SO MANY LIGANDS, SO FEW TRANSCRIPTION FACTORS: A NEW PARADIGM FOR SIGNALING THROUGH THE STAT TRANSCRIPTION FACTORS

SO MANY LIGANDS, SO FEW TRANSCRIPTION FACTORS: A NEW PARADIGM FOR SIGNALING THROUGH THE STAT TRANSCRIPTION FACTORS

doi:10.1006/cyto.2001.0905, available online at http://www.idealibrary.com on REVIEW ARTICLE SO MANY LIGANDS, SO FEW TRANSCRIPTION FACTORS: A NEW PA...
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doi:10.1006/cyto.2001.0905, available online at http://www.idealibrary.com on

REVIEW ARTICLE

SO MANY LIGANDS, SO FEW TRANSCRIPTION FACTORS: A NEW PARADIGM FOR SIGNALING THROUGH THE STAT TRANSCRIPTION FACTORS Prem S. Subramaniam, Barbara A. Torres, Howard M. Johnson

The signal transducers and activators of transcription (STAT) family of transcription factors has seven members (reviewed in Refs 1–3). Most of the STAT proteins form homodimers in response to ligand stimulation.1 Notable exceptions are STAT1/STAT2 mediation of gamma interferon (IFN- activity, STAT1/STAT3 mediation of interleukin-6 (IL-6) activity, and STAT5a/STAT5b mediation of growth hormone activity. Given the limited number of STAT family members and their preference for homodimerization for transcription, it is difficult to explain the various specificities of the over 50 proteins that utilize the STATs in signal transduction (Table 1). Perhaps the specificity of signaling by proteins that activate the same STATs could be explained in part by the nature or prior commitment of the target cell. In the context of a single ligand activating one STAT, specificity models for recognition and differential activation of genes have been proposed.71,72 Such kinetic models with respect to interaction of STATs with promoters based on differing affinities among competing promoters, however, do not wholly explain the unique biological effects on a cell by different ligand/receptors that activate the same STAT. For example, IFN- and interleukin-10 (IL-10) both activate STAT1 in human monocytes to form homodimers, yet the cellular response differs for the two proteins.3,24 IFN- induces an antiviral state and upregulates MHC class II molecules on monocytes, while IL-10 does neither. This scenario is further complicated by the fact that under many conditions a given STAT homodimer recognizes the same promoter element in different genes activated From the Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA Correspondence to: Howard Johnson, Dept of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA. E-mail: [email protected] Received 9 May 2001; accepted for publication 24 May 2001  2001 Academic Press 1043–4666/01/010175+13 $35.00/0 CYTOKINE, Vol. 15, No. 4 (21 August), 2001: pp 175–187

by different ligands. Binding of accessory proteins such as BRCA1, CBP3000, and MCM to the carboxylterminal transcription activator domains (TADs) of STATs in the nucleus also does not explain the unique specificity of a given ligand, unless that ligand plays a more direct role in STAT signaling than is currently known (reviewed in Ref. 1). Before even specificity questions can be considered, there is the question of the nuclear transport of STATs themselves. A common pathway for extracellular signal-activated transcription factors is the well-known Ran/importin pathway. In general, in this pathway, proteins targeted for the nucleus bind to a heterocomplex called importin, which is made up of  and  subunits (importin  and importin , respectively).73 The transcription factor (or chaperone of the transcription factor) binds to importin  via a specific motif called the nuclear localization sequence (NLS).74 NLSs are short amino acid stretches characterized by clusters of polycationic residues occurring together as a single group or two short groups separated by a fixed spacer (bipartite NLS sequence). Following the binding of the NLS-bearing protein, importin  interacts with importin  through its importin  domain. Importin  mediates docking of the complex at the NPC. Translocation into the nucleus requires an NPCassociated GTPase called Ran, as well as other cofactors.73 Thus, transcription factors enter the nucleus via an energy-requiring active transport mechanism. STAT1, the transcription factor for IFN-, has recently been shown to be actively transported to the nucleus via the GTPase activity of Ran/TC3 through the NPC.75,76 It is quite likely that the other types of STATs also use the same nuclear transport mechanism. However, based on current knowledge there is a potential paradox, as STATs like STAT1 do not contain a polycationic or functional NLS.76 As a rule, in the absence of a sequestering protein (discussed later), 175

176 / Subramaniam et al.

CYTOKINE, Vol. 15, No. 4 (21 August, 2001: 175–187)

TABLE 1. Compilation of representative cytokines, growth factors and hormones that utilize STATs and the corresponding proven (where indicated) or putative NLSs found in the ligands or receptor chains. A portion of this list of putative NLSs has been published by others.4 Putative bipartite NLSs clusters in individual sequences have been underlined. Effort has been made to identify all STATs reported to be activated, whether in vitro or in vivo, by the indicated ligand Ligand Interferons hIFN- mIFN- hIFN- Interleukins hIL-1 hIL-1 hIL-2

NLS (proven/putative)

GIKRLRRKD RKIIEKKT LRKRKRSRC (proven; i)† NSNKKKR (proven, i, ii) KTGKRKRS (proven; ii)

KVLKKRRL* (Proven; iii) PKKKMEKRF* QRRQRKSRR PDRRRWN* hIL-3 ARVRVRTSR mIL-4 KHRVKKK hIL-4 HRHKKQLIRFLKRLER mIL-5 KKYIDROKEKCGEERRRTR (proven; iv) hIL-5 KKYIDOOKKKCGEERRRVR (proven; iv) mIL-6 KKPCPDDLKSVDLFKKEK hIL-6 KKPFPEDLKSLDLFKKEK PTVRTKK* mIL-7 KKKYLKKVKHD hIL-7 KKPR* KPRK hIL-8 (None detected) hIL-9 PRVKRIF* hIL-10 RRRKK* mIL-11 KKPCPDDLKSVDLFKKEK hIL-11 KKPFPEDLKS LDLFKKEK PTVRTKK* hIL-12 RRRRR* hIL-13 KRLKIIIFPPIPDPGKIFKK hIL-14 receptor system not fully defined hIL-15 PRRARGCR* hIL-16 receptor system not fully defined hIL-17 PLKPRKV* hIL-18 receptor system not fully defined hIL-19 receptor system not fully defined hIL-20 RKISSLANSFLTIKKDLR hIL-21 KKLKRKPPSTNAGRRQKHRL hIL-22 PKHLRPK Growth factors, hormones, other cytokines hPDGF PRESGKKRKRKR hPDGF RVTIRTVRVRRPPKGKHRK (proven; v) hInsulin RRSYALVSLSFFRKLRL* RKTSSGTGAEDPRPSRKRRS* hGH VRVRSKQRN hEGF RRRHIVRKRTLRRL hEPO RKLFRVYSNFLRGKLK hVEGF KKRH* hFGF-1 NYKKPKL (proven; vi; also see below) hHGF (Hepatocyte Growth Factor) RKRR* hIGF PERKRRD* HRKR* hGM-CSF RNSKRRREIR c-Kit RRKR* Angiotensin KKFKK proven; vii) hLeptin KKSIYYLGVTSIKKRES* uPA (urokinase-type plasminogen activator) RRRP* RRHR* PDNRRRP*

Where NLS found

STATs activated

Ref. nos

1, 2, 3, 4, 5A/B(?), 6

3–8

1 1

3 3

No STAT ?? 3, LIL-STAT 1, 3, 5A/5B

— 9, 10 11, 12

1, 3, 5A/B, 6 3(?), 6 3(?), 6 3, 5A/B 3, 5A/B 1, 3, LIL-STAT 1, 3, LIL-STAT

13–16 3, 16, 17 3, 16, 17 18 18 3, 10, 19 3, 19, 20

3, 5A/5B 3, 5A/5B

20, 21 20, 21

receptor receptor receptor (gp-130) receptor (gp130)

No STAT ?? 1, 3, 5A/5B 1, 3 1, 3 1, 3

— 20, 21 22–25 26–28 —

receptor -chain 2 receptor -chain 1

1, 3, 4, 5 6

3, 29–31 32

1, 3, 5A/5B

32–34

receptor (Acc#XP_009820)

1, 2, 3, 4

35

ligand ligand receptor

Unknown 5A, 5B 1, 3, 5

— 36, 37 38

1, 3 1, 3, 5A/5B 5B

3, 39 3, 40 41

1, 3, 5A/5B 1, 2, 3, 5 1, 3, 5 1, 6 1

3, 42–46 3, 46–49 45, 51, 52 53 54

3 1, 3

55 56

1, 3, 5A/B 1, 5A/5B 1, 2, 3, 5 3, 5, 6

45, 57, 58 59, 60 3, 61–63 64

1, 2, 4

65, 66

ligand (IFN- Consensus) receptor (-chain) ligand ligand ligand ligand (precursor) ligand receptor (-chain) receptor (-chain) receptor (-chain) receptor ligand ligand ligand receptor (gp130) receptor (gp130) receptor receptor -chain receptor -chain

receptor -chain

ligand ligand receptor receptor receptor receptor ligand receptor (R2) ligand ligand receptor (type I) receptor receptor receptor receptor ligand

So many ligands, so few transcription factors / 177

TABLE 1. Continued Ligand TGF hTGF LIF OSM CNTF Viral oncogenes v-abl v-sis (p28sis) v-fps v-src

NLS (proven/putative)

Where NLS found

STATs activated

Ref. nos

RRRHIVRKRTLRRL* KKKP* KKPFPEDLKSLDLFKKEK PTVRTKK* KKPFPEDLKSLDLFKKEK PTVRTKK* KKPFPEDLKSLDLFKKEK PTVRTKK*

receptor (EGFR) receptor (type II) receptor (gp130)

3 ? 1, 3

67

receptor (gp130)

1, 3

3, 28

receptor (gp130)

1, 3

3, 28

PKLLRR (proven; viii) RVTIRTVRVRRPPKGKHRK (proven; v) PRLKMKK* KRKYQEASKDKEREKAK* REHPYGR on possible adapter protein Sam 68 (ix)

— — —

3 3 3

69 70 70

—

3

70

68

†These NLSs have been proven to mediate nuclear translocation of the ligand or receptor concerned: (i) Subramaniam PS, Mujtaba MG, Paddy MR, Johnson HM (1999) J Biol Chem 274:403–407; (ii) Larkin III J, Subramaniam PS, Torres BA, Johnson HM (2001) J Interferon Cytokine Res (21:341–348); (iii) Wessendorf JH, Garfinkel S, Zhan X, Brown S, Maciag T (1993) J Biol Chem 272:28202–205; (iv) Jans DA, Briggs LJ, Gustin SE, Jans P, Ford S, Young IG (1997) FEBS Lett 406:315–320; (v) Lee BA, Maher DW, Hannink M, Donoghue DJ (1987) Mol Cell Biol 7:3527–3537; (vi) Imamura T, Engleka K, Zhan X, Tokita Y, Forough R, Roeder D, Jackson A, Maier JAM, Hla T, Maciag T (1990) Science 249:1567–1570; some studies have suggested that this is a nuclear retention signal rather than an NLS [e.g. Cao Y et al. (1993) J Cell Sci 104:77–87]; (vii) Di L, Yang H, Raizada MK (1998) Endocrinology 139:365–375; (viii) Birchenal-Roberts MC, Ruscetti FW, Kasper JJ, Bertolette DC 3rd, Yoo YD, Bang OS, Roberts MS, Turley JM, Ferris DK, Kim SJ (1995) Mol Cell Biol 15:6088–6099; (ix) Wu J, Zhou L, Tonissen K, Tee R, Artzt K (1999) J Biol Chem 274:29202–29210. *These sequences can be identified using the PSORT motif recognition program (http://psort.nibb.ac.jp/). Note that the program is still being updated, and as such is not yet set up to identify NLSs that deviate even marginally from the PSORT consensus. For example, the already proven bipartite NLS of either FGF-3 or IL-5 are not recognized since the spacer region is longer than the consensus ten amino acid spacer. For putative bipartite NLSs indicated here a spacer of 10–15 amino acids has been considered. This follows from the sequences of bipartite NLSs like those in IL-5.

NLS-containing proteins are synthesized in the cytoplasm and appear to be constitutively transported to the nucleus thereafter. So how is it that preformed cytoplasmic proteins such as the STATs, which require nuclear import for their functions, are actively translocated in the absence of an NLS? It seems reasonable to assume that other NLS-containing molecules must associate in some fashion with STATs in order to use the NPC machinery for transportation to the nucleus. The precedence for the presence of NLS-containing nuclear chaperone proteins assisting in the binding of cytoplasmic non-NLS proteins to importin  has recently been established in the case of the human aryl hydrocarbon receptor.77

IFN- CONTAINS A FUNCTIONAL NLS It has been demonstrated by several investigators that IFN-, which activates STAT1, rapidly translocates to the nucleus following binding to the receptor.78,79 The C-terminus of mouse IFN- contains a polycationic sequence, RKRKRSK, which is similar to the prototypical NLS sequence of SV-40 T antigen.80 This particular sequence has been shown to be essential for the biological activity of IFN-, but its specific function, until recently, was unknown.81–83 Related to this, at least three independent lines of experimentation have identified intracellular IFN- as being biologically active: (1) human IFN- (huIFN-) delivered by lipo-

some vector was able to activate murine macrophages to tumoricidal state; (2) secretion-defective huIFN- expressed in murine fibroblasts triggered antiviral activity; and (3) microinjected IFN- induced Ia expression on murine macrophages.84–86 The activity of huIFN- in these murine cells defies the well-known species-specificity of exogenously supplied huIFN- which does not have activity on murine cells. As the strict species-specificity of IFN- is mediated exclusively by the interaction of exogenous IFN with the extracellular domain of the receptor complex, these cross-species intracellular experiments demonstrate that the intracellular action of IFN- can occur independent of any extracellular interactions. Thus, the IFN- molecule most likely interacts with some intracellular element(s) to induce a biological response. Similarly, intracellularly expressed IFN- has also been shown to be biologically active and can activate the DNA binding activity of the ISGF3 transcription complex of which STAT1 and STAT2 are integral components.87 The formation of an active ISGF3 complex involves the translocation of both STAT1 and STAT2 to the nucleus. Thus, it seems likely that one of the reasons that IFN- is active intracellularly is because it can activate STAT1. Over the last few years, our studies on IFN- binding to its receptor have provided insight into a unique mode by which IFN- can interact directly with intracellular signaling elements via the cytoplasmic domain of the  chain of the IFN- receptor complex.

178 / Subramaniam et al.

The studies outlined below provide several scenarios in which to examine in greater detail the intracellular role of IFN- in signaling by transcription factors like STATs, including the question of maintaining the specificity of signal transduction unique to any given stimulatory ligand. We recently showed that nuclear localization of IFN- is driven by the simple polybasic NLS in its carboxyl terminus, as verified by its ability to specify nuclear import of a heterologous protein allophycocyanin (APC) in standard import assays in digitoninpermeabilized cells.88 Similar to other nuclear import signals, we showed that a peptide representing amino acids 95–132 of mouse IFN-, IFN-(95–132), and containing the polybasic sequence 126RKRKRSR132, was capable of specifying nuclear uptake of the autofluorescent protein, APC, in an energy-dependent fashion that required both ATP and GTP. Nuclear import was abolished when the polybasic sequence was deleted. Moreover, deletions immediately NH2terminal of this sequence did not affect the nuclear import. Thus, the sequence 126RKRKRSR132 was necessary and sufficient for nuclear localization. Furthermore, nuclear import was strongly blocked by competition with the cognate peptide IFN-(95–132) but not the peptide IFN-(95–125), which was deleted in the polybasic sequence, further confirming that the NLS properties were contained in this sequence. A peptide containing the prototypical polybasic NLS sequence of the SV40 large T-antigen was also able to inhibit the nuclear import mediated by IFN-(95–132). This observation suggested that the NLS in IFN- functioned through the components of the Ran/ importin pathway utilized by the SV40 T-NLS. We also showed that intact IFN-, when coupled to APC, was also able to mediate its nuclear import. Again, nuclear import was blocked by the peptide IFN-(95– 132) and the SV40 T-NLS peptide, suggesting that intact IFN- was also transported into the nucleus through the Ran/importin pathway. As expected, human IFN- also contains a functional NLS represented by 128KTGKRKR134; Ref. 89 HuIFN-(1–123), a deletion mutant of wild-type huIFN-(1–146) and lacking the NLS, binds to the extracellular domain of the receptor with the same high affinity as wild-type huIFN-(1–146), but unlike wildtype IFN- translocates only weakly to the nucleus via a minor NLS.90 Introduction of a heterologous NLS, that of the prototypical NLS of the SV40 large T-antigen, into this mutant (at position 123 onwards) is sufficient to restore nuclear translocation and full biological activity similar to wild-type IFN- (PS Subramaniam, MM Green, BA Torres and HM Johnson, manuscript submitted). Thus, the NLS region of IFN- contributes minimally or not at all to extracellular high-affinity receptor/ligand binding, yet exerts

CYTOKINE, Vol. 15, No. 4 (21 August, 2001: 175–187)

a strong functional role in nuclear translocation of IFN-. Interestingly, a C-terminus IFN- peptide that binds to the cytoplasmic domain of the IFN- receptor IFNGR-1 chain and that contains the NLS has biological activity.91 Treatment of murine macrophage cell lines with mouse IFN-(95–133), where the peptide is taken up by pinocytosis, resulted in 10-fold upregulation of MHC class II molecule expression and 106– 109-fold increased resistance to viral infection. Further, a C-terminus peptide corresponding to huIFN-(94– 134) also had activity in murine macrophage cells. Modification of huIFN-(94–134) by addition of a hydrophobic group for cell penetration resulted in duplication of the above results in fibroblast cells.92 Most importantly, IFNGR-1 -/- cells did not respond to the intracellular peptide demonstrating the requirement of the IFNGR-1 cytoplasmic domain.92 These data suggest a direct role for the C-terminus of IFN- in the initiation of intracellular signaling, probably related to its nuclear translocation properties.

DIFFERENTIAL NUCLEAR LOCALIZATION OF THE IFNGR-1 AND IFNGR-2 SUBUNITS OF THE IFN- RECEPTOR COMPLEX FOLLOWING ACTIVATION BY IFN- Uptake of IFN- is a receptor-mediated endocytic process and this raises the question of the fate of both the receptor subunits (IFNGR-1 and IFNGR-2) of the heterodimeric IFN- receptor complex after IFN-/ receptor interaction. Accordingly, human epithelial cells (WISH) were treated with huIFN- and the fate of the receptor complex followed.93 Like the ligand, the IFNGR-1 subunit of the complex on WISH cells was found to translocate to the nucleus on treatment with huIFN-. Using a combination of immunoprecipitation and immunofluorescence techniques, we found the nuclear accumulation of IFNGR-1 to be liganddependent, and it was evident within 10–20 min after ligand stimulation. IFNGR-1 was found to colocalize, in a time-dependent and dose-dependent manner, with the nuclear translocation of the transcription factor STAT1, which is activated by this ligand–receptor system. In addition, STAT1 was found to be complexed with IFNGR-1 over the time period of its nuclear translocation. In marked contrast, IFNGR-2 was not transported to the nucleus. The surface immunofluorescence pattern of IFNGR-2 suggested that, following ligand stimulation, the majority of IFNGR-2 remains at the cell surface, whereas IFNGR-1 is endocytosed and targeted to the cell nucleus. Most interestingly, treatment of WISH cells with an intracellular lipid analog of the agonist peptide

So many ligands, so few transcription factors / 179

muIFN-(95–133), which facilitates transport into cells, is sufficient to induce the nuclear translocation of IFNGR-1 and STAT1 (PS Subramaniam, K Thiam, C Verwaerde and HM Johnson, unpublished observations) strongly suggesting that the intracellular interaction of the C-terminus of IFN- with the cytoplasmic domain of IFNGR-1 drives the nuclear translocation of both IFNGR-1 STAT1. These findings suggest that IFNGR-1 plays an active intracellular role in signal transduction events subsequent to the binding of ligand to the receptor complex. Furthermore, these studies provide the first example of the selective endocytosis and nuclear translocation of a subunit of a multimeric receptor complex. Preliminary studies have identified the plasma membrane microdomains crucial for IFN- signaling through the receptor (Subramaniam and Johnson, unpubl. data). Treatment of human WISH cells with filipin, an inhibitor of caveolae function,94 inhibited receptor endocytosis, STAT1 activation, and nuclear translocation of STAT1, IFNGR-1, and IFN-. By contrast, inhibition of clathrin-coated pits function by acidification of WISH cells followed by treatment with IFN- or treatment of dynamin mutant cells with IFN- did not block receptor endocytosis, STAT1 activation, and nuclear translocation. The mutant is defective in the clathrin pathway.95 Further, similar to previous studies with epidermal growth factor,96 subcellular fractionation using centrifugation techniques showed that IFNGR-1 and IFNGR-2 were constitutively associated with caveolae. Treatment of cells with IFN- resulted in rapid exit of the receptor IFNGR-1 chain from the membrane-associated caveolae. Consistent with differential nuclear translocation of the receptor IFNGR-1 and IFNGR-2 chains, IFNGR-2 remained associated with plasma membrane-associated caveolae following IFN- treatment. Thus, caveolae appear to play the central role in the early events of IFN- signaling at the level of membrane-associated organelles. In the case of EGF, early endosomes played a critical role in signaling at the perinuclear level. Future studies, therefore, will focus on the role of early endosomes in IFN- signaling subsequent to endocytic caveolae-associated events. The cytoplasmic domain of IFNGR-1 is obligatorily required for IFN--induced biological responses and, surprisingly, is not species-restricted.97–100 The region of the cytoplasmic domain that interacts with IFN- encompasses residues 252–291. In the human receptor, this region contains a membrane proximal leucine-isoleucine (LI) sequence (residues 270–271) that may be required for some receptor trafficking events within the cell following receptor endocytosis.101 There is no evidence, however, that this is a functional motif. Immediately upstream of this sequence is an LPKS sequence (residues 266–269) that is required for

the constitutive association of the tyrosine kinase JAK1.102 This association is crucial for the activation of the signal transduction cascade from the receptor following binding of IFN-. In the murine receptor, we have shown that the cytoplasmic domain of IFNGR-1 also contains two sites of interaction for the other tyrosine kinase, JAK2.103 One site encompassing residues 283–309 in the murine receptor overlaps the binding site for the C-terminus of IFN-. In particular, binding of JAK2 at this site on the receptor is enhanced by interaction of the C-terminus of IFN- with the cytoplasmic domain, which may explain why co-immunoprecipitation of JAK2 with the IFNGR-1 chain is seen only after ligand treatment of cells.104 Given the proximity of the ligand-induced JAK2 binding site to binding sites for IFN- and JAK1 on the cytoplasmic domain, it is quite probable that the interaction of the ligand with this domain of the receptor directly contributes to the initial activation of either kinases and consequently initiation of the signal transduction cascade. Finally, phosphorylation of the tyrosine at position 440 generates a binding site on the receptor for STAT1. This site overlaps the second interaction site for JAK2 on the IFNGR-1 chain. Thus, interaction of the NLS-bearing C-terminus of the ligand with the cytoplasmic domain of the receptor could affect processes that include the activation of the signal events that recruit STAT transcription factors within the ligand–receptor complex and the subsequent endocytosis, trafficking, and nuclear translocation of these molecules to their target genes.

PRESENCE OF IFN-/IFNGR-1/STAT1 COMPLEX WITH IMPORTIN  (Npi) The presence of an IFN- binding site in the cytoplasmic domain of the receptor IFNGR-1 chain, the presence of a functional NLS in the C-terminus of IFN-, and the coordinate nuclear translocation of IFN-, IFNGR-1, and STAT1 suggest that such translocation occurs as a complex.105,106 Further, when cells were microinjected with antibodies to the C-terminus of IFN-, followed by external treatment of the cells with IFN-, nuclear translocation of IFN-, IFNGR-1, and STAT1 did not occur.93,105. Treatment of the microinjected cells with the type I IFN, IFN-, by contrast did result in activation and nuclear translocation of STAT1. Thus, the inhibitory effects of the IFN- intracellular antibody were specific. STAT1 itself has been shown to be translocated to the nucleus by the Npi-1 homologue of importin.76 Immunoprecipitation of the importin  analog Npi-1 from WISH cells following huIFN- treatment showed that huIFN-, IFNGR-1, and STAT1 were all complexed to Npi-1.106 Treatment of cells with

180 / Subramaniam et al.

huIFN-(1–123), where the strong NLS is not present, failed to significantly show huIFN-, IFNGR-1, and STAT1 complexed to Npi-1. Reconstitution of the mutant with a heterologous NLS, the NLS of the SV40 large T-antigen (IFN--SV), restored nuclear translocation and the formation of a complex between Npi-1, STAT1 and in this case IFN--SV (PS Subramaniam, MM Green, BA Torres, HM Johnson, manuscript submitted). Thus, these data strongly support the conclusion that it is the IFN- NLS that mediates the complexation of STAT1 and its transporter Npi-1. The results are consistent with the following scenario. IFN- binds to the extracellular domain of the receptor. Endocytosis results in IFN- association with the cytoplasmic domain of the IFNGR-1 receptor chain along with activated STAT1. The complex of IFN-/IFNGR-1/STAT1 in turn binds to importin  via the NLS of IFN-. The complex then binds to importin  and undergoes active transport to the nucleus via the nuclear pore complex (Fig. 1). STAT1 has been shown to bind to the promoter region of genes as a duplex (reviewed in Ref. 1). This is not inconsistent with the picture presented here and the supporting data. It is intriguing to speculate on a potential role of IFN- and/or IFNGR-1 in gene activation beyond the events associated with nuclear transport.

OTHER NLS CANDIDATES AMONG PROTEINS THAT BIND TO STATS Several interferon response factors (IRFs) contain known or putative NLSs.107 A recent study has examined the assembly of the ISGF3 complex involved in IFN- signaling.107 ISGF3 is formed from STAT1, STAT2 and an IRF family member p48 (now designated IRF9). p48 has a bipartite nuclear localization sequence. Overexpressed p48 is constitutively found in the nucleus. However, in normal cells unphosphorylated STAT2 is bound to p48 in the cytoplasm of the cell and blocks its nuclear translocation, creating a cytoplasmic and nuclear pool of p48. Thus, binding of STAT2 to p48 does not induce the nuclear translocation of the p48:STAT2 complex, otherwise STAT2 would be constitutively present in the nucleus. Also, unphosphorylated STAT1 does not bind to p48, but treatment of cells with type I IFN results in formation of the STAT1/STAT2/p48 (ISGF3) complex and subsequent translocation to the nucleus. This suggests that ligand-activation of STAT2 or the ligand-activation and binding of STAT1 is required for nuclear translocation of ISGF3. Thus, these studies indicate that the NLS of p48 in the cytoplasm is not sufficient to induce nuclear translocation of ISGF3, but an as yet undefined NLS needs to be provided that is gained by

CYTOKINE, Vol. 15, No. 4 (21 August, 2001: 175–187)

IFN-activation of STATs. In the light of our studies it is interesting to determine whether the NLS comes from IFN or IFNAR in the IFN/IFNAR/STAT complex formed following ligand activation of STAT1/2. STAT1 and IRF1 have also been shown to be associated with a GAS promoter site in the nucleus of unstimulated cells.108 This complex is responsible for the constitutive expression or suppression, as the case may be, of many genes.108 However, the constitutive nuclear level of STAT1 is several-fold lower than that seen after treatment of cells with IFN-. It has been shown that this constitutive level of STAT1 is itself maintained by the autocrine effect of low levels of IFN- secreted by cells.109 This low level of IFN- (probably accounting for the low levels of STAT1) also modulates the cross-talk between the type I IFN and type II IFN receptor systems in caveolar microdomains of the cells. Thus, the ‘‘constitutive’’ STAT1 expression is actually a ligand-activated (IFN-activated) event, again raising the question of the role of IFN/IFN-receptor mediated nuclear chaperoning of ‘‘constitutive’’ STAT1 nuclear presence. In studies with chimeric receptors, where the intracellular receptor domains are from the IFNGR-1 and IFNGR-2 IFN- receptors and the extracellular domains are from the erythropoietin (EPO) receptor, EPO treatment of the cells resulted in upregulation of MHC class I molecules.110 The cells also contained the intact IFN- receptor IFNGR-1. It would seem that there is no obvious mechanism for nuclear transport of STAT1 in the context of our IFN-/IFNGR-1 model for STAT1 translocation to the nucleus. However, the receptor cytoplasmic domain plays an important role in the chimeric studies, and if it were shown that receptor translocation to the nucleus was required for STAT1 translocation, then our model would provide important insight to signaling in the receptor chimeras. It is possible that EPO complexed to the chimeric receptor provides the NLS for nuclear transport of activated STAT1. A putative bipartite NLS is present in the EPO sequence (see Table 1). This would suggest that an NLS along with the appropriate cytoplasmic domain of the primary signaling subunit of the receptor are necessary and sufficient conditions for the activation and nuclear transport of a given STAT.

FUSION PROTEINS AND MUTANTS An important distinction needs to be made between the process of nuclear translocation and nuclear retention. Although these events are often used synonymously, they represent different properties of STATs. It is well known that the accumulation of STAT in the nucleus is a balance between entry,

So many ligands, so few transcription factors / 181

Figure 1.

A simplistic model for the STAT chaperoning role of the IFN-/IFNGR-1 complex.

A, Binding of IFN- to the IFNGR complex (, IFNGR-1; , IFNGR-2) causes the activation of JAK1 and JAK2 and induces binding of STAT1 to IFNGR-1 as a dimer. Also binding of IFN- initiates the selective endocytosis of IFNGR-1, along with its bound cargo, into the early endocytic vesicle. It is not known if these events are sequential or simultaneous as binding of the C-terminus of IFN- to the IFNGR-1 cytoplasmic domain itself affects some of these events, for example, the binding of JAK2. These events could be initiated as IFN- crosses the plasma membrane, to be transferred into the cytosol (as shown here) or remain traversed across the plasma membrane, to contact the cytoplasmic domain of IFNGR-1. The disposition of IFNGR-1 in the early endocytic vesicle is derived from the known orientation of other endocytosed receptors. B, The exposed NLS on the C-terminus of IFN- serves to allow recognition by the appropriate importin- homologue, which in this case has been shown to be Npi-1.106 Docking of importin- then allows recognition by the Ran GTPase and subsequent import of the IFN-/IFNGR/STAT complex.

182 / Subramaniam et al.

DNA binding (and retention) and exit (see, for example, Ref. 111). The exit of STAT1 is dependent (a) on an intrinsic nuclear export signal (NES), and (b) a nuclear tyrosine phosphatase that dephosphorylates STAT1 and releases it from DNA, triggering its export. In the absence of the ability to bind DNA, STAT1 essentially enters and immediately exits the nucleus via its NES, leading to a situation where very little STAT1 can apparently be detected in the nucleus. This delicate balance is exemplified in a recent study on the role of JAK1 on nuclear cycling of STAT1.112 These studies showed that in the absence of JAK1, STAT1 is not able to bind the transcriptional co-activator CBP in the nucleus, and the otherwise IFN-activated and tyrosine phosphorylated STAT1 is not retained in the nucleus but is transported out. In contrast, inhibition of nuclear export with the CRM1/ exportin inhibitor leptomycin B leads to the detection of nuclear STAT1. Interestingly, JAK1 has a putative bipartite nuclear localization sequence. However, as JAK -/- cells are competent for nuclear transport of STAT1 this putative NLS does not function in STAT1 nuclear import. But the nuclear presence of JAK1, like the previously demonstrated nuclear presence of JAK2,113,114 could regulate the phosphorylation of STAT1 co-activators that can then bind and retain STAT1 in the nucleus. Another study involves STAT5B.115 This study used a green fluorescence protein (GFP)-STAT5B construct and mutational analysis of various domains of STAT5B. Only mutation of a VVVI motif that is involved in binding to DNA (IRF promoter) led to loss of STAT5B signal from the nucleus, although the mutant was tyrosine phosphorylated similarly to wildtype protein. Thus, failure to detect nuclear STAT5B corresponded with a failure to be retained in the nucleus by DNA binding. It is interesting that these studies with STAT5B, similar to those with STAT1,76 also demonstrated that other basic residues present in STAT5B do not contribute to the nuclear localization of STAT5B, showing that STAT5B also does not contain a nuclear localization sequence. Recently, studies with STAT1 and STAT2 point out that dimer duplexes contain arginine/lysine-rich elements in the DNA-binding domain.116 The possibility that these sequences form a non-contiguous NLS in the final dimer or the ISGF3 complex was examined. The authors created point mutations in the implicated arginine/ lysine residues and showed that the mutant STATs were absent from the nucleus. They concluded that the duplexes contained a functional NLS. However, closer examination of these could lead to an alternate interpretation. First, the mutants in these arginine/lysine elements that did not appear in the nucleus were still able to compete with wild-type STATs for the nuclear transport apparatus because

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expression of the mutants blocked the nuclear import of wild-type STATs. That is, mutation of the putative NLS still conferred the ability to compete with wildtype STAT for nuclear import. Further, the mutations were in DNA-binding regions. As described above, such mutations have been shown to result in lack of retention of STAT in the nucleus.115 DNA binding studies on the arginine/lysine mutations in STAT1 and STAT2 homodimers and heterodimers did, in fact, show that the mutations failed to bind to the STAT elements on DNA. Thus, the arginine/lysine STAT mutants were most likely not retained in the nucleus rather than not transported to the nucleus. It would be also instructive to re-examine such mutations with respect to specific interactions with the STAT1 transporter Npi-and their behaviour in nuclear export. Insight into the events contributing to nuclear import of STAT1 can, however, be obtained from the study of the nuclear export of STAT1. In one study involving transfection of cells with STAT1 fused with GFP, STAT1-GFP, it was shown that IFN- treatment of cells was required for nuclear transport.111 Interestingly, transfection of cells with STAT1-GFP containing SV40 NLS, STAT1-NLS-GFP, resulted in constitutive transport of STAT1 to the nucleus in the absence of phosphorylation. There are two important points from these results that impinge on our model: (1) STAT1 is not anchored or sequestered in the cytoplasm, otherwise STAT1-NLS-GFP would not have been translocated to the nucleus constitutively. Anchoring proteins serve the function to block constitutive nuclear transport even in the presence of a functional NLS until ‘‘signaled’’ to release their cargo. (2) As the presence of a heterogeneous NLS on STAT1 results in constitutive transport to the nucleus, a chaperone with an NLS is consistent with nuclear transport of wild-type STAT1 only after activation of cells by IFN-.

CONSTITUTIVE EXPRESSION OF STATS IN VIRAL ONCOGENE TRANSFORMED CELLS A number of viral oncogene products have been shown to produce constitutive activation of the JAK/ STAT pathway, a common target being STAT3. These include, among others, the well-studied v-abl, v-src, and v-fps (of the fer/fes family) oncogenes.117,118 STAT3 is a particularly crucial STAT that affects directly the proliferative and differential capacities of various cells. Consequently, in knockout mice studies deletion of the STAT3 gene is the only STAT-deletion that is associated with embryonic lethality. It is not surprising, thus, that the evolutionary selection of STATs by viral oncogenes has targeted STAT3 as a

So many ligands, so few transcription factors / 183

crucial viral survival factor, and this constitutive activation results in myeloproliferative syndromes and myelodysplasia and acute leukemia. The v-abl oncogene activates the JAK/STAT signaling system in transformed cells.118 V-Abl activates and binds STAT1, STAT3, and STAT5, STATs that are normally activated by IL-4 and IL-7 stimulated cells.118 The SH2 and protein tyrosine kinase domain, as well as myristylation signals at the N-terminus of the protein are required for v-abl-induced transformation.119 Amino acids 858–1080 within the C-terminus of the v-abl protein contained the site of interaction with JAK1.120 A mutant of v-abl, lacking this region, failed to activate JAK1 and STAT proteins. Further, the inducible expression of a kinase inactive JAK1 protein inhibited v-abl activation of the STATs, and resulted in loss of cytokine-independent cell proliferation as well as reduced tumorigenesis.120 Of particular interest, v-abl itself has been shown to contain an NLS (see Table 1).121 Thus, v-abl probably directly chaperones STATs into the nucleus via its intrinsic NLS. Similar to the N-terminal mutations of the SH2 and tyrosine kinase domains, it would be interesting to determine if mutations in the putative NLS regions of v-abl would abrogate STAT nuclear translocation in v-abl transformed cells, as well as abrogation of cytokine-independent cell proliferation and tumorigenesis. The constitutive activation of STAT3 transcription factor has also been reported for v-src transformed cells.122,123 The activation of STAT3 was shown to be due to v-src expression and not as a consequence of cell transformation. Further, v-src bound phosphorylated STAT3 through their respective SH2 domains, and both were translocated to the nucleus. Consistent with STAT3 activation, v-src transformed cells showed activation of the tyrosine kinase JAK1 and JAK2.124,125 It is not known how the v-src/STAT3 complex translocates to the nucleus, as neither possesses a known NLS. The question is does v-src associate with a protein(s) that does contain an NLS? A phosphorylated 68 kDa protein designated SAM68 (Srcassociated in mitosis) for its association with src at mitosis, has been identified.126,127 SAM68 belongs to a family of proteins called STAR (signal transduction and activator of RNA).128 STAR proteins contain an NLS motif that is novel and different from that exemplified by the polycationic sequence of SV40 T antigen. Located in the C-terminus of the molecule, the STAR consensus NLS is RXHPYQ/GR, while that of SAM68 is REHPYGR. All the STAR proteins have been shown to bind RNA.129 Binding to c-src blocks the ability of SAM68 to bind RNA.127 This would suggest that src binding to SAM68 directs SAM68 to carry out an src function.

In the context of our chaperone model of STAT translocation to the nucleus, v-src activated STAT3 and the resultant v-src/STAT3 complex would undergo nuclear translocation by SAM68 which would be a component of the complex via its association with v-src. This is analogous to IFN- providing the NLS for nuclear translocation of the IFN-/IFNGR-1/ STAT1 complex. Co-immunoprecipitation experiments with antibody to SAM68, for example, could determine if indeed SAM68 was complexed to V-src/ STAT3. Peptide competition with STAR motif NLS would determine if SAM68 indeed provided the NLS for STAT3 nuclear translocation. Thus SAM68 could play an important role in trafficking v-src-activated transcription factors into the nucleus, of which STAT3 would be one. This variant of our IFN- nuclear translocation mechanism could provide considerable insight into the mechanism of v-src transformation of cells. An interesting family of tyrosine kinases that are activated by v-src and c-src is that of Btk (reviewed in Ref. 130). These proteins play an important role in hemopoeitic cell function, including B and T lymphocytes. They are involved in many of the complex signaling events in the cell such as MAP, JAK, PI3K, lyn, syk and PKC kinase activity. One of the Btk members, Etk, is involved in v-src activation of STAT3.130 The findings provide evidence that v-src phosphorylates and thus activates Etk, which in turn plays a role in STAT3 phosphorylation. Characteristic of Btk family members, Etk contains an N-terminal pleckstrin homology (PH) domain, a Tec homology domain, SH2 and SH3 domains, and a C-terminal kinase domain.131 Small amounts of Etk are constitutively present in the nucleus as well as in discrete regions of the cytoplasm such as the plasma membrane. Etk contains candidate polycationic amino acid sequences in its PH domain that could potentially play a role in nuclear transport of Etk. PH domain mutants of Etk, lacking the candidate NLSs, however, traffic to the nucleus similar to the wild-type Etk. Thus, Etk does not contain a known NLS. V-Src is required for Etk nuclear translocation, possibly via SAM68 as proposed above for v-src and STAT3. The SAM68 variant of our STAT chaperone model could thus play an important role in cell signaling.

DO LIGANDS AND/OR RECEPTORS PLAY A NUCLEAR ROLE IN STAT SIGNALING? In the cases where examined, ligands that utilize the STAT transcription factors undergo nuclear translocation (see Table 1). The IFN- model presented suggests that nuclear transport of STAT is dependent on a chaperone role for IFN-. Although it remains to

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be established, it is possible that the other ligands that enter the nucleus function similar to IFN- and serve as chaperones for STAT translocation. Further, do ligands/receptors such as IFN- and IFNGR-1 play a role in the nucleus for signaling in association with and/or independent of the STAT signaling? Hints of an active role in the nucleus would be the observation of nuclear modification of ligand and/or receptor such as phosphorylation, methylation, or acetylation. Also, recent studies have suggested that some IFN functions are STAT-independent (e.g. see Refs 132, 133). It would be of interest to determine if the IFN NLS is required for these STAT-independent effects. Certainly, the novel model presented here for cell signaling involving STAT transcription factors could provide an explanation for the uniqueness of ligand effects at the cellular level.

Acknowledgements The authors’ work was supported by NIH grant CA 38587.

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