Src Directly Tyrosine-Phosphorylates STAT5 on Its Activation Site and Is Involved in Erythropoietin-Induced Signaling Pathway

Src directly tyrosine-phosphorylates STAT5 on its activation site and is involved in erythropoietin-induced signaling pathway

Yuichi Okutani1, Akira Kitanaka1, Terukazu Tanaka*,2, Hiroshi Kamano6, Hiroaki Ohnishi3, Yoshitsugu Kubota4, Toshihiko Ishida1 and Jiro Takahara5

1First Department of Internal Medicine, Kagawa Medical University, Kagawa 761-0793, Japan; 2Environmental Health Sciences, Kagawa Medical University, Kagawa 761-0793, Japan; 3Laboratory Medicine, Kagawa Medical University, Kagawa 761-0793, Japan; 4Transfusion Medicine, Kagawa Medical University, Kagawa 761-0793, Japan; 5Kagawa Medical University, Kagawa 761- 0793, Japan; 6Health Science Center, Kagawa University, Kagawa 760-8521, Japan

Signal transducers and activators of transcription Growth and dierentiation of hematopoietic cells are (STAT) proteins are transcription factors activated by regulated by the interaction of cell surface receptors phosphorylation on tyrosine residues after cytokine with their speci®c cytokines or growth factors. While stimulation. In erythropoietin receptor (EPOR)-mediated some of these receptors transmit signals via their signaling, STAT5 is tyrosine-phosphorylated by EPO intrinsic protein tyrosine kinase (PTK) activity, others stimulation. Although Janus Kinase 2 (JAK2) is reported use cytoplasmic non-receptor PTK for mediating to play a crucial role in EPO-induced activation of signals (van der Geer et al., 1994). The binding of STAT5, it is unclear whether JAK2 alone can tyrosine- erythropoietin (EPO) to an erythropoietin receptor phosphorylate STAT5 after EPO stimulation. Several (EPOR) which does not contain any intrinsic PTK studies indicate that STAT activation is caused by activity induces rapid and transient tyrosine phosphor- members of other families of protein tyrosine kinases ylation of intracellular proteins (Constantinescu et al., such as the Src family. We previously reported that 1999). reduction of Src by induction of antisense src RNA Signal transducers and activators of transcription expression suppressed EPO-promoted erythroid dier- (STAT) proteins are transcription factors that play entiation in K562 cells. In the present study, we explored key roles in intracellular signaling after stimulation the function of Src downstream of the EPOR-initiated with various cytokines and growth factors (Darnell et signaling. Reduction of Src diminished tyrosine phos- al., 1994; O'Shea, 1997; Coer et al., 2000). Among phorylation of STAT5 in K562 cells regardless of EPO the seven members of the STAT family, STAT5 is treatment. The tyrosine phosphorylation level of STAT5 known to be activated by a wide variety of cytokines, induced by EPO in F-36P cells was reduced in the including EPO (Wakao et al., 1995; O'Shea, 1997). presence of PP1 or PP2 selective Src inhibitor. In Two closely related STAT5 proteins, STAT5A and addition, the expression of dominant negative Src in F- STAT5B, have been identi®ed with 96% homology at 36P cells reduced the tyrosine phosphorylation of the amino acid sequence level (Liu et al., 1995; Mui et STAT5. When Src and STAT5 were co-expressed in al., 1995). Although a previous study showed that COS7 cells, tyrosine phosphorylation of STAT5 was hematopoietic development in the myeloid and observed, and tyrosine residue 694 (Tyr 694) of STAT5A erythroid lineages was not signi®cantly impaired in was identi®ed as the major phosphorylation site by Src. adult mice de®cient in both STAT5A and STAT5B In vitro kinase assay revealed that GST-STAT5 fusion genes (Teglund et al., 1998), analyses of these mice protein with the conserved C-terminal, but not the C- during fetal development and experiments using cell terminal-truncated mutant which lacks Tyr 694, was lines have suggested that STAT5 plays important roles tyrosine-phosphorylated by Src. Src can thus directly in the proliferation and dierentiation of hematopoie- tyrosine-phosphorylate the activation site of STAT5 (Tyr tic cells, including erythroid progenitors (Iwatsuki et 694 in STAT5A), and Src may contribute to EPO- al., 1997; Nosaka et al., 1999; Socolovsky et al., induced signal transduction via STAT5. Oncogene 1999). (2001) 20, 6643 ± 6650. One or more members of Janus Kinase (JAK) family PTKs have been reported to be activated by a wide Keywords: Src; STAT5; erythropoietin; tyrosine phos- range of cytokines (Ihle, 1995). Although JAK2 has phorylation; antisense been reported to play an important role in the EPO- induced signal transduction which activates STAT5 (Ihle, 1995; Coer et al., 2000), it is still an open question whether JAK2 is the only PTK responsible for the tyrosine phosphorylation of STAT5 after *Correspondence: T Tanaka; E-mail: [email protected] Received 27 December 2000; revised 14 June 2001; accepted 5 July stimulation with EPO. Several reports have recently 2001 suggested that other families of PTKs are involved in Src tyrosine-phosphorylates STAT5 in EPOR signaling Y Okutani et al 6644 a STAT activation. Overexpression of v-Src or v-Abl has been shown to activate various STAT proteins (Danial et al., 1995; Yu et al., 1995; Cao et al., 1996). In a K562 erythroleukemia cell line derived from a patient with CML in blastic crisis, STAT5 was shown to be constitutively activated and to play a crucial role in cellular transformation by the bcr-abl oncogene (Carlesso et al., 1996; de Groot et al., 1999). Furthermore, it was reported that STATs were activated by Src PTK after stimulation with inter- leukin-3 or epidermal growth factor (Chaturvedi et al., 1998; Olayioye et al., 1999). Src, a prototype molecule of Src family PTKs, is expressed in a variety of hematopoietic cells (Kitanaka et al., 1994; Thomas and Brugge, 1997). We previously showed that reduction of Src induced by the antisense src RNA expression suppressed EPO-induced hemo- globin synthesis and glycophorin A expression in K562 cells (Kitanaka et al., 1994). The mechanism by which Src contributes to the EPOR-mediated signaling cascade has not yet been clari®ed. In the present study, we investigated whether Src is one of the components of a signaling cascade that leads to b tyrosine phosphorylation of STAT5 in response to stimulation with EPO. The amount of Src in K562-ASRC cells (see the legend to Figure 1) can be eciently suppressed by treatment with dexamethasone (Dexa) (Figure 1a), as reported previously (Kitanaka et al., 1994). The signaling mechanism downstream of Src in K562- ASRC cells after EPO stimulation was investigated by examining its possible role in tyrosine phosphorylation of STAT5. K562-ASRC cells were stimulated with EPO with or without Dexa treatment. Although constitutive tyrosine phosphorylation of STAT5 has been observed in K562 cells (Carlesso et al., 1996; de Groot et al., 1999), incubation of the cells in serum-free medium for 24 h reduced the baseline level of STAT5 tyrosine phosphorylation (data not shown). Under these experimental conditions, we could detect an increase in the tyrosine phosphorylation of STAT5 in response to EPO treatment (Figure 1b). Down- regulation of Src diminished the increase in the tyrosine phosphorylation of STAT5 caused by EPO stimulation (Figure 1b).

Immunoprecipitated proteins were separated by 7.5% SDS ± PAGE and transferred to a PVDF ®lter. The ®lter was incubated with the anti-Src antibody. The antibody binding was visualized by means of an enhanced chemiluminescence detection system (Amersham). The positions of Src, immunoglobulin heavy chain Figure 1 Induction of antisense src RNA expression suppresses (IgH) and molecular mass markers (in kD) are indicated. (b) tyrosine phosphorylation of STAT5 induced by EPO. Establish- Serum-starved K562-ASRC cells were incubated with Dexa for ment of K562 cells transfected with an antisense src RNA 24 h. The cells were then incubated for 5 min in the presence or expression vector in which antisense src RNA expression is absence of 30 U/ml recombinant human EPO (Kirin Brewery) promoted by Dexa (K562-ASRC cells) was previously described and lysed in the lysis buer. The cell lysates were subjected to (Kitanaka et al., 1994). (a) K562-ASRC cells were incubated with immunoprecipitation with a monoclonal anti-STAT5 antibody or without 2 mM of Dexa for 24 h. Cell lysates were prepared as (Santa Cruz), followed by Western blotting using an anti- described previously (Kitanaka et al., 1998). Cell lysates contain- phosphotyrosine antibody (a-pTyr, upper panel; Santa Cruz). ing equal amounts of proteins were subjected to immunoprecipi- The ®lter was stripped and reprobed with the anti-STAT5 tation with a monoclonal anti-Src antibody (Oncogene Science). antibody (lower panel)

Oncogene Src tyrosine-phosphorylates STAT5 in EPOR signaling Y Okutani et al 6645 a To further investigate the role of Src in EPO-induced tyrosine phosphorylation of STAT5, we took advan- tage of three compounds, the Src PTK inhibitors PP1 and PP2 and the JAK PTK inhibitor AG490. PP1 and PP2 preferentially inhibit Src family PTKs without inhibiting JAK family PTKs, while AG 490 selectively inhibits JAKs (Hanke et al., 1996; Meydan et al., 1996; Figure 2 and data not shown). We ®rst examined whether inhibition of Src PTK activity aects the EPO- induced tyrosine phosphorylation of STAT5 in the human erythroleukemia cell line F-36P, which can be induced to dierentiate into the erythroid lineage in response to EPO (Chiba et al., 1991). As shown in Figure 2a, both PP1 and PP2 dose-dependently reduced the tyrosine phosphorylation of STAT5 as compared with that induced by EPO without inhibitors, whereas PP3, an inactive analog, had no eect. In contrast, both PP1 and PP2 had little eect on the tyrosine phosphorylation of JAK2. These results suggest that Src PTK activity is required for EPO- induced tyrosine phosphorylation of STAT5 and that the PTK activity of JAK2 enhanced by its tyrosine phosphorylation is not adequate for maximal phosphorylation of STAT5. We then examined whether inhibition of JAK PTKs by AG490 aects the extent of tyrosine phosphorylation of STAT5 in F-36P cells. Although the enhancement of JAK2 tyrosine phos- b phorylation was completely inhibited by treatment with 100 mM of AG490, an EPO-promoted increase in STAT5 tyrosine phosphorylation was still detectable (Figure 2b), suggesting that this phosphorylation was induced independently of JAK2. Neither Src PTK inhibitors nor the JAK PTK inhibitor completely blocked the tyrosine phosphorylation of STAT5. Thus, Src and JAK2 cooperatively tyrosine-phosphorylate STAT5. To corroborate the results obtained using PTK inhibitors, we established stable transfectants of F- 36P cells expressing a dominant negative Src (Src-DN) with mutations of Lys 296 to Arg and Tyr 528 to Phe (Mukhopadhyay et al., 1995). In the selected transfec- tant clones, the amounts of Src were much greater than in mock-transfected F-36P cells because of the high expression levels of Src-DN (Figure 3a). In F-36P cells expressing Src-DN, the extent of EPO-induced tyrosine phosphorylation of STAT5 was signi®cantly impaired (Figure 3b). In contrast, the tyrosine phosphorylation of JAK2 in these cells was similar to that observed in mock-transfected cells. These results indicate that Src,

anti-STAT5A antibody (upper 2 panels; Santa Cruz) or an anti- JAK2 antibody (lower 2 panels; Santa Cruz), analysed by Western blotting using the anti-phosphotyrosine antibody (top Figure 2 Both inhibitors of Src and JAK PTKs suppress the panel of each pair). The ®lters were then stripped and reprobed extent of tyrosine phosphorylation of STAT5 induced by EPO. with the anti-STAT5A antibody or the anti-JAK2 antibody, (a) F-36P cells were serum- and EPO-starved for 16 h and then respectively (bottom panel of each pair). (b) F-36P cells were treated with the indicated concentrations of PP1 (Biomol), PP2, pretreated with the indicated concentrations of AG490 (Calbio- PP3 (Calbiochem), or vehicle (DMSO) alone for 1 h followed by chem) or the vehicle alone for 16 h and then stimulated with EPO stimulation with 100 U/ml EPO for 5 min. Cell lysates were for 5 min. Tyrosine phosphorylation of STAT5A and JAK2 was prepared and subjected to immunoprecipitation with a polyclonal examined as in (a)

Oncogene Src tyrosine-phosphorylates STAT5 in EPOR signaling Y Okutani et al 6646 a b

Figure 3 Expression of dominant negative Src reduces EPO-induced tyrosine phosphorylation of STAT5. Either the expression vectors for dominant negative Src (UBI) or the empty vectors without insert were electroporated into F-36P cells as described previously (Kitanaka et al., 1999). (a) Cell lysates of F-36P cells transfected with the empty vectors alone (F-36P-Mock) or the vectors for dominant negative Src (F-36P-Src-DN) were subjected to immunoprecipitation with the anti-Src antibody, and analysed by Western blotting using the anti-Src antibody. (b) F-36P-Mock cells or two clones of F-36P-Src-DN cells were serum- and EPO- starved for 16 h and then incubated for 5 min in the presence or absence of 100 U/ml EPO. The cell lysates were subjected to immunoprecipitation with the anti-STAT5A antibody (upper 2 panels) or the anti-JAK2 antibody (lower 2 panels), and analysed by Western blotting using the anti-phosphotyrosine antibody (top panel of each pair). Filters were then stripped and reprobed with the anti-STAT5A antibody or the anti-JAK2 antibody, respectively (bottom panel of each pair)

at least in part, contributes to EPO-induced tyrosine co-immunoprecipitating considerable amounts of phosphorylation of STAT5. STAT5 with Src from the plasmids-transfected COS7 In order to investigate the ability of Src to tyrosine- cells (data not shown). Of note, from JAK2-co- phosphorylate STAT5, Src and STAT5A or STAT5B transfected COS7 cells in which Src and STAT5 were were transiently expressed in COS7 cells. Co-expression co-expressed, the amount of STAT5 co-immunopreci- of kinase active Src, but not kinase-inactive Src, pitated with Src was increased to such an extent that it markedly enhanced the extent of tyrosine phosphoryla- was detected by Western blotting, suggesting that tion of both STAT5A and STAT5B (Figure 4a). JAK2 plays a role in their assembly and/or stabiliza- Several reports have shown the complex formation tion of the Src-STAT5 complex (data not shown). It is between Src and STAT proteins including STAT5 (Cao interesting to note that others have also failed to detect et al., 1996; Chaturvedi et al., 1998; Kazansky et al., co-immunoprecipitation of v-Src with STAT5 from v- 1999). However, we have not been successful to date in src-transformed 32Dcl3 cells in which STAT5 was

Oncogene Src tyrosine-phosphorylates STAT5 in EPOR signaling Y Okutani et al 6647 a b

Figure 4 In vivo tyrosine phosphorylation of STAT5 by Src. (a) The empty vectors (Vector) or expression vectors for STAT5A, STAT5B, kinase-active (Src-WT) or kinase-inactive (Src-KD) Src (UBI) were introduced into COS7 cells in the indicated combinations using SuperFect transfection reagent (Qiagen). After culture for 36 h, STAT5A and STAT5B were immunoprecipitated from cell lysates with polyclonal antibodies speci®c to each relevant protein and subjected to Western blot analysis using the anti-phosphotyrosine antibody (upper panel). The ®lters were then stripped and reprobed with the anti-STAT5A antibody or the anti-STAT5B antibody (lower panel). (b) Schematic representation of the full-length (STAT5A-WT) and carboxyl- deleted STAT5A-based mutants truncated at amino acids 713 (STAT5A-713) or 693 (STAT5A-693). FLAG epitope tags (closed diamond) were added to the C-terminal ends. The DNA binding domain (DNAB) and SH2 domain as well as the position of Tyr 694 are indicated. These mutants were constructed by means of mutagenesis PCR (Kitanaka et al., 1999) using full-length STAT5A as a template. (c) Eect of carboxyl-truncation of STAT5A on Src-mediated tyrosine phosphorylation. COS7 cells were transfected with expression vectors for wild-type or mutant STAT5 together with those for Src. Proteins immunoprecipitated with an anti- FLAG antibody (Sigma) were subjected to Western blot analysis using either the antibody speci®c to phosphotyrosine (upper panel) or STAT5 (lower panel) tyrosine-phosphorylated and activated (Chaturvedi et DNA binding activity of STAT5A (Gouilleux et al., al., 1997). These discrepancies in detection of the 1994). Thus, we constructed FLAG-tagged protein complex formation between Src and STAT5 may be expression plasmids for wild-type STAT5A and due to their weak association or dierences in cell lysis carboxyl-truncated mutants of STAT5A with or with- conditions in dierent experiments. out Tyr 694 (Figure 4b). The tyrosine phosphorylation The tyrosine residue at position 694 (Tyr 694) in the levels of full-length and carboxyl-truncated STAT5A STAT5A amino acid sequence is known to be the site mutants in COS7 cells were compared in the presence phosphorylated by JAK2 and the regulator of the of co-transfected Src. As illustrated in Figure 4c,

Oncogene Src tyrosine-phosphorylates STAT5 in EPOR signaling Y Okutani et al 6648

b c

Figure 5 In vitro tyrosine phosphorylation of STAT5 by Src. (a) Schematic representation of GST-fusion proteins. Recombinant GST (closed oval) fusion proteins containing amino acid sequences from 494 to 794 (GST-STAT5A-WT) and from 494 to 693 (GST-STAT5A-693) of human STAT5A were constructed. To prepare GST-STAT5A-WT, the fragment of human STAT5A cDNA coding an amino acid sequence from 494 to 794 was subcloned into pGEX-6P-2 vector (Pharmacia). C-terminal-deleted GST- STAT5A fusion protein, which is truncated at amino acid 693 (GST-STAT5A-693), was constructed by mutagenesis PCR. GST- fusion proteins in the bacteria were solubilized by being boiled in SDS ± PAGE sample buer, separated by SDS ± PAGE and electroeluted using a Whole Gel Eluter (Bio-Rad). Protein ± protein interaction between GST ± fusion proteins and endogenous proteins in the bacteria was not expected to survive under this denaturing condition. (b) GST-STAT5A-WT or GST-STAT5A-693 was incubated with or without 3 U of anity puri®ed recombinant human Src expressed in Sf9 insect cells (UBI) in a kinase buer containing 10 mM ATP at 258C for 5 min. After the reaction was stopped, samples were subjected to Western blot analysis using the anti-phosphotyrosine antibody (upper panel) followed by reprobing with an anti-GST antibody (lower panel; Santa Cruz). (c) COS7 cells transiently transfected with STAT5A were lysed and immunoprecipitated with the anti-STAT5A antibody. Kinase reactions were performed as in (b) using anti-STAT5A immunoprecipitates as substrates. Samples were subjected to Western blot analysis using the anti-phosphotyrosine antibody (upper panel), polyclonal anti-phospho-STAT5A/B antibody (a-pSTAT5; middle panel; UBI), or anti-STAT5 antibody (bottom panel)

Oncogene Src tyrosine-phosphorylates STAT5 in EPOR signaling Y Okutani et al 6649 FLAG-tagged wild-type STAT5A co-expressed with nant negative Lyn. However, the importance of Lyn to Src exhibited a high level of tyrosine phosphorylation. other erythroid cell lines and normal erythroid Src-induced tyrosine phosphorylation of STAT5A progenitor cells has not yet been clari®ed. Our ongoing carboxyl-truncated at Ala 713 (STAT5A-713) was also study revealed that antisense oligonucleotides against readily detected. In contrast, carboxyl-truncation at src but not lyn inhibited EPO-induced erythroid Gly 693 of STAT5A (STAT5A-693), from which Tyr dierentiation of K562 cells and EPO-induced devel- 694 was deleted, diminished the tyrosine phosphoryla- opment of erythroid colony formation of the cells tion of STAT5A. This suggests that Tyr 694 at the derived from normal human bone marrow (Y Kubota carboxyl-terminus of STAT5A is a main phosphoryla- et al., submitted). These ®ndings indicate that Src may tion site induced by Src. play a more universal role in EPO-induced erythroid It may be possible to speculate that Src activates dierentiation than Lyn. endogenous PTK(s) in COS7 cells, which in turn Recently, Src-induced tyrosine phosphorylation and tyrosine-phosphorylate(s) STAT5. To rule out this DNA binding of STAT5 have been reported (Kazan- possibility and to elucidate the kinase-substrate status sky et al., 1999). In this study, Src was shown to in Src-mediated STAT5 phosphorylation, we per- associate with STAT5. However, the researchers failed formed in vitro kinase assays. We prepared recombi- to detect an increase in tyrosine phosphorylation of nant GST-fusion proteins which were intact (GST- STAT5 by in vitro kinase assay using the immunopre- STAT5A-WT) and carboxyl-truncated STAT5A (GST- cipitated Src-STAT5 complex. Although we have not STAT5A-693) (Figure 5a). GST-fusion proteins were yet succeeded in detecting the tight complex formation incubated in the presence of ATP with or without between Src and STAT5, a direct kinase-substrate recombinant Src. Western blot analysis using the anti- relationship between them was clearly demonstrated by phosphotyrosine antibody showed marked tyrosine the in vitro kinase assay using GST-STAT5A fusion phosphorylation of GST-STAT5A-WT (Figure 5b, left proteins and recombinant Src under a condition free of upper panel). In contrast, no tyrosine phosphorylation mammalian cellular proteins. was observed in GST-STAT5A-693 after kinase In conclusion, our results reveal that STAT5 is a reaction with Src (Figure 5b, right upper panel). Src- direct target of Src. This implies the existence of Src- mediated tyrosine phosphorylation of STAT5A ob- mediated STAT5-activating machinery downstream of tained from STAT5A-transfected COS7 cells was also EPOR. This conclusion is in good agreement with the detected by Western blotting using either the anti- recently proposed JAK-Src-STAT model which sug- phosphotyrosine antibody (Figure 5c, top panel) or an gests the involvement of Src family PTKs in the anti-phospho-STAT5A/B (Y694/Y699) antibody (Fig- cytokine-mediated signaling pathways (Reddy et al., ure 5c, middle panel). Similar results were observed in 2000). Additional studies undertaken in order to the kinase assay in which anti-STAT5B immunopreci- determine the role of Src in the STAT5 activation pitates were used as substrates (data not shown). These and the contribution of JAK PTK(s) may provide results are consistent with those of the in vivo insight into the function of this pathway in EPOR- phosphorylation on Tyr 694 of STAT5A by Src shown mediated signal transduction. in Figure 4 and strongly support the direct kinase- substrate relationship between Src and STAT5. Several investigators have demonstrated the involvement of Lyn, a member of the Src family PTKs widely Acknowledgments expressed in hematopoietic cells, in EPO-induced signal We would like to thank Dr T Kitamura for providing the transduction (Tilbrook et al., 1997; Chin et al., 1998). STAT5 cDNAs, Dr JN Ihle for providing the JAK2 cDNA The ability of J2E cells to dierentiate in response to and Dr M Kotb for a critical reading of the manuscript. EPO was severely suppressed by treatment with We would also like to thank Kirin Brewery for providing antisense lyn oligonucleotides or expression of domi- recombinant human EPO.

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Oncogene