited by the broad-spectrum serine protease inhibitor. proteolytic cleavage of Stat5 is an important physiological mechanism in regulating myeloid cell differentiation. M) and Stat5b between Y724 and M725 [17]. 3. Analysis of the truncated Stat5 proteins using N.20]. phenylmethanesulfonyl fluoride (PMSF). studies of human peripheral blood mononuclear cells (PBMCs) indicate that naı¨ ve T cells in the peripheral immune system possess a similar mechanism for regulating Stat5 as myeloid progenitor cells. Cleavage of the FLAG-Stat5a input protein was obtained specifically with fresh PBMC extrcats and not with extracts made from PHABlasts. Ongoing studies indicate that the truncated Stat5 protein is generated by the activity of a protease. lane 3). mediated by the action of immunologically important cytokines. 3). which has been shown to occur in a cytokine-dependent and independent manner for STAT proteins [22. Importantly. Unlike myeloid progenitor cells. Cellular fractionation and chromatography studies indicate that the protease has an approximate molecular mass of 25 kDa and cleaves murine Stat5a between amino acids 719 (tyrosine. The functional significance of truncated Stat5 proteins in maintaining an undifferentiated immature phenotype of myeloid cells was convincingly demonstrated by studies using stable enforced expression of mutant. It was noted that distinct forms of Stat5 proteins were activated upon IL-3 treatment of specific myeloid cell lineages. C-terminally truncated isoform of Stat5a (77 kDa) and Stat5b (80 kDa).and C-terminal Stat5 antibodies revealed that the truncation is at the C-terminus of the Stat5 protein.16]. GM-CSF or erythropoietin activates a shorter. Stat5a protein is cleaved to Stat5c by the activity of a protease present in peripheral blood mononuclear cell (PBMC) extract. were incubated with FLAG-tagged Stat5a protein at 37 °C for 15 min. The presence of Stat5-proteolytic activity was evaluated by coincubation assay. which signal via Type I and Type II cytokine receptors. is an important regulator of cell proliferation and survival [19. Extracts prepared from either PBMC (lane 2) or PBMC mitogenically stimulated with phytohaemagglutinin (PHA-Blasts.17]. Future biochemical characterization and purification of the protease(s) and the identification of the exact cleavage site on Stat5 will be important in enhancing our understanding of the regulation of Stat5 function by proteolytic cleavage in peripheral T cells. the expression of truncated Stat5a and Stat5b proteins disappears and is replaced by the expression and activation of the full-length Stat5 proteins [21]. Activation of naı¨ ve T cells by antigenic or mitogenic stimulation leads to cell proliferation and differentiation into effector T cells. nucleus of naı¨ ve PBMC/T cells [21].15]. Recently. the normal regulation of truncated vs. the Stat5-proteolytic activity was absent in mature myeloid cells suggesting that either the expression of protease is down-regulated or alternatively inactivated upon myeloid cell differentiation [10.Ó FEBS 2004 STAT signalling by proteolytic processing (Eur. The mutant cell lines developed a partially differentiated phenotype and were resistant to further differentiation by cytokine treatment. in myeloid progenitor cell lineages stimulation with IL-3. Mutant Stat5 proteins bearing amino acid substitutions at these positions were resistant to cleavage by the protease. Samples were then analyzed by Western blot analysis using an anti-FLAG IgG. Schindler and colleagues were unable to demonstrate an analogous situation for lymphoid cell development in murine thymic T cells [17]. The Stat5c proteins in myeloid progenitor cells are generated by a putative Stat5 protease. although at present we cannot exclude the possibility that the truncated protein is exclusively generated in the nucleus but is present in the cytoplasmic fraction due to protein shuttling.

While IL-4 induced Stat6 signalling is an activating signal in murine B and T cells. phosphorylated form of Stat3a [25]. which are activated by a wide variety of cytokines and growth factors. 271) Ó FEBS 2004 phonuclear neutrophils [24–27]. The Stat5 protease from myeloid cells does not cleave Stat6 and is not inhibited by ONO-5046. as calpain is potently activated by increased intracellular calcium concentrations following cellular Regulation of Stat6 activity by proteolytic cleavage in mast cells Unlike Stat3 and Stat5.38]. whereas Stat6 is expressed as a 100 kDa full-length protein in B and T cells. the Stat3 protease. Furthermore. as Stat6-deficient BMMC also contained Stat6-specific proteolytic activity [35. but neither was effective at inhibiting the protease in vitro [25]. More recently. the stable expression of these Stat6 mutants in cell lines results in prolonged nuclear accumulation of Stat6 and enhanced IL-4-induced apoptosis and growth inhibition of the mutant mast cell lines [35]. enforced coexpression of truncated Stat6 with Stat6 D685A reverses the functional effect of the latter mutant indicating that the truncated Stat6 protein can potentially function as a dominant-negative in BMMC [35]. No evidence was found for alternative splicing of Stat5 in these cells and instead truncated Stat5 proteins were generated by the activity of a nuclear.4616 L. Stat6 deficient mice reveal defects in such crucial aspects of normal immune function as Th2 cell differentiation. which are both potent modulators of neutrophil activity [29]. Thus. induced by granulocyte colony stimulating factor (G-CSF). J. The Stat6 protease cleaves Stat6 between amino acids 685 (aspartic acid. at least one other cellular protease is known to specifically cleave certain STAT proteins. Despite the finding that both the Stat5 protease and the Stat6 protease are serine proteases. the main STAT that is activated is Stat3 and it is predominantly expressed as Stat3c. PMSF-sensitive serine protease. The relationship between the Stat3 protease from mature neutrophils and the Stat5 protease from immature myeloid cells is also unknown at present. Stat6 was cleaved upon activation of calpain by dibucaine treatment of BMMC [37]. its role in bone marrow-derived mast cells (BMMC) is less clear [31. The exact specificity of the Stat3 protease appears to be less clear. Stat5 and Stat6 by calpain While the most common mechanism of proteolytic processing of STAT proteins is mediated by the action of serine proteases. In a now familiar theme. consistent with the inability of these cytokines to induce proliferation of these cells [26]. these cytokines activate nuclear expression of a C-terminally truncated form of Stat5 in neutrophils. The physiological importance of STATc isoforms generated by calpain is unknown at present but. Unlike the progenitor myeloid Stat5 protease. While no evidence for alternative splicing of Stat6 was obtained in mast cells.37]. M). Analysis of Stat6 expression in murine BMMC provided a clue to these apparent cellular differences in response to IL-4. Stat5 is activated in human neutrophils by the cytokines IL-2 and GM-CSF. B cell isotype switching and the loss of contact hypersensitivity [30]. such as osm and pim-1. More recently Iwamoto and colleagues have further characterized the serine protease to be inhibited by an elastase inhibitor ONO-5046. Similarly. the truncated Stat6 protein that is generated as a result of cleavage by calpain is a 70 kDa protein as compared to the 65 kDa protein generated by the Stat6 protease. human PBMC and mature neutrophils awaits identification by future molecular cloning studies. However. Furthermore. as the proteolytic activity was shown to be inhibited by diisopropylfluorophosphate and not PMSF in living cells. but the activation of these proteases in different developmental contexts may suggest that they are distinct proteases. the activity of the Stat6 protease is not dependent on the expression of Stat6.36]. Biochem. During terminal differentiation of neutrophils. the generation of the 70 kDa but not the 65 kDa Stat6 protein is inhibited by the calpain inhibitor calpeptin [37]. providing an explanation for the lack of observation of truncated Stat6 in human mast cells. Studies revealed that Stat6 is truncated at its Cterminus and is lacking the transactivation domain in murine BMMCs [33]. the serine proteases that regulate STAT activity show STAT and cell-type specificity. Thus. PMSF and 4-(2-aminoethyl)-benzenesulfonylfluoride [35]. Processing of Stat3. Brown and colleagues first observed that. Stat6 is very selectively activated by IL-4 and the related cytokine. John (Eur.28]. the similarity apparently does not extend any further. which results in a failure to induce expression of known Stat5-regulated genes. The amino acid sequences surrounding the cleavage site are not conserved in the human Stat6 protein. multiple different STATc isoforms can be generated by the activation of different cellular proteases in BMMCs. several groups have established that truncated Stat6 protein is generated by proteolytic processing in these cells [33–35]. suggesting that this protease may belong to an elastase family [36. A similarly truncated Stat6 protein has not been identified in human mast cells and it is possible that this mechanism of regulation of Stat6 has been lost during evolution. While cleavage-resistant point mutants of Stat6 (Stat6 D685A and M686A) have similar transcriptional activity as their wild-type counterpart in cell transfection assays. it is expressed as a 65 kDa protein in murine BMMC [33]. The activity of the murine Stat6 protease is exclusively nuclear and can be inhibited by the serine protease inhibitors. D) and 686 (methionine. investigators have shown that Stat5 is also similarly regulated by proteolytic processing in mature human neutrophils [26]. Activation of intracellular calpain by thrombin treatment of platelets resulted in a significant increase in the levels of C-terminally truncated Stat3 and Stat5 [38]. Moreover. generated by proteolytic cleavage of Stat3a [25. Hendry and S. The calciumdependent cysteine protease calpain was demonstrated to cleave Stat3 and Stat5 in platelets and Stat6 in mast cells to generate C-terminally truncated proteins [37. IL-13 [30]. 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. and the Stat6 protease from BMMC does not cleave Stat5 [35–37]. activated by G-CSF can only cleave the active.32]. The exact relationship between the various Stat5-serine proteases derived from myeloid progenitors. .

and express Th2-like cytokines (IL-4. which display a memory activated phenotype. 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]. Stat5 activation is inducible [21. revealed that a proteolytic activity was expressed in these samples. Hendry and S. and include mycosis fungoides and its leukaemic variant Sezary syndrome (SS) [48]. it seems likely that the regulation of other important target genes. AML blasts can proliferate in response to haematopoietic cytokines such as GM-CSF. it is not known whether the truncated Stat5 protein in patient cells is generated by proteolytic activity or by alternative splicing. unpublished observations). However. Thus. Analysis of PBMC from SS patients showed that. IL-5 and IL-10) [49]. However. the preferential DNA binding of truncated Stat5 proteins and the concomitant loss of Stat5-dependent gene expression in SS patients demonstrates that truncated Stat5 proteins can behave as physiological dominant-negatives. Biochemical characterization of the AML samples that contained truncated STAT proteins. However. suggesting that the expression of truncated Stat3 and Stat5 proteins may contribute adversely to disease progression [44]. Nevertheless. Moreover. which signal via the JAK-STAT pathway [42]. Given the clearly established dominant-negative functions of C-terminally truncated STAT proteins. Analysis of a number of bone marrow samples from pretreatment AML patients revealed that  20–30% of AML blasts expressed constitutively activated full-length Stat3 and Stat5 proteins but a much higher proportion ( 80%) expressed C-terminally truncated Stat3 and Stat5 proteins [43]. unlike in healthy controls.50]. unlike normal myeloid cells.Ó FEBS 2004 STAT signalling by proteolytic processing (Eur. Cutaneous T cell Lymphoma Primary CTCLs are one of the most frequent extranodal lymphomas affecting the skin. thrombopoietin and IL-3. which could selectively cleave Stat3 and Stat5. Consistent with these findings. the Stat5-regulated gene CD25 was still inducible by IL-2. as previously observed for progenitor myeloid cells. truncated Stat5 proteins are activated upon IL-2 stimulation and preferentially bind to known Stat5 binding sites. the loss of this pathway has important . J. Like their normal counterparts. it is plausible that calpain mediated processing of STAT proteins may be an important mechanism for regulating STAT-dependent gene expression [39]. 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]. While the Jak3-Stat3 pathway is constitutively activated in SS. consistent with findings from other studies. which undergo differentiation in response to specific cytokine treatment. Given the critical role of IL-2 induced Stat5 signalling in normal immune homeostasis and the maintenance of peripheral tolerance. while at the same time preventing cellular differentiation [18]. truncated Stat5 proteins in T cells will enable us to better understand the functional differences between the different forms of Stat5 and their potential dysregulation in SS. 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. Biochem. but not Stat6 [47]. even in patients where a mixture of full-length and truncated Stat5 proteins are expressed. Thus. The serine protease inhibitor PMSF was able to inhibit the activity of the Stat3/5 protease from AML blasts. John. These studies suggest that the relative ratio of fulllength : truncated STAT protein may influence the outcome of disease progression. the leukaemic cells proliferate but do not differentiate. cis. 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. Recent studies of acute myeloid leukaemia (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.41]. the active protease in AML blasts may either represent yet another member of the STAT-serine protease family or alternatively may be aberrantly post-translationally modified. 271) 4617 activation. DNA binding studies revealed that in SS patients. there was elevated or exclusive expression of the C-terminally truncated Stat5 protein even in potently activated cells. G-CSF. it is plausible to speculate that the selective expression of truncated Stat5 and Stat3 proteins may enhance survival of leukaemic blasts cells in AML. Tumour cells are typically CD4 T cells. there was a loss of IL-2-induced Stat5dependent gene expression of target genes such as pim-1. Future studies investigating the repertoire of target genes activated by full-length vs. However. Dysregulated expression of proteolytically processed STAT proteins in human diseases The constitutive activation of full-length Stat3 and Stat5 is a common feature of many primary human tumours of haematopoietic and nonhaematopoeitic origins and is extensively reviewed elsewhere [40. the aberrant constitutive expression of truncated Stat3 and Stat5 proteins in AML blasts has important physiological implications for the pathology of the disease. suggesting that crucial signalling pathways that regulate cell proliferation and differentiation may be dysregulated in this disease. As mentioned earlier. which are shared by Stat5 and Stat3 may be similarly dysregulated in SS. As cleavage-resistant mutant Stat5 proteins induce differentiation and apoptosis of myeloid cells when artificially expressed. 94% of patients in relapse expressed truncated STAT proteins compared to 35% of patients with constitutively active fulllength STAT proteins. Stat5 is regulated by proteolytic processing in normal PBMC. Nevertheless. Recently. a study of SS patients with advanced stage disease identified a different form of dysregulation of Stat5 [21]. and bcl-2 in patient samples. which indicated that constitutively activated Stat3 aberrantly regulates CD25 expression in SS [50].

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