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* Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston;
Department of Biochemistry and Molecular Biology, Medical and Health Science Center, The University of Debrecen, Hungary;
Isis Pharmaceuticals Inc., Molecular Pharmacology, Carlsbad, California;
IRSP, SAIC Frederick Cancer Research and Development Center, Maryland;
|| Division of Immunology and Organ Transplantation, Department of Surgery, University of Texas Medical School at Houston; and
# Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, Maryland
Correspondence: Dr. Robert A. Kirken, University of Texas Health Science Center at Houston, Department of Integrative Biology, MSB Room 4.218, Houston, TX 77030. E-mail: Robert.A.Kirken{at}uth.tmc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: T lymphocytes • signal transduction • Janus tyrosine kinase • oligodeoxynucletide • mTor
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, IL-17, lymphotoxin, IL-12, or IL-18 for cell-mediated immunity [1
]. T cells belonging to the Th2 class can secrete and bind IL-4, IL-10 and IL-13 to drive lymphocyte differentiation for humoral-based immunity [2
]. Many of these cytokines activate Janus tyrosine kinases (Jak), which indirectly govern gene transcription by activating several effector molecules including a family of transcription factors known as signal transducer and activator of transcription (Stat) [3
]. Gene deletion studies have helped define the role of these Stats, as Stat1- or Stat2-deficient mice fail to respond to proinflammatory cytokines IFN- or IFN-
and are highly sensitive to viral or bacterial infection [4
5
6
]. Mice devoid of Stat4 or Stat6 display losses of Th1 or Th2 cell function, driven primarily by proinflammatory cytokines such as IL-12 or T cell regulatory cytokines such as IL-4/13, respectively [7
, 8
]. Prevailing evidence suggests that Stat5a/b is critically important for T cell-mediated mitogenic signals and immune function, as Stat5a/b-/- mice are immune suppressed with T cells unable to proliferate in response to IL-2 and displaying reduced protein levels of cyclin A, D2, D3, E, and Cdk6 [9
, 10
]. Current models suggest that activation of Stats is mediated by two distinct kinases, a tyrosine kinase such as Jak and/or Src enzymes and one of several Stat serine kinases [11 ]. Only the dual-phosphorylated Stat would be competent to disengage from the receptor, dimerize, nuclear-translocate, bind select DNA sequences, and associate with transcriptional molecules necessary for maximal gene transcription [3 ]. The Stat phosphoacceptor sites are represented by a highly conserved tyrosine residue, and the serine is typically located within one or more proline-rich motifs of the transactivation domain [12 ]. For Stats 1, 3, and 4, this site has been mapped to S727 and is required for full gene transcription [11 , 13 14 15 16 17 ]. In T cells, noncytokine-dependent signals can also regulate this site with studies showing that TCR engagement can drive S727 phosphorylation of Stat1 and Stat3 [14 , 15 ]. CD28 ligation also increased S727 phosphorylation within Stat1 [18 ], whereas cross-linking the Fc receptor for immunoglobulin G (IgG)IIa stimulated Stat3 S727 phosphorylation in B cells [19 ]. Several serine kinases have been demonstrated to phosphorylate the Pro-Met-Ser-Pro (PMSP) motif, a consensus mitogen-activated protein kinase (Mapk) phosphorylation sequence [20 ] that has been further substantiated by coprecipitation of extracellular-regulated kinase (Erk)1/2 with Stats [16 , 21 ] and inhibited by Mapk kinase (Mek)1/2 poisons [15 , 16 , 22 ]. Noncell surface receptor activation of Erk1/2 by phorbol esters, which use protein kinase C (PKC)-Raf intermediates, also increased Stat3 serine phosphorylation [15 , 23 ]. However, new reports have identified phosphatidylinositol-3 kinase (PI-3K) [18 , 19 ] and mammalian target of rapamycin (mTOR) [24 ] as candidate Stat serine kinases, although p38 and Jnk [22 , 25 , 26 ] have been mostly discounted.
Unlike Stat1, -3, and -4, less is known about the Stat5a/b serine kinase regulation. Although Stat5 tyrosine phosphorylation sites of Stat5a/b have been mapped to Y699/701 [27
], respectively, the serine phosphorylation sites and putative kinase are less clear. Increasing evidence suggests this site lies within the transactivation domain located within a Pro-Ser-Pro (PSP) motif that lacks the invariant methionine residue and thus, a weak Mapk target [20
]. Earlier work by our group used alanine scanning mutagenesis, reconstitution assays, and phosphoamino acid analyses of prolactin-treated Cos-7 cells to identify these phosphoacceptor sites in murine Stat5a S725 (human S726) and Stat5b S730 (human S731) [28
]. Supportive evidence in the form of functionally relevant pro-B cell and T cell lines lends credence to our findings, as delivery of carboxyl terminal-deleted Stat5 variants lacking these serine residues inhibits cytokine-driven cell proliferation and cell cycle gene expression [29
, 30
]. Recently published findings by Park et al. [31
] suggest these Stat5a/b serine sites are critical for maximal growth hormone-regulated gene transcription. To determine putative regulation of this PSP motif in Stat5a/b of immunocompetent cells, phosphospecific antibodies to this site were generated. Herein, we report that phorbol esters and several T cell growth factors competent to recruit the common IL-2 receptor chain (c) mediate phosphorylation of these proline-juxtaposed serine residues in human Stat5a (S726) and Stat5b (S731). Unexpectedly, phosphorylation and dephosphorylation kinetics of the highly homologous Stat5a and Stat5b proteins differed qualitatively and temporally. In addition to providing a first account that IL-2 can activate all three Raf isoforms (A, B, and C), Raf-specific antisense oligonucleotides did not inhibit Stat5 serine phosphorylation or inhibitors to PI-3K, mTOR, and Mek1/2, unlike Stat3, which was sensitive. This new evidence suggests that the Stat5a/b serine kinase regulated in immunocompetent cells by proliferative cytokines (e.g., IL-2, IL-7, IL-9, and IL-15) is mechanistically distinct from other Stats including Stat3.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Solubilization of membrane proteins and immunoprecipitation
Cells were solubilized in 1% Triton X-100 lysis buffer (108 cells/ml) and clarified by centrifugation as previously described [33
]. For immunoprecipitation, supernatants were incubated with 5 µl/ml polyclonal rabbit antiserum raised against peptides derived from the extreme carboxyl termini of murine forms of Stat5a (LDARLSPPAGLFTSARSSLS) or Stat5b (MDSQWIPHAQS) conjugated to keyhole limpet hemocyanin and used as immunogen in rabbits. Site-specific anti-Stat5-phosphoserine antibodies were similarly produced to the corresponding phosphopeptide sequence DQAP[pS]PAVC [28
]. Blots were Westerned with monoclonal mouse antiphosphotyrosine or antiphosphotyrosine Stat5a/b antibodies (Upstate Biotechnology Inc., Waltham, MA; 4G10; cat. nos. 05-321 and 05-595, respectively) or monoclonal anti-Stat5 (Transduction Laboratories, Lexington, KY; S21520-050) at 1:1000 as previously indicated [32
]. For TCR activation experiments, 20 µl p56Lck antibody was used for immunoprecipitation per sample (Santa Cruz Biotechnology, Santa Cruz, CA; cat. no. SC-433), and reblotting was performed with anti-Lck (Transduction Laboratories; cat. no. L156200) and blotted as described above. Immunoprecipitation of Raf isoforms was performed as described below. For serine-kinase-inhibitor experiments, cells were preincubated for 1 h with Me2SO as a mock control or with varying concentrations of PD98059 (New England Biolabs, Inc., Beverly, MA; cat. no. 9900L), rapamycin (Calbiochem, San Diego, CA; cat. no. 553210-Q), and wortmannin (Calbiochem; cat. no. 681675-Q) and were then lysed and immunoprecipitated as described in figure legends. For all samples, total protein was determined by the bicinchoninic acid method (Pierce, Rockford, IL).
Raf antisense oligodeoxynucleotide (ODN) treatment and viability assay
YT or CTLL2 cells were then treated with selective antisense to each Raf isoform. The phosphothioate backbone was synthesized using phosphorothioate chemistry, and 2'-methoxyethyl modification of the five terminal nucleotides (underlined) used to increase stability (underlined base) of the oligonucleotide sequences used was as follows: human A-Raf (ISIS 15489) 5'-CTAAGGCACAAGGCGGGCTG-3', mouse/human B-Raf (ISIS 15344) 5'-CTGCCTGGATGGGTGTTTTT-3', human C-Raf (ISIS 13650) 5'-TCCCGCCTGTGACATGCATT-3', mouse A-Raf (ISIS 15493) 5'-CTAAGGCACAAGGCGGGCTG-3', mouse C-Raf (ISIS 15770) 5'-ATGCATTCTGCCCCCAAGGA-3'. Nonsense control sequence for all (ISIS) is described previously [34
]. YT cells in exponential growth phase were resuspended at 7.5 x 107/ml in ice-cold OPTI-MEM-I medium. A 400 µl sample of these cells was placed in a cuvettes with ODN at varying concentrations ranging from 5 to 20 µM and was then electroporated at 220V and 960 µF using a Bio-Rad Gene PulserTM (Bio-Rad, Hercules, CA), cultured for 48 h in growth medium and then stimulated with IL-2 (100 nM) as described above. Viability was assessed by trypan blue dye exclusion.
Raf in vitro kinase assay
IL-2-treated and -untreated cells were lysed, and a normalized protein amount was immunoprecipitated with A-, B (Santa Cruz Biotechnology; cat. nos. SC-408 and SC-166, respectively)-, or C-Raf antibodies (BD Pharmingen, San Diego, CA; cat. no. R19120) as described above. Immunocomplexes were washed with lysis buffer three times followed by washing with kinase buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol. Kinase reaction was carried out in the presence of 50 µM adenosine 5'-triphosphate (ATP) and 10 µCi [32P]-ATP (NEN DuPont, Wilmington, DE) for 10 min at 37°C. Samples were washed three times with lysis buffer, boiled for 5 min, and subjected to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. For substrate assays, 0.25 µg/µl kinase-dead Mek1-glutathione-S-transferase (GST) was used for each sample. In both experiments, reactions were terminated by adding 10 µl 4 x SDS sample buffer, separated by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membrane, and exposed to X-ray film (X-Omat, Kodak, Rochester, NY) at -70°C.
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared from IL-2-, IL-7-, IL-9-, or IL-15-stimulated T cells (described above) and were then pelleted by centrifugation (20,000 g for 1 min at 4°C), and nuclear extracts were prepared as previously described [35
]. For supershift assays, the nuclear extracts were preincubated with 1 µg normal rabbit serum or antisera specific to Stat5a, 5b, and phosphoserine Stat5 as indicated in the corresponding legend at 4°C for 20 min. Samples were then incubated with a [32P]-end-labeled oligonucleotide corresponding to the ß-casein gene sequence (5'-AGATTTCTAGGAATTCAATCC-3') for 15 min at room temperature and were subjected to gel electrophoresis as described [35
].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To address this question, site-specific phosphoserine antibodies to this domain were generated, and specificity was verified using phosphopeptide competition assays (data not shown). Next, quiescent PHA-activated human T cells were stimulated with IL-2 from 0 to 120 min, and Stat5a or Stat5b was immunoprecipitated, separated on SDS-PAGE, and ultimately blotted with phosphospecific Stat5a/b antibodies. Stat5a and Stat5b displayed nearly identical tyrosine phosphorylation kinetics, peaking at approximately 10 min when blotted with antiphosphotyrosine antibody (Fig. 1A , upper panel). IL-2 promoted Stat5a/b serine phosphorylation within its PSP motif, which was detected as doublets for each transcription factor (Fig. 1A , middle panel). However, densitometric analysis revealed Stat5a serine phosphorylation was slightly protracted, maximal levels reached between 10 and 30 min, and serine phosphorylation peaked after approximately 60 min for Stat5b. At 60 min post-IL-2 stimulation, the slower migrating form of Stat5a showed pronounced dephosphorylation and was weakly detectable at 120 min. In contrast, both phosphorylated forms of Stat5b were detectable with little reduction in signal after 120 min. Stat5a/b immunopurified from non-IL-2-treated T cells did not display any levels of tyrosine or serine phosphorylation (Fig. 1A , upper and middle panels). Reprobing both blots with a pan Stat5 antibody confirmed equivalent loading (Fig. 1A , lower panel).
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c cytokines induce serine phosphorylation of Stat5a/b in T cells, which serves as a common receptor for several cytokines including IL-2, IL-7, IL-9, and IL-15. Jak3, which is primarily expressed in lymphocytes, binds to the c and is necessary for T cell development and function [37
38
39
40
41
]. To determine if the Stat5 proline-directed serine kinase was uniquely activated by IL-2 or also a shared serine kinase by this family of T cell growth factors, quiescent human T cells were stimulated with 100 nM of IL-2, IL-7, IL-9, or IL-15 for 10 min at 37°C. Stat5a or Stat5b proteins were immunoprecipitated from corresponding cell lysates, separated by SDS-PAGE, and probed with -phosphotyrosine (PY; Fig. 1B
, upper panel), -phosphoserine Stat5a/b (PS; Fig. 1B
, middle panel), or -Stat5 (Fig. 1B
, lower panel). Each growth factor showed comparable ability to promote Stat5a/b tyrosine and serine phosphorylation of each conserved residue. Current crystallographic results of Stat1, -3, and -4 dimers are based on carboxyl-truncated forms that lack the transactivation domain, which harbors the PMSP motif, and are not known if the putative phosphoserine protrudes away from each monomer to the milieu to interact with transcriptional enhancing machinery, complementary Stat dimmer, or DNA [17 , 42 ]. To test the notion that the native PSP site in Stat5a/b might be exposed and accessible to transcriptional cofactors, antiphosphoserine Stat5 antibodies were used in an EMSA to supershift the complex. As shown in Figure 2 , nuclear cell extracts (5 µg/well) were mixed with a [32P]-labeled ß-casein probe that is readily bound by activated Stat5a/b (Fig. 2 , lanes b and i, indicated by an arrow) and could be verified by supershifting with antibodies to Stat5a (Fig. 2 , lanes c and j), Stat5b (Fig. 2 , lanes d and k), or both (Fig. 2 , lanes e and l). However, the phosphoserine Stat5a/b antibody was also able to partially supershift the Stat5 complex (Fig. 2 , lanes f and m). The specificity was confirmed when normal rabbit sera failed to supershift the Stat5/DNA complexes (Fig. 2 , lanes g and n). Competition with phosphoserine Stat5 immunizing peptide could partially recover the Stat5a/b supershifted complex (data not shown). These findings suggest that the phosphoserine site is exposed, albeit weakly on Stat5a/b, or that a limited pool of unoccupied Stat5a/b phosphoserine (S726 or S731) is accessible to the phosphoserine Stat5a/b antibody.
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CD3 treatment of human T cells induce serine phosphorylation of Stat5a and Stat5b in human T cellsCD3 stimulation of T cells rapidly activates Stat5a/b tyrosine phosphorylation and is critical for antigen-induced T cell proliferation [45
]. To test whether engagement of the TCR complex with CD3 antibodies can activate a Stat5a/b proline-directed serine kinase, PHA-activated T cells were made quiescent and then activated for 10 min in the presence of 5 µg/ml CD3 (Fig. 3A
, lanes b, d, and j) or with 100 nM IL-2 (Fig. 3A
, lanes f and h). Three independent experiments failed to detect CD3-mediated serine phosphorylation of Stat5a/b (Fig. 3A
, middle panel) as well as phosphotyrosine Stat5 (Y701) using a Stat5 Y701-specific monoclonal antibody (Fig. 3A
, upper panel) even if cells were stimulated for 30 min (data not shown). Activation of the TCR complex was verified by CD3 induction of p56Lck tyrosine phosphorylation (Fig. 3A
, lanes i and j), whereas IL-2-mediated phosphorylation of the conserved Stat5a/b tyrosine (Y699/701; Fig. 3A
, upper panel) and serine (S726/731; Fig. 3A
, middle panel) residues was detected. Reprobing both blots with a pan-Stat5 or p56Lck verified equivalent protein loading (Fig. 3A
, lower panel).
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IL-2-mediated Stat3 but not Stat5a/b serine phosphorylation is sensitive to inhibition of several IL-2-activated Stat serine kinases
IL-2 is a prominent activator of several serine-threonine kinases including Erk1/2, PI-3K, and mTor, all established Stat1/3 activators [18
, 19
, 24
]. Herein, we examined their role in promoting Stat3 and Stat5 serine phosphorylation in human T cells or the IL-2-responsive YT cell line (shown). First, YT cells were stimulated in the presence of 100 nM IL-2 (Fig. 4A
, lanes b–e) or 100 nM PMA (Fig. 4A
, lanes g and h), and immunoprecipitated Stat3 blotted for phosphotyrosine (PY; Fig. 4A
, upper panel) or phosphoserine (PS; Fig. 4A
, middle panel) was found increased by either agent. Inhibition of Stat3 serine phosphorylation was detected following IL-2-stimulated cells pretreated with 50 µM PD98059 (Fig. 4A
, lane c), 20 nM rapamycin (Fig. 4A , lane d), or 100 nM wortmannin (Fig. 4A
, lane e) compared with controls (Fig. 4A
, lane b). Similarly, PMA-induced Stat3 S727 phosphorylation was inhibited following inhibition of Mek1 with PD98059 (Fig. 4A
, middle panel, lane h).
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-phosphotyrosine Stat5 (PY; Fig. 4B
, upper panels), -phosphoserine Stat5 (PS; Fig. 4B
, middle panels), and -Stat5 (Fig. 4B
, lower panels). Compared with untreated control samples (Fig. 4B
, lanes a and b or h and i), pretreatment with each selective inhibitor displayed no significant, deleterious effects on IL-2-mediated Stat5a/b tyrosine or serine phosphorylation. It is concluded from these experiments that IL-2-stimulated Stat5a/b serine kinase(s) act independent of these signaling pathways. Similar treatment of human T cells with c cytokines (IL-2, -7, -9, and -15), pretreated with the highest dose of each inhibitor, also failed to ablate Stat5a/b serine phosphorylation (data not shown). As previously mentioned, PMA can directly stimulate PKC, which in turn activates c-Raf and subsequently Erk1/2 [46 ]. Because PMA activates Stat5a/b serine phosphorylation (Fig. 3) , likely independent of Mek1/2 based on PD98059 studies (Fig. 4) , we tested a possible model in which the Stat5a/b serine kinase bifurcates from Raf to an alternate proline-directed Stat5a/b serine kinase. Although the Raf protein kinases consist of three isoforms (A-, B-, and C-Raf) to regulate cell proliferation, differentiation, and apoptosis, IL-2 signaling studies have primarily focused on C-Raf [47 , 48 ].
IL-2 promotes the autokinase activity of A-, B-, and C-Raf isoforms
To address this question, human YT cells were stimulated with IL-2 for 10 min, and each Raf isoform was immunoprecipitated from equivalent protein (1 mg) using a Raf-specific or isotype control antibody and autokinase assay performed using -[32P]-ATP (see Materials and Methods for details). Depicted in Figure 5A
is a representative autoradiograph (n=3) that showed A-Raf (68 kDa) and C-Raf (74 kDa) had no basal activity but were inducible upon IL-2 stimulation (Fig. 5A
, lanes b and h). B-Raf exhibited a high level of basal enzyme activity, as previously reported [49
], and migrates at two positions within the gel (74 and 95 kDa) but can be further autophosphorylated following IL-2 treatment (Fig. 5A
, lane e). Reprobing these membranes suggested each Raf isoform is equally expressed within YT cells (Fig. 5A , lower panel).
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To investigate whether each Raf antisense disrupts p44/42 Erk1/2 activity, total cell extracts from each treated sample were subjected to separation by SDS-PAGE (10% acrylamide gels), transferred to PVDF membrane, and blotted with phosphoErk1/2 antibodies. Cell lysates obtained from Figure 5B were next examined for a loss in Mek1/2 phosphorylation of the Thr-Glu-Tyr (TEY) motif in Erk1/2 using antiactive antibodies (Fig. 5C) . In contrast to nonsense oligonucleotide treatment (Fig. 5C , lane f), each antisense oligonucleotide reduced Erk phosphorylation by greater than 70% when normalized against p42, identified using a pan-Erk antibody. Western blots and densitometric analysis of normalization of Erk1/2 phosphorylation yielded an I.I. of 70%. Based on these results, we conclude that activation of each of three Raf isoforms contributes to promote Erk1/2 activation.
Inhibition of A-, B-, and C-Raf with antisense oligonucleotides does not affect Stat5a and Stat5b serine phosphorylation
As PMA or IL-2 activates Raf kinases, we next examined whether inhibition of each Raf isoform would block Stat5a/b serine phosphorylation (Fig. 6
). For this study, corresponding lysates from YT cells (Fig. 5B
and 5C)
were used to generate data shown in Figure 6 . Briefly, Stat5a and Stat5b immune complexes were isolated from equivalent protein cell lysates and blotted with -phosphotyrosine (PY; Fig. 6
, upper panel), -phosphoserine Stat5 (PS; Fig. 6
, middle panel), and -panStat5 (Fig. 6
, lower panel), which were harvested from YT cells electroporated with 5 µM A-, B-, or C-Raf antisense oligonucleotides (lanes i and j, k and l, and m and n, respectively), a mixture of 9 µM Raf antisense oligonucleotides (lanes g and h), 9 µM nonsense control (lanes e and f), and no oligonucleotides (lanes c and d), or were left untreated. Neither Stat5a nor Stat5b showed a decrease in IL-2-induced serine/tyrosine phosphorylation upon the Raf antisense oligonucleotide treatment, suggesting that the IL-2-activated Stat5 serine kinase does not require all three Raf isoforms, Mek1/2, or Erk1/2 proteins/pathways.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Current cytokine-signaling models hold that ligation of cytokine receptors recruit and activate Jak enzymes, which subsequently mediate phosphorylation of Stat proteins of a conserved tyrosine residue [51 ]. Herein, we observed rapid tyrosine phosphorylation kinetics of Stat5a/b, which closely mimic IL-2-induced Jak3 autophosphorylation in human T lymphocytes (5–10 min) post-TCGF stimulation (Fig. 1A) [52 53 54 ]. The limited region of sequence divergence existing within Stat5a/b is localized to their transactivation domains, which accommodate the conserved PSP motif. Although Stat5a/b share 95% sequence identity, they display differences in biological activity, as mice void of Stat5a develop mammary gland defects [55 ], and Stat5b null mice manifested as a runted phenotype [56 ]. In addition to two phosphoacceptor sites in Stat5a [57 ], the current work adds to a series of Stat5a versus Stat5b dissimilarities also localized within the transactivation domain, as both displayed different serine phosphorylation kinetics. IL-2 treatment induced a serine-phosphorylated Stat5a doublet (Fig. 1A , middle panel) with both phosphorylated forms attaining maximal levels within 10–30 min. Stat5b showed more protracted kinetics with serine phosphorylation peaking at 30–60 min and remained largely unchanged after 120 min, in contrast to Stat5a, which was significantly reduced at the same time point.
The main implication of this dissimilarity in phosphorylation for Stat5a/b is that they operate differently, explaining in part the lack of compensatory gene expression displayed in Stat5 single knockout mice. However, differences in immunoregulatory cells isolated from Stat5a or Stat5b null mice fail to display obvious cell defects [9 ]. Taken together, these findings suggest each transcription factor is redundant or may act to "fine-tune" T cell gene expression. Based on reconstitution assays using the prolactin receptor and serine mutants of Stat5a (S726) and Stat5b (S731), we failed to identify a significant loss in Stat5a/b transactivation potential [28 ]. However, reconstitution of the positionally conserved Stat1 (S727) mutant in functional, relevant cells yielded definitive changes in gene transcription potential that could be correlated to association with MCM5 [17 ]. Crystallographic analysis of Stat1, -3, and -4 is truncated within the transactivation domain, making the position of the conserved serine site unknown. However, here, using our phospho-Stat5a/b antibodies and EMSA analysis, we were able to partially supershift either Stat5 protein (Fig. 1B) , suggesting at least partial availability of this domain and not interacting within the Stat5 dimer or DNA. Extensive attempts to detect coassociation of MCM5 with unstimulated or cytokine-activated, fully serine-phosphorylated Stat5a/b protein failed (data not shown), perhaps highlighting distinct roles for the Stat5a/b PSP motif compared with the PMSP motif shared by Stat1, -3, and -4.
The cellular location where Stat5a and Stat5b become serine-phosphorylated is not clear but does not appear to be entirely dependent on an activated receptor complex, as PMA alone could induce this event (Fig. 3B) . These findings parallel earlier findings of phorbol ester activation of Stat3 [19 , 23 ]. The identity of the Stat5 proline-directed serine kinase is not immediately known, yet based on earlier work from our group, we have shown that no membrane distal domains of the IL-2Rß were required to activate the kinase [58 ]. The only prerequisite for this event was a functional Jak3, as inactivation of this enzyme with Jak3 inhibitors or deletion of the Box1 domain of the IL-2Rß failed to promote Stat5a/b serine phosphorylation [58 , 59 ]. We are left to conclude that the Stat5a/b serine kinase is not directly associated with TCGF receptor complexes but activated distal to the receptor.
Based on studies presented herein and evidence by several other laboratories, an IL-2 model depicted in Figure 7
can be conceived in which the IL-2R, comprised of three distinct, single membrane-spanning receptors, directs the activation of Jak1 via the IL-2Rß and Jak3 by the c. Activated Jaks phosphorylate key tyrosine residues on IL-2Rß, which serve as docking sites for downstream signaling molecules, including Mapk adapter protein Shc, Src enzymes, as well as Stat5a/b [51
]. Shc would subsequently recruit Grb-2/Sos, activating the Ras/Erk pathway, and Grb-2/Gab-2 would engage the PI-3K and mTOR signaling pathway. The mitogenic signaling properties of Ras are linked to activation of the Erk signaling cascade, driving phosphorylation of several transcription factors (e.g., Elk-1, Ets-1, Fos, and NF-AT) [15
16
17
]. Our findings suggest that Mek1/2, PI-3K, mTOR, and Raf (A, B, and C) pathways act to encourage antiapoptotic pathways, and Stat5a/b may play a more central role in proliferative gene expression [13
, 30
].
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/ß, IFN-, IL-12) or regulatory cytokines (e.g., IL-4, IL-12, IL-13) to modulate cytokine release and cell differentiation, respectively. These findings have direct therapeutic consequences since inhibition of this putative proline directed Stat5a/b serine kinase could be used to manipulate T cell expansion as found within graft versus host disease. Recent findings that Stat6 is also serine phosphorylated and void of a PSP or PXSP motif (60), suggest that its selective uncoupling might prove useful in combating Th2 diseases such as allergy or airway hypersensitivity.
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ACKNOWLEDGEMENTS |
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Received March 3, 2002; revised May 8, 2002; accepted May 23, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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