Serine Phosphorylation of Stats

Thomas Decker*,1 and Pavel Kovarik1

1Vienna Biocenter, Institute of Microbiology and Genetics, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria

Tyrosine phosphorylation regulates the dimerization of eect of STAT serine phosphorylation have been made STATs as an essential prerequisite for the establishment and will be summarized and discussed below. of a classical JAK-STAT signaling path. However, most vertebrate STATs contain a second phosphorylation site Known STAT serine phosphorylation sites are located within their C-termini. The phosphorylated residue in this within the C-terminus case is a serine contained within a P(M)SP motif, and in the majority of situations its mutation to alanine alters Evidence for serine phosphorylation of STATs 1 and 3 transcription factor activity. This review addresses recent had initially been obtained by isolating the proteins from advances in understanding the regulation of STAT serine 32P-labeled cells after appropriate stimulation with phosphorylation, as well as the kinases and other signal cytokines and subjecting them to phospho-amino acid transducers implied in this process. The biochemical and analysis (Eilers et al., 1995; Wen et al., 1995; Zhang et al., biological consequences of STAT serine phosphorylation 1995). Moreover, cytokine treatment in the presence of are discussed. Oncogene (2000) 19, 2628 ± 2637. the serine kinase inhibitor H7 partially reversed an SDS ± PAGE mobility shift of STATs 3, 4 and 5b Keywords: STAT; signal transduction; phosphoryla- isolated from cells after stimulation with IL-6, IL-12 and tion; MAP kinase IL-2, respectively (Beading et al., 1996; Boulton et al., 1995; Cho et al., 1996; Lutticken et al., 1995). Sequence alignments of dierent vertebrate STATs Introduction reveal blocks of conserved amino acids in virtually every domain except the last approx. 50 ± 150 amino Cytokine receptor signal transduction utilizes STATs acids, i.e., the portion beyond the phosphorylated both as messengers to the cell nucleus and as activators tyrosine residue, where conserved amino acids are very of many genes whose products orchestrate the infrequent (Schindler and Darnell Jr, 1995). The C- physiological changes within a cytokine-treated cell. terminal domains are tightly linked to the transactiva- While STATs share the dual function as signal tion function of STATs because their removal transducers and activators of transcription with other generates transcriptionally inactive proteins, frequently intracellular mediators of cytokine responses like dominant-negative alleles when overexpressed (Calden- SMADs, NFkB or Notch, the mechanism converting hoven et al., 1996; Mui et al., 1996; Shuai et al., 1993). STATs from latent cytoplasmatic proteins into their Comparing the C-termini of STATs 1, 3 and 4 it was active forms is unique. This uniqueness derives from noted that unlike the majority of amino acids, a PMSP two structural components not found in other families motif between positions 720 and 730 is perfectly of transcription factors: an SH2 domain and a C- conserved (Wen et al., 1995, Figure 1a). In addition, terminal tyrosine residue which becomes phosphory- STAT5a and STAB5b contain a conserved PSP motif lated in cytokine-stimulated cells, most often by in an equivalent position (Yamashita et al., 1998; receptor-associated Janus kinases (JAKs). Tyrosine Figure 1a; for reasons of simplicity we will collectively phosphorylation and subsequent SH2 domain- refer to these STATs as P(M)SP-STATs). Since PMSP mediated homo- or heterodimerization of STATs and PSP sequences are potential serine phosphoryla- provide the essential prerequisite for biological activity tion sites for proline-directed kinases, particularly the because dimerization shifts the subcellular localization MAP family kinases (Clark-Lewis et al., 1991; to the nucleus and it allows STATs to bind DNA. Gonzalez et al., 1991), their recognition sparked an It appeared initially that a single STAT tyrosine immediate interest in the question whether a second residue is all that becomes phosphorylated as a STAT phosphorylation site had been uncovered. consequence of cytokine stimulation. For STATs 2 Conclusive evidence for this was ®rst provided for and 6 this may still be true. However, all other STATs STAT1 and STAT3. Using phospho-peptide mapping were found phosphorylated also on serine residues in a and phospho-amino acid analysis, a cytokine-regulated stimulus-regulated manner. With a second phosphor- increase of phospho-serine could be demonstrated, ylation event providing the possibility to modulate assigned to a C-terminal tryptic peptide, and shown STAT activity, the question arose which aspect of to be abrogated by mutation of S727 (Wen et al., STAT function (if any) was being regulated and with 1995). Subsequently the phosphorylation of the what consequences for cytokine responses. Moreover, P(M)SP motifs of STATs 1, 3, 4, 5a and 5b was STAT serine kinases were being sought. For individual established or con®rmed using phosphospeci®c anti- STATs these issues are far from being resolved, but bodies (Frank et al., 1997; Ng and Cantrell, 1997; advances towards understanding the cause for and Kovarik et al., 1998; Visconti et al., submitted; Yamashita et al., 1998). Immunoreactivity with such antibodies strongly increased after treatment with appropriate cytokines and where tested it was absent *Correspondence: T Decker when assaying the respective S-A mutations (Kovarik STAT serine phosphorylation T Decker and P Kovarik 2629

Figure 1 Amino acid sequence comparisons of STAT C-termini. The P(M)SP motif is highlighted in bright red, the tyrosine phosphorylation site in dark red. Upper case letters denote amino acids conforming to the consensus sequence (allowing for conservative changes) while lower case letters denote amino acids deviating from the consensus sequence. (a) Comparison of human STATs containing the P(M)SP phosphorylation site. Consensus amino acids are indicated at positions where four of the ®ve STATs contain identical amino acids or a conservative change in amino acids. (b and c) Comparison of zebra®sh STAT1 (b) and STAT3 (c) with mammalian homologues. Consensus amino acids are indicated at positions where the zebra®sh and at least three out of the four mammalian STATs contain identical amino acids or a conservative change in amino acids et al., 1998; Yamashita et al., 1998; Visconti et al., known whether a B-type STAT gene is encoded by, or submitted). In case of STATs 3 and 4, the H7-sensitive was lost in, insect genomes. Therefore, ®nal proof is SDS ± PAGE mobility shift decreased upon mutation lacking for the assumption that the ancestral STAT of, respectively, S727 and S721 to alanine (Chung et gene duplication occurred before the separate evolution al., 1997; Visconti et al., submitted). of vertebrates and insects (Barillas-Mury et al., 1999). The occurrence of serine phosphorylation sites All vertebrate homologues of STAT1, STAT3 and distinct from the C-terminal P(M)SP is suggested by STAT4 sequenced to this date include a C-terminal the fact that some (or perhaps all) STATs still contain PMSP serine phosphorylation motif and all STAT5s phospho-serine when mutated to P(M)AP (Chung et contain the PSP sequence. Thus, the PMSP sequence is al., 1997; Yamashita et al., 1998). Since nothing is characteristic for all B-type STATs except STAT2 and known about such sites except that they may exist, this is found in species as distantly related to humans as review will entirely be concerned with serine phosphor- zebra®sh or Xenopus laevis (Figure 1b,c). Comparing ylation of the C-terminal P(M)SP. the zebra®sh and human STAT1 underscores the relevance of PMSP conservation, because only 19 out of 49 amino acid positions in the C-terminus beyond Evolutionary conservation of the C-terminal serine Y701 (human STAT1) are identical and another three phosphorylation site positions contain conservative changes. The sequence During the evolution of organisms, STATs appear at analyses therefore suggest that the PMSP motif was the metazoan boundary (Darnell, 1997). The most acquired shortly after the original STAT gene duplica- primitive multicellular organism containing STATs tion and the emergence of B-type STATs. Since STAT2 with a characteristic SH2 domain and phosphotyrosine is of the B-type, but does not contain a PMSP motif, residue is Dictyostelium discoideum (Kawata et al., we speculate that the absence of the phosphorylation 1997). An evolutionary relationship has been proposed, site is due to a secondary loss. This assumption is not according to which the STAT genes in Dictyostelium far-fetched in light of the fact that the C-terminus in and Coenorhabditis elegans represent ancestral forms. STAT2 from dierent species is very divergent (Park et However, an early duplication of the ancestral gene al., 1999; Paulson et al., 1999) and that STAT2 may must have occurred, eventually giving rise to A- and B- also have lost or changed its DNA-binding domain. It type STATs (Barillas-Mury et al., 1999; Copeland et may represent a special case among STATs. al., 1995). Insect STATs from Drosophila melanogaster The situation is much less clear in case of A-type and Anopheles gambiae, D-STAT and Ag-STAT, STATs. There is no potential site for a proline-directed respectively, belong with A-type STATs that include kinase (i.e. a SP sequence) in the C-terminus of D- also vertebrate STATs 5a, 5b and 6. Vertebrate STATs STAT (Hou et al., 1996; Yan et al., 1996). The Ag- 1, 2, 3 and 4 are B-type STATs. It is currently not STAT contains a QS681PS sequence (Barillas-Mury et

Oncogene STAT serine phosphorylation T Decker and P Kovarik 2630 al., 1999). Vertebrate STAT6 contain SS756PD (human) stems from data showing that the entire STAT3 C- or SS747PE (mouse) sequences in a position similar to terminus appears to be dispensable for activating the the B-STAT PMSP. As in the case of Ag-STAT there a2-macroglobulin acute phase promoter in hepatoma is no available information whether the serines within cells, but not in immortalized simian kidney ®broblasts them are phosphorylated. Vertebrate STAT5 is so far (COS cells; Sasse et al., 1997). the only example of an A-type STAT where phosphor- Signal transduction pathways causing phosphoryla- ylation of the C-terminus on serine has been tion of STAT3 S727 can decrease STAT3-dependent documented. transcriptional responses (Jain et al., 1998, 1999; Lim The available information suggests that duplication and Cao, 1999; Sengupta et al., 1998). The interpreta- of STAT genes and functional diversi®cation occurred tion of these data in molecular terms is rendered in primitive vertebrates or their evolutionary ancestors. dicult by the fact that such pathways also aect The separate evolution of A- and B-type STATs as well STAT3 tyrosine phosphorylation. As will be discussed as the acquisition of a C-terminal serine phosphoryla- below, S727-independent and S727-dependent routes of tion motif are dierent facets of the diversi®cation down-modulating STAT3 tyrosine phosphorylation process, which caused STATs to play diverse functions have been described. in multiple aspects of cell biology and immunity. As in the case of STATs 1 and 3, mutation of S721 to alanine decreased the transcription factor activity of STAT4 when assayed in transfected, IL-12-stimulated Biochemical and biological implications of STAT serine cells (Visconti et al., submitted). By contrast, mutation phosphorylation of S725 or S730 in STAT5a and STAT5b, respectively, Information concerning the consequences of serine did not aect transcriptional activation of a prolactin- phosphorylation of the P(M)SP-STATs is either responsive promoter in COS cells (Yamashita et al., derived from the analysis of S-A mutants or from the 1998). There are several potential explanations for this. correlation of a phosphorylation event with a parti- Most simply, phosphorylation of these STAT5 residues cular downstream eect. There are as yet no mice with may not aect transcription. Alternatively, the require- S-A mutations in the endogenous STAT loci, but the ment for a phosphorylated serine could be promoter or generation of such animals is currently under way. cell type-dependent, as may also be the case for STAT3. Activation of transcription The ®rst analysis of STAT1 and STAT3 S-A mutations showed a reduc- DNA binding Some reports indicate a correlation of tion of cytokine-stimulated transcription factor activ- STAT1 or STAT3 serine phosphorylation with in- ity. With an approx. 80% reduction of interferon- creased DNA-binding (Eilers et al., 1995; Ng and gamma (IFN-g)-induced transcription factor activity Cantrell, 1997; Zhang et al., 1995). Mutation of either the eect of the mutation was very pronounced for STAT1 or STAT3 did not produce any evidence for an the STAT1 dimer. It was observed either in transient eect of S727 phosphorylation on DNA-binding. transfection assays or after complementation of Mutation of S725 and S730 in STAT5a and STAT5b, STAT1-de®cient U3A cells with STAT1 S727A and respectively, was similarly without an eect on the assaying the endogenous interferon regulatory factor-1 ability of these proteins to bind DNA (Yamashita et (IRF-1) target gene (Wen et al., 1995). The link al., 1998). Therefore, if serine phosphorylation indeed between phosphorylation of STAT1 on S727 and aects DNA binding, other serine residues must be increased transcription was strengthened by studies involved. Rigorous proof for this assumption has not showing that stimuli causing serine phosphorylation been provided. increase the expression of a Stat1 target gene upon further treatment with IFN-g (Goh et al., 1999; Nuclear translocation There is currently no evidence Kovarik et al., 1998, 1999; Stoiber et al., 1999). for an in¯uence of serine phosphorylation on the STAT1 S727 phosphorylation is not required for nuclear translocation of STATs. C-terminal truncations transcriptional activity of the ISGF3. This complex removing the P(M)SP motif have no apparent eect on of STAT1-STAT2-IRF-9 proteins is assembled in the subcellular localization of STATs. IFN-a-treated cells. ISGF3 functions in U3A cells after complementation with either STAT1b that lacks Interdependence of serine and tyrosine phosphory- the entire C-terminus or with a S727A mutant lation The enhancement of transcription by serine (Bromberg et al., 1996; Muller et al., 1993). In spite phosphorylation could potentially be explained by a of this, a fraction of ISGF-3 complexes was recently positive in¯uence of serine phosphorylation on STAT shown to contain S727-phosphorylated STAT1 (Goh tyrosine phosphorylation, i.e. an increased anity for et al., 1999). The signi®cance of this ®nding is unclear. activated receptors and/or JAKs. However, available STAT3 S727A-mediated activation of a transfected information argues against this possibility. Reconstitu- IRF-1 promoter in response to IFN-a was reduced by tion of STAT1-de®cient U3A cells with the STAT1 about 50% (Wen et al., 1995). The S-A mutation S727A mutant had no eect on IFN-g-mediated caused a similar eect on the IRF-1 promoter in IL-6- tyrosine phosphorylation (Zhu et al., 1997). Moreover, treated hepatoma cells (Schuringa et al., 1999), but in a the serine kinase inhibitor H7 inhibited IFN-g- comparable situation wtSTAT3 and the S727A mutant mediated serine phosphorylation without a concomi- were reported to have similar activity in mobilizing the tant eect on tyrosine phosphorylation (Kovarik et al., haptoglobin acute phase promoter (Kim and Bau- 1998). mann, 1997). Therefore, the requirement for a As in the case of STAT1, tyrosine phosphorylation phosphorylated S727 may vary with the promoter of STAT3 is not enhanced by a phosphorylated S727. and/or cellular context. Support for this assumption In fact, serine phosphorylation may decrease the rate

Oncogene STAT serine phosphorylation T Decker and P Kovarik 2631 of tyrosine phosphorylation. Chung et al. (1997) report about a phospho-S727-enhanced protein asso- reported an increase in EGF-induced tyrosine phos- ciation. The human MCM5 protein, previously known phorylation of a transfected STAT3 S727A mutant for its role in DNA replication, was isolated by a compared to wt STAT3. This suggests that S727 biochemical approach using the STAT1 C-terminus as phosphorylation either inhibits tyrosine phosphoryla- an anity matrix and its association was reduced if the tion or that it increases tyrosine dephosphorylation. To matrix contained either the S727A or an L724A reconcile this ®nding with the enhancement of mutation (Zhang et al., 1998). Transcription factor transcription suggested by analysis of the S-A mutant, activity of STAT1 varied during the cell cycle and one has to assume that phospho-S727-dependent events correlated with the level of nuclear MCM5, which is (cofactor association?) overcompensate the negative high in G1 and low during S phase. Thus, MCM5 may eect on tyrosine phosphorylation. Moreover, since couple the transcription factor activity of STAT1 (and down-modulation of STAT3 tyrosine phosphorylation other STATs?) to the cell cycle, but its role concerning through a MEK/ERK pathway can also occur the enhancement caused by serine phosphorylation in independently of S727 phosphorylation (Sengupta et resting cells is unclear. Several other proteins co- al., 1998), the relative contribution of phospho-S727 to puri®ed with MCM5 on the STAT5 anity matrix and the regulation of STAT3 tyrosine phosphorylation in we may expect that the nature and signi®cance of these vivo is presently unclear. other proteins will soon be revealed. The inverse relationship, the impact of tyrosine Most, or even all STATs associate with the coactiva- phosphorylation on serine phosphorylation has also tors p300 and CBP and this interaction results in a been tested in STAT1-de®cient U3A cells after recon- moderate increase of transcription factor activity in stitution with STAT1 Y701F (Zhu et al., 1997). The transient transfection assays (Bhattacharya et al., 1996; authors concluded that constitutive or IFN-g-mediated Gingras et al., 1999; McDonald and Reich, 1999; serine phosphorylation occur without the need for a Paulson et al., 1999; P®tzner et al., 1998; Zhang et al., phosphorylated tyrosine. That the two phosphorylation 1996). Association of STAT1 with p300/CBP has been events can, in principle, be uncoupled is further carefully studied and no evidence for an in¯uence of supported by studies showing serine phosphorylation phospho-S727 was found (Zhang et al., 1996). The by surface stimuli that do not cause concomitant tyrosine impact of P(M)SP phosphorylation on protein interac- phosphorylation of STAT1 or STAT3 (Table 1; Ceresa tions with the STAT C-termini is therefore largely and Pessin, 1996; Gotoh et al., 1996; Kovarik et al., 1998, unexplained and awaits the further identi®cation of 1999; Kuroki and O'Flaherty, 1999; Lim and Cao, 1999; partner molecules. Ng and Cantrell, 1997). Moreover, STAT1 dimerized via a fused estrogen receptor hormone-binding domain Biological implications of STAT serine phosphory- moves to the nucleus and activates transcription, but lation A number of cell surface receptors are capable transcriptional activity is hampered by mutation of S727. of stimulating the serine phosphorylation of STATs This further suggests that a non-tyrosine-phosphory- without activating a STAT tyrosine kinase (Table 1). lated STAT becomes phosphorylated on S727 in vivo On the other hand, we are not aware of a receptor (Milocco et al., 1999). stimulating tyrosine phosphorylation, but not serine Studying the IFN-g-response in macrophages we phosphorylation of a P(M)SP-STAT. Therefore, a noted that about 90% of the serine-phosphorylated unidirectional exploitation of the possibility to un- STAT1 is found in the population of molecules that is couple tyrosine and serine phosphorylation predomi- also tyrosine phosphorylated (Kovarik et al., 1998). nates in biological systems. This suggests that serine This suggests a hitherto unde®ned supportive role of phosphorylation alone is a means of priming STATs the tyrosine phosphate on serine phosphorylation, or a for an altered transcriptional response once a second preferential serine-dephosphorylation of the non-tyr- stimulus causing tyrosine phosphorylation is received. osine-phosphorylated STAT1. In conclusion, there is From the current literature we would infer primed no mechanistic impediment for serine and tyrosine STATs to cause increased transcription in most cases, phosphorylation of STATs to occur independently. but the negative eect reported for STAT3 complicates However, we have reason to further consider a positive the issue and demands further clari®cation. in¯uence of tyrosine phosphorylation on serine phos- The signi®cance of STAT1 serine phosphorylation in phorylation in the context of an IFN-g response. vivo has been studied in U3A cells complemented with STAT1 S727A. The lack of growth inhibition or the Protein association It is reasonable to assume that the establishment of the antiviral state by IFN-g in these cells increased transcription factor activity of serine-phos- implies that STAT1 dimers with the S727A mutation are phorylated STATs, particularly STAT1, is due to biologically inactive (Bromberg et al., 1996; Horvath and protein interactions occurring preferentially after the Darnell, 1996). The critical importance of S727 phos- phosphorylation of S727. Transcriptional cofactors or phorylation for the biological impact of IFN-g is further coactivators (corepressors) are attractive candidates for supported by studies with mediators of in¯ammation such STAT-interacting proteins (reviewed in Decker (LPS, TNF-a), which cooperate with IFN-g in the and Kovarik, 1999). To this date there is a single activation of macrophages. These prime STAT1 for enhanced transcription factor activity through phosphorylation of S727 (Kovarik et al., 1998, 1999). Table 1 Signals uncoupling serine from tyrosine phosphorylation of IFN-a/b displays normal biological activity in U3A STATs cells expressing STAT1 S727A, suggesting that type I Ser727 of STAT1 LPS, UV, TNFa IFN employs predominantly the ISGF3 complex Ser727 of STAT3 LPS, UV, TNFa, GM-CSF, Steel factor, IL-3, (STAT1-STAT2-IRF-9) for the expression of critical sodium arsenide, anisomycin, hyperosmolarity target genes rather than the STAT1 dimer (Bromberg

Oncogene STAT serine phosphorylation T Decker and P Kovarik 2632 et al., 1996). As mentioned in a preceding section, the STATs by signals from the same cell surface receptor ISGF3 complex does not require the STAT1 C- can be routed through distinct signaling paths. This terminus and appears to employ the STAT2 transacti- has been documented for bacterial LPS or for UV- vating domain for transcriptional activity. irradiation which causes SB203580-sensitive S727 STAT3-de®cient cells are only beginning to become phosphorylation in case of STAT1, but SB203580- available and there are no published studies with STAT3 insensitive S727 phosphorylation in case of STAT3 S727A-reconstituted cells so far. Therefore, it is not yet (Kovarik et al., 1999; Lim and Cao, 1999). (3) possible to judge the importance of serine phosphoryla- Mitogen-activated protein kinase (MAPK) pathways tion for STAT3-dependent responses, e.g. to EGF/ {ERK, c-Jun kinases (JNK) or p38MAPK} play an PDGF, IL-6 family cytokines or to IL-10. In spite of important role in the phosphorylation of the PMSP this, recent data support the notion that STAT3 serine motif. By using pathway-speci®c inhibitors and/or by phosphorylation is important for biological responses. forced activation of MAPK pathways in transient STAT3 is constitutively phosphorylated in v-Src-trans- transfection, STATs 1, 3, 4 and 5 proved to be targets formed cells and participates in v-Src-mediated ®bro- of either growth factor regulated MAPK (ERK1/2) or blast transformation (Bromberg et al., 1998; Turkson et stress-regulated MAPK (p38, JNKs), or both (refer- al., 1998, 1999). Constitutively active STAT3 was shown ences provided below). (4) The P(M)SP motif may also to display oncogenic potential (Bromberg et al., 1999). be phosphorylated through pathways not involving Overexpressed STAT3 S727A exerted a dominant- MAPK. This probably applies for those phosphoryla- negative eect on v-Src-mediated transformation (Brom- tion events, which are sensitive to H7, but insensitive to berg et al., 1998). Furthermore, inhibition of STAT3 PD98059 and SB203580. One such example is STAT3 serine phosphorylation resulted in reduced transforma- S727 phosphorylation in response to IL-6 (Chung et tion by the v-src, but not the v-ras oncogene (Turkson et al., 1997; Schuringa et al., 1999). (5) In-vitro kinase al., 1999). Constitutive STAT3 serine phosphorylation activity is dicult to evaluate with regard to P(M)SP was also reported for B-cell-derived tumors (Frank et al., kinase activity in-vivo. Particularly in case of ERKs, 1997). Taken together, these ®ndings suggest an JNKs and p38MAPK, in-vitro phosphorylation of the important role for STAT3 and the phosphorylation of consensus PMSP motif is an expected result, but sheds S727 in cellular growth control. little light on the nature of the STAT kinase(s) acting Unlike STAT1 and STAT3, there are currently no in cells, unless complemented by additional data. The available data to estimate the importance of STAT4 or enzymatic parameters for the reaction between a STAT5 serine phosphorylation for their biological candidate kinase and a STAT substrate should be activity. Uncovering the relevance of serine phosphor- comparable to those obtained with known in-vivo ylation for cellular responses or the integrity of substrates and a tight correlation should exist between organisms is a major challenge for future research. activation of a candidate kinase and STAT phosphorylation in vivo. If these criteria are rigorously applied to all cellular responses causing STAT serine phosphor- Kinases and other signal transducers that participate in ylation, a question mark must often remain behind the the serine phosphorylation of STATs proposed STAT P(M)SP kinases. Inconsistent results Indications as to which signaling pathways target the have been obtained with dierent experimental proto- STAT P(M)SP motif were ®rst (and mostly) obtained cols concerning the phosphorylation of STATs by using more or less speci®c inhibitors of serine kinases. dierent MAPK in-vitro or with regard to the eects of These include the PD98059 inhibitor of MEK1/2 (also particular signaling pathways on serine phosphoryla- called MKK1/2), upstream kinases of the extracellular tion of, and transcriptional activation by STATS [e.g. signal-regulated kinases (ERK1/2), the p38MAPK Lim and Cao (1999) and Schuringa et al. (1999), or inhibitor SB203580, the PI3 kinase inhibitor Wortman- Chung et al. (1997) and Turkson et al. (1999), or nin, and H7, a compound with a broader spectrum of Schuringa et al. (1999) and Turkson et al. (1999)]. inhibitable serine kinases. As will be described below, pharmacological studies were in some cases comple- The ERK pathway Compelling evidence that ERKs, mented by experiments with dominant-negative, con- particularly ERK2, are STAT kinases or upstream of a stitutively active, or inhibitor-resistant kinase alleles, as STAT P(M)SP kinase has been provided for STAT3. well as with in-vitro phosphorylation assays (Table 2). Inhibition of phosphorylation by PD98059 is thought Before proceeding to a more detailed description of to be a quite speci®c criterion for an involvement of individual signaling routes we would like to point out the ERK pathway and this compound interferes with the following more general aspects of the relationship STAT3 serine phosphorylation by receptor tyrosine between signal transducers and STAT serine phosphor- kinases, IL-2, and lymphocyte antigen receptors ylation: (1) Serine phosphorylation of one STAT can (Chung et al., 1997; Ng and Cantrell, 1997; Su et al., occur through dierent signal transduction paths. For 1999). The general picture emerges that STAT3 is example, STAT1 S727 phosphorylation in macro- usually phosphorylated on S727 whenever ERK2 is phages is sensitive to SB203580 in stress of LPS active in a cell. ERKs phosphorylate STAT3 S727 in responses, but insensitive to this inhibitor in the IFN-g vitro with unknown reaction kinetics (Chung et al., response (Kovarik et al., 1999). Similarly, STAT3 S727 1997; Turkson et al., 1999). ERK2 and STAT3 can phosphorylation in response to EGF, PDGF, insulin, also be co-immunoprecipitated from cells (Jain et al., IL-2, BCR or TCR stimulation is sensitive to PD98059 1998). All facts considered ERK2 is a bona ®de STAT3 and, where tested, insensitive to H7, whereas the serine kinase. opposite is true for IL-6 or IFN-a (Chung et al., As mentioned before, ERK activity has been linked to 1997; Ng and Cantrell, 1997; Stephens et al., 1998; Su the negative regulation of STAT3 activity. We would et al., 1999). (2) S727 phosphorylation of dierent again like to stress the fact that this is due to S727-

Oncogene STAT serine phosphorylation T Decker and P Kovarik 2633 Table 2 Stimuli and signals causing serine phosphorylation of STATs Stat Phosphorylated Pathway Phosphorylating protein residue Stimulus involved kinase Evidence and reference STAT1 Ser727 IFNg, PDGF n.d. n.d. Wen et al., 1995 STAT1 Ser727 IFNg JAK2-dependent; partially purified, . overexpression of dominant-negative and Ras-independent unknown kinase constitutively active Ras . in vitro kinase assay (Zhu et al., 1997) STAT1 Ser727 IFNg, IFNa, LPS H7-sensitive n.d. (Kovarik et al, 1998) STAT1 Ser727 IFNg p38- and JNK1- n.d. . no activation of p38 and JNK1 by IFNg (macrophages) independent . lack of sensitivity to SB203580 . normal IFNg-induced phosphorylation of Ser727 in JNK1 7/7 cells (Kovarik et al., 1999) STAT1 Ser727 IFNa, IFNg p38 n.d. . weak activation of p38 by IFNs (HeLa cells) . inhibition of phosphorylation at Ser727 by overexpression of dominant-negative p38 . increased phosphorylation at Ser727 by overexpression of constitutively active MKK6 (Goh et al., 1999) STAT1 Ser727 combination of p38-dependent n.d. . activation of p38 by combination of IL-2 and IL-12 IL-2 and IL-12 . sensitivity to SB203580 (Gollob et al., 1999) STAT1 Ser727 LPS, UV, TNFa p38-dependent; p38 . sensitivity to SB203580 JNK1-, ERK1- . strong correlation of p38 activation with and JAK2- phosphorylation of Ser727 independent . absence of sensitivity to SB203580 in cells expressing SB203580-resistant p38 . resistance to PD98059 . phosphorylation of recombinant STAT1 by p38 (Kovarik et al., 1999) STAT1 Ser727 B cell receptor n.d. n.d. (Su et al., 1999) cross-linking STAT3 Ser727 EGF n.d. n.d. (Wen and Darnell Jr, 1997) STAT3 n.d. insulin n.d. n.d. (Ceresa and Pessin, 1996) STAT3 n.d. Steel factor, GM-CSF n.d. n.d. (Gotoh et al., 1996) STAT3 Ser727 IL-2, T cell MEK/ERK- n.d. . sensitivity to PD98059 receptor activation dependent (Ng and Cantrell, 1997) STAT3 Ser727 EGF MEK/ERK- ERK . sensitivity to PD98059 dependent . induction of phosphorylation at Ser727 by activation of B-Raf:ER (estrogen receptor-Raf fusion protein) . phosphorylation assays with recombinant STAT3 (Chung et al., 1997) STAT3 Ser727 IL-6 MEK/ERK- n.d. . no stimulation of ERK by IL-6 independent (Chung et al., 1997) STAT3 n.d. EGF MEK/ERK- n.d. . phosphorylation at serines by overexpression of dependent constitutive MEK1 . co-immunoprecipitation of ERK2 with Stat3 (Jain et al., 1998) STAT3 Ser727 insulin, OSM, LIF MEK/ERK- n.d. . sensitivity to PD98059 dependent (Stephens et al., 1998) STAT3 Ser727 combination of p38-dependent n.d. . activation of p38 by combination of IL-2 and IL-12 IL-2 and IL-12 . sensitivity to SB203580 (Gollob et al., 1999) STAT3 Ser727 GM-CSF, G-CSF MEK/ERK- n.d. . sensitivity to PD98059 complemet C5a dependent (Kuroki and O'Flaherty, 1999) STAT3 n.d./Ser727 IL-6 PKCd- PKCd . activation of PKCd by IL-6 dependent . co-immunoprecipitation of PKCd with STAT3 . increase of IL-6 induced phosphorylation at Ser727 by overexpression of PKCd . decrease of IL-6 induced phosphorylation at Ser727 by overexpression of kinase defective PKCd . kinase assays showing phosphorylation of recombinant STAT3 (Jain et al., 1999) STAT3 n.d./Ser727 UV, TNFa, JNK-dependent; JNK1 . correlation of phosphorylation at Ser727 with anisomycin p38-and ERK- activation of JNKs sodium arsenide independent . induction of phosphorylation by co-transfection with hyperosmolarity MEKK1 and JNK1 . resistance to SB203580 and PD98059 . kinase assays showing phosphorylation of recombinant STAT3 (Lim and Cao, 1999) STAT3 n.d./Ser727 activation by v-Src p38-and JNK- JNK and p38 . increased phosphorylation at Ser727 in v-Src transformation dependent transformed cells . correlation of phosphorylation at Ser727 with activation of JNK and p38 in v-Src transformed cells Continued

Oncogene STAT serine phosphorylation T Decker and P Kovarik 2634 Table 2 (Continued ) Stat Phosphorylated Pathway Phosphorylating protein residue Stimulus involved kinase Evidence and reference . kinase assays showing phosphorylation of recombinant STAT3 (Turkson et al., 1999) STAT3 Ser727 IL-6 SEK1-, Rac1- n.d. . correlation of IL-6 induced phosphorylation at Ser727 and Vav- with activation of SEK1, Rac1 and Vav dependent; . reduction of phosphorylation at Ser727 by MEK/ERK-, overexpression of dominant-negative forms of SEK1, p38- and JNK- Vav, Rac1 and MEKK independent . no activation of JNK by IL-6 . no activation of p38 by IL-6 . resistance to SB203580 (Schuringa et al., 1999) STAT4 n.d. IL-12, IFNa n.d. n.d. (Cho et al., 1996) STAT4 Ser721 IL-12 p38-dependent; . weak activation of p38 by IL-12 MEK/ERK- and . no activation of ERK and JNK by IL-12 JNK-independent . induction of phosphorylation at Ser721 by co-transfection with MKK6 and p38d (Visconti et al., submitted) STAT5a Ser725 unregulated event MEK/ERK- n.d. . sensitivity to PD98059 dependent (Yamashita et al., 1998) STAT5b Ser730 prolactin MEK/ERK- n.d. . resistance to PD98059 independent n.d. (Yamashita et al., 1998) n.d.=not determined

dependent and independent down-regulation of tyrosine 2 was insensitive to the inhibition with either PD98059 or phosphorylation and does not indicate a negative eect Wortmannin, suggesting that MEK-dependent as well as of phospho-S727 per se on STAT3-mediated transcrip- PI3 kinase and MEK-independent pathways control, tion. To our knowledge it has not been tested whether the respectively, constitutive and induced S725 and S730 induction of the SOCS proteins, STAT-induced feed- phosphorylation of the STAT5 isoforms (Beadling et al., back inhibitiors of JAKs (Hilton, 1999), depends on 1996; Kirken et al., 1997a; Yamashita et al., 1998; ERK activity. This might explain MAPK-dependent reviewed in Grimley et al., 1999). down-regulation of STAT tyrosine phosphorylation. ERK2 was also connected to serine phosphorylation The JNK pathway The only STAT linked to the JNK of STAT1. The enzyme co-immunoprecipitated with pathway until now is STAT3. Similar to STAT1, STAT1 and a dominant-negative ERK allele decreased STAT3 S727 phosphorylation is caused by stressing transcription in response to IFN-a (David et al., 1995). cells with UV irradiation or hyperosmolarity, or by In case of IFN-g, a dominant-negative allele of the Pyk2 exposing them to in¯ammatory signals like TNF-a and tyrosine kinase suppressed ERK activation and at the LPS. Unlike STAT1, however (see below), the UV- same time the S727 phosphorylation of STAT1 activated signaling path for STAT3 phosphorylation is (Takaoka et al., 1999). While these data support a role resistant to SB203580 (Lim and Cao, 1999). Transfec- for ERKs as STAT1 serine kinases, several lines of tion of cells with the JNK kinase kinase MEKK1 evidence argue against a general validity of the concept. caused S727 phosphorylation of STAT3 and produced First, particularly IFN-g is a poor activator of ERKs in-vitro STAT3 S727 kinase activity in JNK-immuno- compared to e.g. ligands of tyrosine kinase receptors in precipitates, a ®nding consistent with the in-vitro most cell types and the activation of ERKs in dierent phosphorylation of STAT3 by puri®ed JNK (Turkson situations does not correspond to the level of STAT1 et al., 1999), but inconsistent with yet another study serine phosphorylation (Kovarik et al., 1998). Second, ®nding very little STAT3 kinase activity in anisomycin- the MEK inhibitor PD98059 was reported to have no or treated cell extracts which contain high levels of JNK only a partial eect on IFN-induced STAT1 S727 activity (Schuringa et al., 1999). Similar to the ERK phosphorylation (Goh et al., 1999; Ala-Aho et al., pathway, activation of a MEKK1-MKK7-JNK signal- 2000). Third, up- or downregulation of ERK activity ing path caused inhibition of STAT3 tyrosine phos- had no eect on STAT1 serine phosphorylation in the phorylation and, therefore, an inhibition of gene IFN-g response (Zhu et al., 1997). Fourth, STAT1 is a transcription (Lim and Cao, 1999). STAT3 S727 very poor in-vitro substrate for ERKs (Chung et al., phosphorylation in response to IL-6 was inhibited by 1997); Kovarik P and Decker T (2000), unpublished dominant-negative alleles of the Vav guanine nucleo- results). These ®ndings present considerable evidence tide exchange factor, the small G-protein Rac-1 and against ERKs being STAT1 S727 kinases. the second putative JNK kinase MKK4 (Schuringa et STAT5, particularly the STAT5a isoform, was found al., 1999). Surprisingly however, the authors of this to be constitutively phosphorylated on serine in Cos report found very little JNK activation by IL-6 and cells, Nb2 lymphocytes and HC11 mammary gland concluded that despite a requirement for MKK4, JNK epithelial cells (Kirken et al., 1997a,b; Wartmann et al., was not the relevant STAT3 kinase. It is also puzzling 1996). Constitutive phosphorylation of STAT5a S725, or that the study by Schuringa et al. (1999) implies a STAT5b S730 was reduced by treating cells with stimulatory role for MKK4 regarding STAT3-depen- PD98059 (Yamashita et al., 1998). In marked contrast, dent transcriptional responses to IL-6, whereas its the enhanced serine phosphorylation of STAT5a and presumptive functional analogue MKK7 inhibits STAT5b observed after stimulation with prolactin or IL- STAT3 tyrosine phosphorylation (Lim and Cao, 1999).

Oncogene STAT serine phosphorylation T Decker and P Kovarik 2635 JNK activity was also suggested to be required for ylation of STAT1 is unlikely to aect the transcrip- STAT3-dependent ®broblast transformation. As in the tional activity of ISGF3. Goh et al. (1999) found a case of IL-6, dominant-negative MKK4 inhibited requirement for p38MAPK also in ISGF3 assembly, STAT3 transcriptional activity in cells expressing v-Src. but Uddin et al. (1999) did not con®rm this result. The Ras and Rac-1 GTPases as well as a member of the Therefore, the precise role of p38MAPK in transcrip- mixed-lineage kinase family were placed upstream of tional responses to IFN-a remains an open question. MKK4-JNK in this situation (Turkson et al., 1999). As in the case of JNKs, the p38MAPK pathway was suggested to be important for STAT3 activity in the The p38MAPK pathway STAT serine phosphorylation context of v-Src-mediated cell transformation. v-Src- caused by a variety of stimuli is sensitive to the inhibition induced phosphorylation of STAT3 S727, the transcrip- of p38MAPK. An involvement of the kinase is further tional activity of STAT3, and cell transformation were suggested by transfection experiments employing domi- inhibited by SB202190, another speci®c p38MAPK nant-negative or constitutively active p38MAPK kinase inhibitor (Turkson et al., 1999). Like the MKK4-JNK (MKK6, Goh et al., 1999; Visconti et al., submitted), or module, the MKK6-p38MAPK module was suggested to an inhibitor-resistant p38MAPK (Kovarik et al., 1999). localize in a signaling path downstream of the Ras and p38MAPK phosphorylates S727 of Stat1 and Stat3 in Rac-1 GTPases, and of (a) member(s) of the mixed- vitro (Kovarik et al., 1999; Turkson et al., 1999), but, is lineage family of MAPK kinase kinases. unclear whether this applies to cells as well. STAT4 is phosphorylated on S721 in cells trans- STAT1 S727 phosphorylation in response to cellular fected with a constitutively active MKK6, suggesting it stress (UV irradiation) or in¯ammatory signals (LPS, too can be a target of the p38MAPK pathway TNF-a) is inhibited by SB203580 (Kovarik et al., 1999). (Visconti et al., 1999, submitted). IL-12 activates Cells expressing an SB203580-resistant p38MAPK dis- p38MAPK, at a rather low level (Gollob et al., 1999; play SB203580-resistant UV-induced S727 phosphoryla- Visconti et al., 1999, submitted) This may suce to tion, which clearly positions p38MAPK upstream of mediate the phosphorylation of Stat4 S721 seen after STAT1 in situations of stress or in¯ammation. The IL-12 treatment, but alternative signaling routes cannot kinase speci®cally phosphorylates STAT1 S727 in vitro, be ruled out. however, with a signi®cantly lower rate of turn-over Finally, it should be noted that the p38MAPK compared to the known in-vivo substrate MAPKAP-K2. pathway might also have an impact on STAT tyrosine Activation of p38MAPK by signals of stress or phosphorylation. Hyperosmolarity treatment of certain in¯ammation correlates well with STAT1 S727 phos- cell types leads to the phosphorylation of STATs 1 and phorylation, but the kinase is activated about 5 ± 10 min 3 on tyrosine and this eect can be inhibited by earlier than the STAT. This may indicate intermediate SB203580 or by dominant-negative p38MAPK or steps or a need for relocation of p38MAPK from the MKK6 (Bode et al., 1999). Activation of JAKs does nucleus to the cytoplasm. In pursuit of the former not appear to be aected and the question arises how a hypothesis, cells de®cient for the p38MAPK substrate p38MAPK-dependent pathway causing tyrosine phos- kinase MAPKAP-K2 were tested for STAT1 S727 phorylation of STATs might be shaped. phosphorylation in response to UV and no signi®cant dierence to wt cells was found (Kovarik et al., 1999). Protein kinase C delta In search of an H7-sensitive, While SB203580 also clearly inhibits the synergistic IL-6 activated STAT3 serine kinase, Jain and cow- S727 phosphorylation of STAT1 by IL-12 and IL-2 orkers came upon PKCd (Jain et al., 1999). The and there is a good correlation to p38MAPK enzyme associated with STAT3 in an IL-6-dependent activation in this situation (Gollob et al., 1999), there fashion, phosphorylated STAT3 in-vitro, and a domi- is a controversy with regard to a role for p38MAPK in nant-negative form blocked S727 phosphorylation in IFN-g-mediated STAT1 S727 phosphorylation. Study- response to IL-6. Like MAPK pathways, PKCd was ing macrophages we did not detect an eect of reported to have a negative impact on STAT3 DNA- SB203580 on S727 phosphorylation in response to binding (tyrosine phosphorylation?) and on STAT3 IFN-g and p38MAPK activation by the cytokine in transcriptional activity. To link these data with other macrophages and some other cell types was insignif- studies on the same subject, PKCd would have to be icant (Kovarik et al., 1999). By contrast, Goh et al. connected to the Vav-Rac-MKK4 pathway proposed (1999) placed p38MAPK upstream of STAT1 in IFN- by Schuringa et al. (1999). g-signaling, based on a twofold activation by IFN-g in HeLa cells and a rather small inhibitory eect of mTOR kinase The IL-6 family cytokine CNTF causes SB203580 on STAT1 phosphorylation. While we STAT3 activation in neuroblastoma cells. Yokogami et hypothesize the existence of a p38MAPK-independent al. (2000) reported that the drug rapamycin inhibits STAT1 kinase in the IFN-g response based on our CNTF-induced STAT3 S727 phosphorylation and macrophage results, we do not rule out the existence of Stat3-mediated reportergene transcription to the same alternative pathways in other cell types. extent as co-transfection of the STAT3 S-A mutant. Two groups independently reported the activation of The mTor kinase, a phosphatidylinositol kinase-related the p38MAPK pathway by type I IFN (IFN-a/b; Goh enzyme, is sensitive to rapamycin treatment. Immuno- et al., 1999; Uddin et al., 1999). In the presence of precipitated mTOR phosphorylated a STAT3 substrate SB203580 STAT1 serine phosphorylation in response on S727 in vitro. Based on these results, the authors of to IFN-a was inhibited and a reduced IFN-a- this study suggested mTOR to be a CNTF-responsive stimulated transcriptional activity of an ISGF3 STAT3 serine kinase. (STAT1-STAT2-IRF-9 complex)-dependent reporter gene was noted (Goh et al., 1999; Uddin et al., Protein kinase R (PKR) STAT1-dependent transcrip- 1999). For reasons explained above, serine phosphor- tion in IFN-g-treated ®broblasts is strongly reduced

Oncogene STAT serine phosphorylation T Decker and P Kovarik 2636 upon targeted disruption of the pkr gene. Consistent analysis of conditions under which STAT serine with this result, Ramana et al. (2000) reported PKR- phosphorylation at the C-terminal P(M)SP motif de®ciency to cause the virtual absence of S727 occurs. Therefore, considerable information has been phosphorylation in response to IFN-g. The position gathered about biological stimuli that cause serine of PKR in an IFN-g-responsive signaling path phosphorylation. While it is clear at least in some cases targeting STAT1 is unknown. Based on earlier that serine phosphorylation in¯uences the transcrip- ®ndings, a direct enzymatic activity of PKR on STAT1 tional activity of STATs, the mechanisms by which this appears very unlikely (Wong et al., 1997). occurs, as well as the biological impact it causes, are unclear in most situations. Signal transduction pathways are beginning to emerge, but much more needs to Dephosphorylation of STAT serine residues be learned about the kinases phosphorylating STATs Phosphatase 2A treatment in-vitro was shown to reduce on serine in dierent situations. This review is a the SDS ± PAGE mobility shift of STAT4 which is at snapshot documenting the current situation in a least partially due to the phosphorylation of S727 (Cho dynamic process aimed at providing more de®nitive et al., 1996). Moreover, treatment of cells with answers about causes and eects of STAT serine inhibitors of PP2A causes STAT3 S727 phosphoryla- phosphorylation. tion (Woetmann et al., 1999). If physiologically relevant these results suggest that permanent PP2A activity be required to keep STATs dephosphorylated on S727 and that an increase in phosphorylation demands the inhibition of this phosphatase. However, our knowledge of STAT serine dephosphorylation is at a very preliminary stage. Acknowledgments We thank colleagues for providing preprints of manu- scripts for communication of unpublished results. We are Concluding remarks grateful to Manuela Baccarini for critical reading of the manuscript. Research in our laboratory is funded by the The generation of phosphospeci®c antisera recognizing Austrian research foundation (FWF) through grants serine-phosphorylated STATs has greatly facilitated the P13640-GEN and P-12946-GEN to T Decker.

References

Ala-Aho R, Johansson N, Grenman R, Fusenig NE, Lopez- Darnell Jr JE. (1997). Proc. Natl. Acad. Sci. USA, 94, Otin C and Kahari VM. (2000). Oncogene, 19, 248 ± 257. 11767 ± 11769. Barillas-Mury C, Han YS, Seeley D and Kafatos FC. (1999). DavidM,PetricoinER,BenjaminC,PineR,WeberMJand EMBO J., 18, 959 ± 967. Larner AC. (1995). Science, 269, 1721 ± 1723. Beadling C, Ng J, Babbage JW and Cantrell DA. (1996). Decker T and Kovarik P. (1999). Cell. Mol. Life Sci., 55, EMBO J., 15, 1902 ± 1913. 1535 ± 1546. Bhattacharya S, Eckner R, Grossman S, Oldread E, Arany Eilers A, Georgellis D, Klose B, Schindler C, Ziemicki A, Z, D'Andrea A and Livingston DM. (1996). Nature, 383, Harpur AG, Wilks AF and Decker T. (1995). Mol. Cell. 344 ± 347. Biol., 15, 3579 ± 3586. Bode JG, Gatsios P, Ludwig S, Rapp UR, Haussinger D, Frank DA, Mahajan S and Ritz J. (1997). J. Clin. Invest., Heinrich PC and Graeve L. (1999). J. Biol. Chem., 274, 100, 3140 ± 3148. 30222 ± 30227. Gingras S, Simard J, Groner B and P®tzner E. (1999). BoultonTG,ZhongZ,WenZ,DarnellJrJE,StahlNand Nucleic Acids Res., 27, 2722 ± 2729. Yancopoulos GD. (1995). Proc. Natl. Acad. Sci. USA, 92, Goh KC, Haque SJ and Williams BRG. (1999). EMBO J., 6915 ± 6919. 18, 5601 ± 5608. Bromberg JF, Horvath CM, Besser D, Lathem WW and Gollob JA, Schnipper CP, Murphy EA, Ritz J and Frank Darnell Jr JE. (1998). Mol. Cell. Biol., 18, 2553 ± 2558. DA. (1999). J. Immunol., 162, 4472 ± 4481. Bromberg JF, Horvath CM, Wen Z, Schreiber RD and Gonzalez FA, Raden DL and Davis RJ. (1991). J. Biol. Darnell Jr JE. (1996). Proc. Natl. Acad. Sci. USA, 93, Chem., 266, 22159 ± 22163. 7673 ± 7678. GotohA,TakahiraH,MantelC,LitzJacksonS,BoswellHS Bromberg JF, Wrzeszczynska MH, Devgan G, Zhao Y, and Broxmeyer HE. (1996). Blood, 88, 138 ± 145. Pestell RG, Albanese C and Darnell Jr JE. (1999). Cell, 98, Grimley PM, Dong F and Rui H. (1999). Cytokine Growth 295 ± 303. Factor Rev., 10, 131 ± 157. Caldenhoven E, van Dijk TB, Solari R, Armstrong J, Hilton DJ. (1999). Cell. Mol. Life Sci., 55, 1568 ± 1577. Raaijmakers JAM, Lammers JWJ, Koenderman L and Horvath CM and Darnell Jr JE. (1996). J. Virol., 70, 647 ± de Groot RP. (1996). J. Biol. Chem., 271, 13221 ± 13227. 650. Ceresa BP and Pessin JE. (1996). J. Biol. Chem., 271, 12121 ± Hou XS, Melnick MB and Perrimon N. (1996). Cell, 84, 12124. 411 ± 419. Cho SS, Bacon CM, Sudarshan C, Rees RC, Finbloom D, Jain N, Zhang T, Fong SL, Lim CP and Cao X. (1998). Pine R and O'Shea JJ. (1996). J. Immunol., 157, 4781 ± Oncogene, 17, 3157 ± 3167. 4789. Jain N, Zhang T, Kee WH, Li W and Cao X. (1999). J. Biol. Chung J, Uchida E, Grammar TC and Blenis J. (1997). Mol. Chem., 274, 24392 ± 24400. Cell. Biol., 17, 6508 ± 6516. Kawata T, Shevchenko A, Fukuzawa M, Jermyn KA, Totty Clark-Lewis I, Sanghera JS and Pelech SL. (1991). J. Biol. NF, Zhukovskaya NV, Sterling AE, Mann M and Chem., 266, 15180 ± 15184. Williams JG. (1997). Cell, 89, 909 ± 916. Copeland NG, Gilbert DJ, Schindler C, Zhong Z, Wen Z, Kim H and Baumann H. (1997). J. Biol. Chem., 272, 14571 ± DarnellJrJE,MuiALF,MiyajimaA,QuelleFW,IhleJN 14579. and Jenkins NA. (1995). Genomics, 29, 225 ± 228.

Oncogene STAT serine phosphorylation T Decker and P Kovarik 2637 Kirken RA, Malabarba MG, Xu J, Dasilva L, Erwin RA, Stephens JM, Lumpkin SJ and Fishman JB. (1998). J. Biol. Liu XW, Hennighausen L, Rui H and Farrar WL. (1997a). Chem., 273, 31408 ± 31416. J. Biol. Chem., 272, 15459 ± 15465. Stoiber D, Kovarik P, Cohney S, Johnston JA, Steinlein P Kirken RA, Malabarba MG, Xu J, Liu XW, Farrar WL, and Decker T. (1999). J. Immunol., 163, 2640 ± 2647. Hennighausen L, Larner AC, Grimley PM and Rui H. Su L, Rickert RC and David M. (1999). J. Biol. Chem., 274, (1997b). J. Biol. Chem., 272, 14098 ± 14103. 31770 ± 31774. Kovarik P, Stoiber D, Eyers PA, Menghini A, Neininger A, Takaoka A, Tanaka N, Mitani Y, Miyazaki T, Fujii H, Sato Gaestel M, Cohen P and Decker T. (1999). Proc. Natl. M, Kovarik P, Decker T, Schlessinger J and Taniguchi T. Acad. Sci. USA, 96, 13956 ± 13961. (1999). EMBO J., 18, 2480 ± 2488. Kovarik P, Stoiber D, Novy M and Decker T. (1998). EMBO Turkson J, Bowman T, Adnane J, Zhang Y, Djeu JY, J., 17, 3660 ± 3668. Sekharam M, Frank DA, Holzman LB, Wu J, Sebti S and Kuroki M and O'Flaherty JT. (1999). Biochem. J., 341, 691 ± Jove R. (1999). Mol. Cell. Biol., 19, 7519 ± 7528. 696. Turkson J, Bowman T, Garcia R, Caldenhoven E, De Groot Lim CP and Cao X. (1999). J. Biol. Chem., 274, 31055 ± RP and Jove R. (1998). Mol. Cell. Biol., 18, 2545 ± 2552. 31061. Uddin S, Majchrzak B, Woodson J, Arunkumar P, Alsayed Lutticken C, Coer P, Yuan J, Schwartz C, Caldenhoven E, Y, Pine R, Young PR, Fish EN and Platanias LC. (1999). Schindler C, Kruijer W, Heinrich PC and Horn F. (1995). J. Biol. Chem., 274, 30127 ± 30131. FEBS Lett., 360, 137 ± 143. Visconti R, Gadinea M, Chiariello M, Chen EH, Gutkind S McDonald C and Reich NC. (1999). J. Interferon Cytokine and O'Shea JJ. (2000). Submitted. Res., 19, 711 ± 722. Wartmann M, Cella N, Hofer P, Groner B, Liu X, Milocco LH, Haslam JA, Rosen J and Seidel HM. (1999). Hennighausen L and Hynes NE. (1996). J. Biol. Chem., Mol. Cell. Biol., 19, 2913 ± 2920. 271, 31863 ± 31868. Mui AL-F, Wakao H, Kinoshita T, Kitamura T and Wen Z, Zhong Z and Darnell Jr JE. (1995). Cell, 82, 241 ± Miyajima A. (1996). EMBO J., 15, 2425 ± 2431. 250. MullerM,LaxtonC,BriscoeJ,SchindlerC,ImprotaT, Wen ZL and Darnell Jr JE. (1997). Nucleic Acids Res., 25, Darnell Jr JE, Stark GR and Kerr IM. (1993). EMBO J., 2062 ± 2067. 12, 4221 ± 4228. Woetmann A, Nielsen M, Christensen ST, Brockdor J, Ng J and Cantrell D. (1997). J. Biol. Chem., 272, 24542 ± KaltoftK,EngelAM,SkovS,BrenderC,GeislerC, 24549. Svejgaard A, Rygaard J, Leick V and Odum N. (1999). Park C, Lecomte MJ and Schindler C. (1999). Nucleic Acids Proc. Natl. Acad. Sci. USA, 96, 10620 ± 10625. Res., 27, 4191 ± 4199. Wong AH, Tam NW, Yang YL, Cuddihy AR, Li S, Paulson M, Pisharody S, Pan L, Guadagno S, Mui L and Kirchho S, Hauser H, Decker T and Koromilas AE. Levy DE. (1999). J. Biol. Chem., 274, 25343 ± 25349. (1997). EMBO J., 16, 1291 ± 1304. P®tzner E, Jahne R, Wissler M, Stoecklin E and Groner B. Yamashita H, Xu J, Erwin RA, Farrar WL, Kirken RA and (1998). Mol. Endocrinol., 12, 1582 ± 1593. Rui H. (1998). J. Biol. Chem., 273, 30218 ± 30224. Ramana CV, Grammatikakis N, Chernov M, Nguyen H, Yan R, Small S, Desplan C, Dearolf CR and Darnell Jr JE. Goh KC, Williams BR and Stark GR. (2000). EMBO J., (1996). Cell, 84, 421 ± 430. 19, 263 ± 272. Yokogami K, Wakisaka S, Avruch J and Reeves SA. (2000). Sasse J, Hemmann U, Schwartz C, Schniertshauer U, Heesel Curr. Biol., 10, 47 ± 50. B, Landgraf C, Schneider-Mergener J, Heinrich PC and Zhang JJ, Vinkemeyer U, Gu W, Chakravarti D, Horvath Horn F. (1997). Mol. Cell. Biol., 17, 4677 ± 4686. CM and Darnell Jr JE. (1996). Proc. Natl. Acad. Sci. USA, Schindler C and Darnell Jr JE. (1995). Annu.Rev.Biochem., 93, 15092 ± 15096. 64, 621 ± 651. Zhang JJ, Zhao Y, Chait BT, Lathem WW, Ritzi M, Schuringa J-J, Jonk LJC, Dokter WHA, Vellenga E and Knippers R and Darnell Jr JE. (1998). EMBO J., 17, Kruijer W. (2000). Biochem. J., in press. 6963 ± 6971. Sengupta TK, Talbot ES, Scherle PA and Ivashkiv LB. Zhang X, Blenis J, Li HC, Schindler C and Chen Kiang S. (1998). Proc. Natl. Acad. Sci. USA, 95, 11107 ± 11112. (1995). Science, 267, 1990 ± 1994. ShuaiK,StarkGR,KerrIMandDarnellJrJE.(1993). ZhuX,WenZL,XuLZandDarnellJrJE.(1997).Mol. Cell. Science, 261, 1744 ± 1746. Biol., 17, 6618 ± 6623.

Oncogene