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• W2009098875 abstract "Previous work has shown that inhibition of Jak2 via the pharmacological compound AG490 blocks the angiotensin II (Ang II)-dependent activation of ERK2, thereby suggesting an essential role of Jak2 in ERK activation. However, recent studies have thrown into question the specificity of AG490 and therefore the role of Jak2 in ERK activation. To address this, we reconstituted an Ang II signaling system in a Jak2–/–cell line and measured the ability of Ang II to activate ERK2 in these cells. Controls for this study were the same cells expressing Jak2 via the addition of a Jak2 expression plasmid. In the cells expressing Jak2, Ang II induced a marked increase in ERK2 activity as measured by Western blot analysis and in vitro kinase assays. ERK2 activity returned to basal levels within 30 min. However, in the cells lacking Jak2, Ang II treatment resulted in ERK2 activation that did not return to basal levels until 120 min after ligand addition. Analysis of phosphatase gene expression revealed that Ang II induced mitogen-activated protein kinase phosphatase 1 (MKP-1) expression in cells expressing Jak2 but failed to induce MKP-1 expression in cells lacking Jak2. Therefore, our results suggest that Jak2 is not required for Ang II-induced ERK2 activation. Rather Jak2 is required for Ang II-induced ERK2 inactivation via induction of MKP-1 gene expression. Previous work has shown that inhibition of Jak2 via the pharmacological compound AG490 blocks the angiotensin II (Ang II)-dependent activation of ERK2, thereby suggesting an essential role of Jak2 in ERK activation. However, recent studies have thrown into question the specificity of AG490 and therefore the role of Jak2 in ERK activation. To address this, we reconstituted an Ang II signaling system in a Jak2–/–cell line and measured the ability of Ang II to activate ERK2 in these cells. Controls for this study were the same cells expressing Jak2 via the addition of a Jak2 expression plasmid. In the cells expressing Jak2, Ang II induced a marked increase in ERK2 activity as measured by Western blot analysis and in vitro kinase assays. ERK2 activity returned to basal levels within 30 min. However, in the cells lacking Jak2, Ang II treatment resulted in ERK2 activation that did not return to basal levels until 120 min after ligand addition. Analysis of phosphatase gene expression revealed that Ang II induced mitogen-activated protein kinase phosphatase 1 (MKP-1) expression in cells expressing Jak2 but failed to induce MKP-1 expression in cells lacking Jak2. Therefore, our results suggest that Jak2 is not required for Ang II-induced ERK2 activation. Rather Jak2 is required for Ang II-induced ERK2 inactivation via induction of MKP-1 gene expression. Extracellular signal-regulated kinase 2 (ERK2) 1The abbreviations used are: ERK, extracellular signal-regulated kinase; Ang II, angiotensin II; HA, hemagglutinin; MKP-1, mitogen-activated protein kinase phosphatase 1; AT1, angiotensin II type 1; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; VSMC, vascular smooth muscle cell; DN, dominant negative; WT, wild type; PBS, phosphate-buffered saline; mAb, monoclonal antibody; STAT, signal transducers and activators of transcription; Jak, Janus tyrosine kinase; PP2A, protein phosphatase 2A; EGFR, epidermal growth factor receptor.1The abbreviations used are: ERK, extracellular signal-regulated kinase; Ang II, angiotensin II; HA, hemagglutinin; MKP-1, mitogen-activated protein kinase phosphatase 1; AT1, angiotensin II type 1; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; VSMC, vascular smooth muscle cell; DN, dominant negative; WT, wild type; PBS, phosphate-buffered saline; mAb, monoclonal antibody; STAT, signal transducers and activators of transcription; Jak, Janus tyrosine kinase; PP2A, protein phosphatase 2A; EGFR, epidermal growth factor receptor. is a member of the mitogen-activated protein kinase family of serine/threonine protein kinases. These proteins become activated by phosphorylation on tyrosine and threonine residues in response to a variety of ligands binding their cognate receptors at the cell surface (1.Anderson N.G. Maller J. Tonks N.K. Sturgill T.W. Nature. 1990; 343: 651-653Crossref PubMed Scopus (793) Google Scholar, 2.Crews C. Alessandrini A. Erikson R.L. Science. 1992; 258: 478-480Crossref PubMed Scopus (736) Google Scholar, 3.Kosako H. Gotoh Y. Matsuda S. Ishikawa M. Nishida E. EMBO J. 1992; 11: 2903-2908Crossref PubMed Scopus (146) Google Scholar, 4.Wu J. Harrison J.K. Vincent L.A. Haystead C. Haystead T.A.J. Michel H. Hunt D.F. Lynch K.R. Sturgill T.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 173-177Crossref PubMed Scopus (117) Google Scholar). Angiotensin II is one such ligand; it exerts many of its mitogenic effects by binding to the angiotensin II type 1 (AT1) receptor and thus activating ERK1/2. This occurs via rapid angiotensin II-dependent phosphorylation of the dual specificity kinase MEK, which in turn phosphorylates ERK1/2 on both threonine and tyrosine residues (5.Takahasi T. Yasuhiro K. Masanori O. Hikaru U. Akira T. Mitsuhiro Y. J. Biol. Chem. 1997; 272: 16018-16022Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar).ERK activity is tightly regulated. The duration of ERK activation is regulated by the intracellular signals that phosphorylate and dephosphorylate it (6.Pearson G. Robinson F. Beers Gibson T. Xu B.E. Karandikar M. Berman K. Cobb M.H. Endocr. Rev. 2001; 22: 153-183Crossref PubMed Scopus (3485) Google Scholar). While ERKs are activated by ligand binding at the cell surface, they are inactivated by several dual specificity phosphatases (7.Keyse S.M. Curr. Opin. Cell Biol. 2000; 12: 186-192Crossref PubMed Scopus (702) Google Scholar). One of these, mitogen-activated protein kinase phosphatase 1 (MKP-1), associates with and is phosphorylated by activated ERK2 and thus protected from proteasomal degradation. The phosphorylated MKP-1 then dephosphorylates ERK2, thereby inactivating it (8.Brondello J.M. Pouyssegur J. Mckenzie F.R. Science. 1999; 286: 2514-2517Crossref PubMed Scopus (363) Google Scholar).Evidence shows that Jak2 forms a membrane complex with the intermediate signaling molecules Ras and Raf1 and may therefore play a role in the regulation of ERK activity (9.Wang X.-Y. Fuhrer D.K. Marshall M.S. Yang Y.-C. J. Biol. Chem. 1995; 270: 27999-28002Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 10.Winston L.A. Hunter T. J. Biol. Chem. 1995; 270: 30837-30840Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 11.Xia K. Mukhopadhyay N.K. Inhorn R.C. Barber D.L. Rose P.E. Lee R.S. Narsimhan R.P. D'Andrea A.D. Griffin J.D. Roberts T.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11681-11686Crossref PubMed Scopus (73) Google Scholar). In fact, previous work has suggested that inhibition of Jak2 using the pharmacological compound AG490 blocks the angiotensin II-dependent activation of ERK2 (12.Marrero M.B. Schieffer B. Li B. Sun J. Harp J.B. Ling B.N. J. Biol. Chem. 1997; 272: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). One problem, however, with using AG490 to study the function of Jak2 is its lack of specificity for Jak2. While AG490 is a potent Jak2 inhibitor, it is known to inhibit other tyrosine kinase signaling pathways as well. AG490 inhibits activation of cyclin-dependent kinases and causes growth arrest of cells in G1 phase (13.Kleinberger-Doron N. Shelah N. Capone R. Gazit A. Livitzki A. Exp. Cell Res. 1998; 241: 340-351Crossref PubMed Scopus (43) Google Scholar). It also inhibits calf serum-inducible cell growth and DNA synthesis (14.Oda Y. Renaux B. Bjorge J. Saifeddine M. Fujita D.J. Hollenberg M.D. Can. J. Physiol. Pharmacol. 1999; 77: 606-617Crossref PubMed Scopus (74) Google Scholar) and is a partial blocker of c-Src activity (13.Kleinberger-Doron N. Shelah N. Capone R. Gazit A. Livitzki A. Exp. Cell Res. 1998; 241: 340-351Crossref PubMed Scopus (43) Google Scholar). Most critically, AG490 inhibits epidermal growth factor receptor autophosphorylation more potently than it inhibits Jak2 kinase activity (15.Osherov N.A. Gazit C. Gilon C. Levitzki A. J. Biol. Chem. 1993; 268: 11134-11142Abstract Full Text PDF PubMed Google Scholar). In our experiments, AG490 is certainly a potent inhibitor of Jak2. However, we have found that alternate methods such as transfection of dominant negative Jak2, introduction of Jak2 antisense oligonucleotides, or some other method must be used when working with AG490 (16.Sayeski P.P. Ali M.S. Safavi A. Lyles M. Kim S.O. Frank S.J. Bernstein K.E. J. Biol. Chem. 1999; 274: 33131-33142Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 17.Ali M.S. Sayeski P.P. Bernstein K.E. J. Biol. Chem. 2000; 275: 15586-15593Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Clearly new strategies are needed to study Jak2 kinase function.In this work, we utilized the γ2A cell line, which contains a Jak2 null mutation (18.Kohlhuber F. Rogers N.C. Watling D. Feng J. Guschin D. Briscoe J. Witthuhn B.A. Kotenko S.V. Pestka S. Stark G.R. Ihle J.N. Kerr I.M. Mol. Cell. Biol. 1997; 17: 695-706Crossref PubMed Scopus (175) Google Scholar). On this background, stable cell lines were created that expressed either the AT1 receptor alone (γ2A/AT1) or the AT1 receptor and Jak2 (γ2A/AT1+Jak2) via stable integration of cDNA plasmids. Using these cells, we measured the ability of angiotensin II to activate ERK2 as a function of Jak2 expression. We show that, in cells expressing Jak2, angiotensin II induced a marked increase in ERK2 activity, which returned to basal levels within 30 min. However, in cells lacking Jak2, angiotensin II treatment resulted in sustained ERK2 activation, which did not return to basal levels until 120 min after ligand stimulation. We hypothesized that Jak2 was playing a role in the inactivation of ERK2 by mediating the expression of one or more phosphatase proteins that target ERK2 for inactivation. Here we provide evidence that angiotensin II stimulates expression of MKP-1 and that Jak2 is essential for this event. Moreover, we provide evidence that Jak2 is essential for angiotensin II-induced co-association of ERK2 and MKP-1. Collectively, these studies show that Jak2 is not required for angiotensin II-dependent ERK2 activation but rather that Jak2 is required for angiotensin II-induced inactivation of ERK2. Furthermore, these studies demonstrate the need for novel strategies to study the function of Jak2 beyond the use of AG490.EXPERIMENTAL PROCEDURESCell Culture—γ2A/AT1 and γ2A/AT1+Jak2 cells were created as described below. These cells were cultured in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose and 10% fetal bovine serum and growth arrested in serum-free Dulbecco's modified Eagle's medium for 24 h prior to experiments. The vascular smooth muscle cells (VSMCs) expressing either a Jak2 dominant negative allele (Jak2 DN) or a neomycin resistance cassette (control) have been described previously (16.Sayeski P.P. Ali M.S. Safavi A. Lyles M. Kim S.O. Frank S.J. Bernstein K.E. J. Biol. Chem. 1999; 274: 33131-33142Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Cell culture reagents were obtained from Invitrogen. Inhibitor compounds were from either Calbiochem or LC Laboratories. All other reagents were purchased from Sigma or Fisher.Generation of Plasmid Constructs and Stable Cell Lines—The HA-tagged AT1 receptor cDNA cloned into the pcDNAI vector was kindly provided by Dr. Robert J. Lefkowitz (19.Oppermann M. Freedman N.J. Alexander R.W. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13266-13272Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). The cDNA was excised from pcDNAI and cloned into pcDNA3 via HindIII and NotI restriction digestion. A fragment encoding the amino-terminal HA tag and the 5′ sequence of the AT1 receptor cDNA up through the internal EcoRI site was then removed via HindIII and EcoRI restriction digestion and cloned into pZeo/WT (20.Ali M.S. Sayeski P.P. Dirksen L.B. Hayzer D.J. Marrero M.B. Bernstein K.E. J. Biol. Chem. 1997; 272: 23382-23388Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) that had also been cut with these same enzymes. The resulting construct, pZeo/HA-AT1-WT, contained an amino-terminal HA tag inserted just after the first methionine, ∼1100 bp encoding the rat AT1 receptor open reading frame, and ∼850 bp of rat AT1 receptor 3′ untranslated region in a zeocin-resistant plasmid.To generate the stable cell lines, γ2A cells were transfected with 20 μg of pZeo/HA-AT1-WT and 10 μg of pBOS-Jak2-WT encoding the wild type Jak2 cDNA (21.Zuhuang H. Patel S.V. He T. Sonsteby S.K. Niu Z. Wojchowski D.M. J. Biol. Chem. 1994; 269: 21411-21414Abstract Full Text PDF PubMed Google Scholar). Control cells received 20 μg of pZeo/HA-AT1-WT and 10 μg of empty vector control plasmid. Two days after transfection, cells were switched to medium supplemented with 500 μg/ml zeocin to select for stable transfectants. Surviving colonies were eventually ring cloned, and binding assays were performed using [125I-Sar1,Ile8]angiotensin II (where Sar is sarcosine) (PerkinElmer Life Sciences) as described previously (20.Ali M.S. Sayeski P.P. Dirksen L.B. Hayzer D.J. Marrero M.B. Bernstein K.E. J. Biol. Chem. 1997; 272: 23382-23388Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Nonspecific binding was defined as binding in the presence of 1.0 μm angiotensin II. Scatchard analysis was used to identify respective γ2A clones in which the binding parameters (Kd and Bmax) were similar. To measure the level of expressed Jak2 protein, 20 μg of whole cell lysate from each clone was Western blotted with anti-Jak2 antibody as described below.Immunoprecipitation—Cells were washed with 2 volumes of ice-cold PBS containing 1 mm Na3VO4 and lysed in 1.0 ml of ice-cold radioimmune precipitation assay buffer containing protease inhibitors (22.Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, New York1997Google Scholar). The samples were immunoprecipitated exactly as described previously (23.Sayeski P.P. Ali M.S. Harp J.B. Marrero M.B. Bernstein K.E. Circ. Res. 1998; 82: 1279-1288Crossref PubMed Scopus (56) Google Scholar). The immunoprecipitating anti-ERK2 mAb and anti-HA mAb (clone F-7) were from Santa Cruz Biotechnology. The anti-Tyr(P) mAb (PY20) was from BD Transduction Laboratories.Western Blotting—Proteins were detected using enhanced chemiluminescence exactly as described previously (23.Sayeski P.P. Ali M.S. Harp J.B. Marrero M.B. Bernstein K.E. Circ. Res. 1998; 82: 1279-1288Crossref PubMed Scopus (56) Google Scholar). The anti-Jak2, anti-STAT1, anti-STAT3, and anti-ERK2 blotting antibodies were from Santa Cruz Biotechnology. The anti-phospho-ERK2 antibody was from Promega. The anti-PP2A antibody was from Upstate Biotechnology, Inc. The anti-paxillin antibody was from BD Transduction Laboratories. The anti-MKP-1 antibody was from Sigma.In Vitro Kinase Assay—ERK2 immunoprecipitates were washed twice with wash buffer followed by two washes in kinase reaction buffer (25 mm HEPES, pH 7.4, and 20 mm MgCl2). The precipitates were resuspended in 50 μl of the same kinase buffer containing 50 μm ATP, 2 μCi of [γ-32P]ATP and 5 μg of myelin basic protein. The samples were incubated for 15 min at 30 °C. Reactions were terminated by adding sample buffer. The samples were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and subjected to autoradiography.Immunostaining—Cells were grown on 8-well microscope slides. After ligand treatment, cells were washed twice with K+-free PBS and fixed for 60 min at room temperature with 4% paraformaldehyde. Following fixation, cells were washed four times with K+-free PBS, permeabilized for 10 min at room temperature with 0.2% Triton X-100 in K+-free PBS (v/v), washed an additional four times, and then blocked with 5 mg/ml bovine serum albumin in K+-free PBS for 4 h at room temperature. Following blocking, cells were incubated with anti-phospho-ERK2 primary antibody overnight at 4 °C in K+-free PBS containing 5 mg/ml bovine serum albumin. The following day, cells were washed five times and incubated with a goat anti-rabbit antibody conjugated to Texas Red for 4 h at room temperature. The cells were then dehydrated through increasing concentrations of ethanol, dipped into xylene, and mounted. The following day, the cells were visualized with a fluorescence microscope.Luciferase Assay—γ2A/AT1 and γ2A/AT1+Jak2 cells were transfected with the indicated DNA in 20 μl of Lipofectin exactly as described previously (24.Sayeski P.P. Ali M.S. Frank S.J. Bernstein K.E. J. Biol. Chem. 2001; 276: 10556-10563Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Following angiotensin II treatment, luciferase activity was measured from detergent extracts in the presence of ATP and luciferin using the Reporter Lysis Buffer system (Promega) and a luminometer (Monolight Model 3010). Luciferase values were recorded as relative light units.RESULTSγ2A+AT1 Cells Lack Functional Jak2-dependent Signaling—The γ2A cell line was originally created by irradiating HT-1080 human fibrosarcoma cells to create mutations within single genes and then selecting for cells containing a mutation that ablated the expression of Jak2 (18.Kohlhuber F. Rogers N.C. Watling D. Feng J. Guschin D. Briscoe J. Witthuhn B.A. Kotenko S.V. Pestka S. Stark G.R. Ihle J.N. Kerr I.M. Mol. Cell. Biol. 1997; 17: 695-706Crossref PubMed Scopus (175) Google Scholar). To constitute angiotensin II signaling in these cells, the AT1 receptor, which was cloned into a zeocin-resistant vector, was stably transfected into cells either alone (γ2A/AT1) or with a wild type Jak2 cDNA plasmid (γ2A/AT1+Jak2). We performed binding assays, and respective γ2A clones were identified in which the binding parameters were similar. γ2A/AT1 (clone 4) had a Kd of 0.44 nm and a Bmax of 201 fmol/mg of protein, while γ2A/AT1+Jak2 (clone 1) had a Kd of 0.41 nm and a Bmax of 226 fmol/mg of protein. To demonstrate that Jak2 is expressed in γ2A/AT1+Jak2 cells, but not in γ2A/AT1 cells, both cell types were grown to about 70% confluency and lysed, and protein was extracted. The protein extracts were immunoblotted with an anti-Jak2 antibody (Fig. 1, top). The blot shows that Jak2 is only expressed in the γ2A/AT1+Jak2 cells. By stripping the membrane and reprobing with an anti-STAT1 antibody, we demonstrated equal sample loading by detecting endogenous STAT1 protein (Fig. 1, bottom). Thus, while both clones expressed AT1 receptor at similar affinity and abundance, only the γ2A/AT1+Jak2 clone expressed Jak2 protein.We next wanted to determine whether angiotensin II-stimulated, Jak2-dependent signaling was in fact lost in the cells lacking Jak2 but intact in the cells expressing Jak2. Upon angiotensin II stimulation, Jak2 becomes phosphorylated and associates with the AT1 receptor (20.Ali M.S. Sayeski P.P. Dirksen L.B. Hayzer D.J. Marrero M.B. Bernstein K.E. J. Biol. Chem. 1997; 272: 23382-23388Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). To show that these events do not occur in γ2A/AT1 cells but do occur in γ2A/AT1+Jak2 cells, both sets of cells were stimulated with 100 nm angiotensin II for 0, 5, and 15 min. The cells were then lysed, and protein was extracted. The protein extracts were immunoprecipitated with an anti-phosphotyrosine antibody and then immunoblotted with an anti-Jak2 antibody to detect phosphorylated Jak2 protein. In the γ2A/AT1 cells, angiotensin II treatment failed to induce Jak2 tyrosine phosphorylation (Fig. 2A). However, in γ2A/AT1+Jak2 cells, angiotensin II stimulated rapid tyrosine phosphorylation of Jak2.Fig. 2Angiotensin II-mediated signaling in the γ2A/AT1 cells and γ2A/AT1+Jak2 cells. γ2A/AT1 cells and γ2A/AT1+Jak2 cells were treated with 100 nm angiotensin II for the indicated times, and lysates were prepared. A, lysates were immunoprecipitated with anti-Tyr(P) mAb and Western blotted with anti-Jak2 mAb to detect tyrosine-phosphorylated Jak2. B, lysates were immunoprecipitated with anti-HA mAb to immunoprecipitate the HA-tagged AT1 receptor and then Western blotted with anti-Jak2 polyclonal antibody to detect Jak2/AT1 co-association. C, lysates were immunoprecipitated with anti-Tyr(P) mAb and Western blotted with anti-STAT1 mAb to detect tyrosinephosphorylated STAT1. D, lysates were immunoprecipitated with anti-Tyr(P) mAb and Western blotted with anti-STAT3 mAb to detect tyrosine-phosphorylated STAT3. E, lysates were immunoprecipitated with anti-Tyr(P) mAb and Western blotted with anti-paxillin mAb to detect tyrosine-phosphorylated paxillin. Shown is one of four (A, C, and D) or three (B and E) independent results for each. IP, immunoprecipitation; IB, immunoblot.View Large Image Figure ViewerDownload Hi-res image Download (PPT)After Jak2 is activated, it binds to the AT1 receptor in response to angiotensin II (20.Ali M.S. Sayeski P.P. Dirksen L.B. Hayzer D.J. Marrero M.B. Bernstein K.E. J. Biol. Chem. 1997; 272: 23382-23388Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). To detect Jak2 co-association with the AT1 receptor, γ2A/AT1 and γ2A/AT1+Jak2 cells were next stimulated with 100 nm angiotensin II for 0, 2, 4, and 8 min. The cells were lysed, protein was extracted, and the protein extracts were immunoprecipitated with an anti-HA antibody to immunoprecipitate the HA-tagged AT1 receptor. The immunoprecipitates were then immunoblotted with an antiJak2 antibody to assess Jak2/AT1 receptor co-association. Angiotensin II-dependent Jak2/AT1 receptor co-association was absent in the γ2A/AT1 cells but was seen in the γ2A/AT1+Jak2 cells (Fig. 2B).After becoming phosphorylated and associating with the AT1 receptor following angiotensin II stimulation, Jak2 recruits STAT proteins to the receptor and mediates their tyrosine phosphorylation (25.Marrero M.B. Scheiffer B. Paxton W.G. Heerdt L. Berk B.C. Delafontaine P. Bernstein K.E. Nature. 1995; 375: 247-250Crossref PubMed Scopus (649) Google Scholar). Therefore we next examined the ability of angiotensin II to stimulate tyrosine phosphorylation of STAT1 and STAT3 as a function of Jak2 expression. First, γ2A/AT1 and γ2A/AT1+Jak2 cells were stimulated with 100 nm angiotensin II for 0, 5, and 15 min. The cells were then lysed, and protein was extracted. The protein extracts were immunoprecipitated with an anti-phosphotyrosine antibody and immunoblotted with an anti-STAT1 antibody to detect tyrosine-phosphorylated STAT1 (Fig. 2C). The results show that while angiotensin II failed to induce a marked increase in STAT1 tyrosine phosphorylation in the γ2A/AT1 cells, it did promote increased STAT1 tyrosine phosphorylation in the γ2A/AT1+Jak2 cells. The same membrane was then stripped and immunoblotted with an anti-STAT3 antibody to detect tyrosine-phosphorylated STAT3 (Fig. 2D). Again the results show that STAT3 becomes tyrosine-phosphorylated in response to angiotensin II in the γ2A/AT1+Jak2 cells but not in the γ2A/AT1 cells. Collectively, these data show that, in γ2A/AT1+Jak2 cells, phosphorylation of the downstream target proteins of Jak2, namely STAT1 and STAT3, occurs in response to angiotensin II but that phosphorylation of STAT1 and STAT3 is virtually absent in cells lacking Jak2.Upon angiotensin II treatment of cells, the cytoskeletal protein paxillin becomes tyrosine-phosphorylated (26.Leduc I. Meloche S. J. Biol. Chem. 1995; 270: 4401-4404Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). This event happens very rapidly and occurs independently of Jak2. We therefore expected that paxillin signaling would be intact in both the γ2A/AT1 and γ2A/AT1+Jak2 cells. To test this, both cell types were stimulated with 100 nm angiotensin II for 0, 1, and 5 min. The cells were lysed, and protein was extracted. The protein extracts were immunoprecipitated with an anti-phosphotyrosine antibody and immunoblotted with an anti-paxillin antibody to detect tyrosine-phosphorylated paxillin (Fig. 2E). The results show that in both cell types, paxillin was rapidly tyrosine-phosphorylated in response to angiotensin II treatment, thereby indicating that angiotensin II signaling events, which are independent of Jak2, function normally in both cell types.Collectively, the data in Fig. 2 demonstrate that, in γ2A/AT1 cells, functional Jak2-dependent signaling has been lost, while Jak2-independent signaling events have been preserved. As such, these cells are good vehicles for studying Jak2-dependent signaling.ERK2 Activity Is Sustained in γ2A/AT1 Cells Compared with γ2A/AT1+Jak2 Cells following Angiotensin II Treatment—Previous work demonstrated that inhibition of Jak2 phosphorylation using AG490 blocks angiotensin II-dependent activation of ERK2, thus implicating Jak2 as essential in ERK2 activation (12.Marrero M.B. Schieffer B. Li B. Sun J. Harp J.B. Ling B.N. J. Biol. Chem. 1997; 272: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Recently, however, the specificity of AG490 for Jak2 has come under scrutiny (13.Kleinberger-Doron N. Shelah N. Capone R. Gazit A. Livitzki A. Exp. Cell Res. 1998; 241: 340-351Crossref PubMed Scopus (43) Google Scholar, 14.Oda Y. Renaux B. Bjorge J. Saifeddine M. Fujita D.J. Hollenberg M.D. Can. J. Physiol. Pharmacol. 1999; 77: 606-617Crossref PubMed Scopus (74) Google Scholar). We therefore sought to determine the role that Jak2 plays in angiotensin II-dependent ERK2 activity using the γ2A-derived cells. γ2A/AT1 and γ2A/AT1+Jak2 cells were stimulated with 100 nm angiotensin II for 0, 6, 12, 18, 24, and 30 min. The cells were lysed, and protein was extracted. The protein extracts were immunoblotted with an anti-phospho-ERK2 antibody that detects phosphorylated ERK2 protein. In the cells lacking Jak2, angiotensin II stimulation resulted in a rapid and sustained increase in ERK2 activation that persisted for 30 min (Fig. 3A, top). However, in the γ2A/AT1+Jak2 cells, angiotensin II caused an increase in ERK2 activity that peaked at 6–12 min and returned to basal levels 18–24 min after angiotensin II stimulation. The membrane was subsequently stripped and reprobed with an anti-ERK2 antibody to show constant ERK2 expression at all time points (Fig. 3A, bottom).Fig. 3Angiotensin II-dependent ERK2 activity in γ2A/AT1 cells and γ2A/AT1+Jak2 cells. A, γ2A/AT1 and γ2A/AT1+Jak2 cells were treated with 100 nm angiotensin II for the indicated times. Whole cell lysates were prepared and Western blotted with anti-phospho-ERK2 antibody to detect activated ERK2. The membrane was subsequently stripped and reprobed with anti-ERK2 antibody to confirm total ERK2 protein levels. B, the three membranes representing A were subjected to densimetric analysis. Anti-phospho-ERK2 signal was plotted as a function of both angiotensin II treatment and Jak2 expression. *, p < 0.01; **, p < 0.05. C, γ2A/AT1 cells and γ2A/AT1+Jak2 cells were treated with 100 nm angiotensin II for the indicated times. Lysates were prepared and immunoprecipitated with anti-ERK2 mAb and then resuspended in kinase reaction buffer. Phosphorylation of myelin basic protein (MBP) was detected by autoradiography. Shown is one of three independent results for A and C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The three different membranes representing Fig. 3A were scanned for densimetric analysis, and ERK2 phosphorylation was plotted as a function of angiotensin II treatment (Fig. 3B). The graph shows that in the cells expressing Jak2, ERK2 phosphorylation transiently increased, peaking 6 min after angiotensin II treatment. However, in cells lacking Jak2, ERK2 phosphorylation was significantly elevated 30 min after angiotensin II stimulation. Longer time course studies using the γ2A/AT1 cells demonstrated that ERK2 phosphorylation did not return to basal level until about 120 min after angiotensin II treatment (data not shown). Thus, the data in Fig. 3A suggest that loss of Jak2 expression via a null mutation results in sustained ERK2 phosphorylation in response to angiotensin II. This is contrary to previously published data, which suggested that inhibiting Jak2 kinase function using AG490 results in diminished angiotensin II-dependent ERK2 phosphorylation (12.Marrero M.B. Schieffer B. Li B. Sun J. Harp J.B. Ling B.N. J. Biol. Chem. 1997; 272: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). To verify our results using an alternate protocol, in vitro kinase assays were performed using myelin basic protein as a substrate for ERK2 phosphorylation. In γ2A/AT1 cells, phosphorylation of myelin basic protein remained elevated 20 min after angiotensin II treatment, suggesting that ERK2 was catalytically active at this time point (Fig. 3C). However, in γ2A/AT1+Jak2 cells, angiotensin II stimulated ERK2 activity with peak activity occurring around 5–10 min. By 15–20 min, the 32P-labeled myelin basic protein signal was similar to that of basal conditions. Collectively the results in Fig. 3 clearly show that loss of Jak2 expression via a Jak2 null mutation results in enhanced ERK2 activation in response to angiotensin II when compared with Jak2-expressing control cells.AG490 Suppresses Angiotensin II-dependent ERK2 Activation Independently of Jak2—We next wanted to determine whether treating the Jak2-e" @default.
• W2009098875 created "2016-06-24" @default.
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• W2009098875 creator A5038232326 @default.
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• W2009098875 creator A5054584375 @default.
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• W2009098875 date "2004-01-01" @default.
• W2009098875 modified "2023-10-18" @default.
• W2009098875 title "Jak2 Tyrosine Kinase Mediates Angiotensin II-dependent Inactivation of ERK2 via Induction of Mitogen-activated Protein Kinase Phosphatase 1" @default.
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