Dependence on the Motif YIPP for the Physical Association of Jak2 Kinase with the Intracellular Carboxyl Tail of the Angiotensin II AT1 Receptor
1997; Elsevier BV; Volume: 272; Issue: 37 Linguagem: Inglês
10.1074/jbc.272.37.23382
ISSN1083-351X
AutoresMohammad Ali, Peter P. Sayeski, Laurie B. Dirksen, David J. Hayzer, Mario B. Marrero, Kenneth E. Bernstein,
Tópico(s)Urticaria and Related Conditions
ResumoAngiotensin II is the effector molecule of the renin-angiotensin system. Virtually all of its biochemical actions are mediated through a single class of cell-surface receptors called AT1. These receptors contain the structural features of the seven-transmembrane, G-protein-coupled receptor superfamily. Angiotensin II, acting through the AT1 receptor, also stimulates the Jak/STAT pathway by inducing ligand-dependent Jak2 tyrosine phosphorylation and activation. Here, we show that a glutathione S-transferase fusion protein containing the carboxyl-terminal 54 amino acids of the rat AT1A receptor physically binds to Jak2 in an angiotensin II-dependent manner. Deletional analysis, using both in vitro protocols and cell transfection analysis, showed that this association is dependent on the AT1Areceptor motif YIPP (amino acids 319–322). The wild-type AT1A receptor can induce Jak2 tyrosine phosphorylation. In contrast, an AT1A receptor lacking the YIPP motif is unable to induce ligand-dependent phosphorylation of Jak2. Competition experiments with synthetic peptides suggest that each of the YIPP amino acids, including tyrosine 319, is important in Jak2 binding to the AT1A receptor. The binding of the AT1A receptor to the intracellular tyrosine kinase Jak2 supports the concept that the seven-transmembrane superfamily of receptors can physically associate with enzymatically active intracellular proteins, creating a signaling complex mechanistically similar to that observed with growth factor and cytokine receptors. Angiotensin II is the effector molecule of the renin-angiotensin system. Virtually all of its biochemical actions are mediated through a single class of cell-surface receptors called AT1. These receptors contain the structural features of the seven-transmembrane, G-protein-coupled receptor superfamily. Angiotensin II, acting through the AT1 receptor, also stimulates the Jak/STAT pathway by inducing ligand-dependent Jak2 tyrosine phosphorylation and activation. Here, we show that a glutathione S-transferase fusion protein containing the carboxyl-terminal 54 amino acids of the rat AT1A receptor physically binds to Jak2 in an angiotensin II-dependent manner. Deletional analysis, using both in vitro protocols and cell transfection analysis, showed that this association is dependent on the AT1Areceptor motif YIPP (amino acids 319–322). The wild-type AT1A receptor can induce Jak2 tyrosine phosphorylation. In contrast, an AT1A receptor lacking the YIPP motif is unable to induce ligand-dependent phosphorylation of Jak2. Competition experiments with synthetic peptides suggest that each of the YIPP amino acids, including tyrosine 319, is important in Jak2 binding to the AT1A receptor. The binding of the AT1A receptor to the intracellular tyrosine kinase Jak2 supports the concept that the seven-transmembrane superfamily of receptors can physically associate with enzymatically active intracellular proteins, creating a signaling complex mechanistically similar to that observed with growth factor and cytokine receptors. The analysis of cytokines and their receptors has implicated the intracellular Jak family of kinases as critically important for the intracellular signaling initiated in response to ligand (1Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4950) Google Scholar, 2Briscoe J. Kohlhuber F. Muller M. Trends Cell Biol. 1996; 6: 336-340Abstract Full Text PDF PubMed Scopus (73) Google Scholar, 3Ihle J.N. Philos. Trans. R Soc. Lond. B Biol. Sci. 1996; 351: 159-166Crossref PubMed Scopus (66) Google Scholar). Cytokines induce receptor dimerization and the activation, via tyrosine phosphorylation, of the associated Jak kinases. The Jak kinases phosphorylate the cytokine receptors, leading to the binding and eventual activation of intermediate signaling molecules referred to as STAT (signal transducers andactivators of transcription). The STAT proteins are a family of transcription factors that migrate to the nucleus and induce gene transcription (4Schindler C. Darnell Jr., J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1640) Google Scholar). The Jak/STAT pathway was first elucidated through the study of interferon signaling, but it is now known that this pathway participates in the signaling initiated by a wide variety of cytokines and growth factors. Recently, the vasoactive peptide angiotensin II was also found to activate the Jak/STAT pathway (5Marrero M.B. Schieffer B. Paxton W.G. Heerdt L. Berk B.C. Delafontaine P. Bernstein K.E. Nature. 1995; 375: 247-250Crossref PubMed Scopus (649) Google Scholar).Angiotensin II is the effector molecule of the renin-angiotensin system. It is an 8-amino acid peptide that induces several physiologic responses that act to raise blood pressure. Virtually all of its biochemical actions are mediated through a single class of cell-surface receptors called AT1 (6Timmermans P.B. Wong P.C. Chiu A.T. Herblin W.F. Benfield P. Carini D.J. Lee R.J. Wexler R.R. Saye J.A. Smith R.D. Pharmacol. Rev. 1993; 45: 206-251Google Scholar). Whereas humans have a single AT1 receptor gene, rodents possess two genes encoding highly homologous receptor isoforms termed AT1A and AT1B. These proteins are 95% identical and appear to bind ligand and to signal in an identical fashion (7Bernstein K.E. Berk B.C. Am. J. Kidney Dis. 1993; 22: 745-754Abstract Full Text PDF PubMed Scopus (87) Google Scholar, 8Tian Y. Baukal A.J. Sandberg K. Bernstein K.E. Balla T. Catt K.J. Am. J. Physiol. 1996; 270: E831-E839PubMed Google Scholar). All AT1 receptors contain the structural features of the seven-transmembrane, G-protein-coupled receptor superfamily and are structurally quite different from cytokine receptors. However, studies by our group (5Marrero M.B. Schieffer B. Paxton W.G. Heerdt L. Berk B.C. Delafontaine P. Bernstein K.E. Nature. 1995; 375: 247-250Crossref PubMed Scopus (649) Google Scholar) and by Baker and co-workers (9Bhat G.J. Thekkumkara T.J. Thomas W.G. Conrad K.M. Baker K.M. J. Biol. Chem. 1994; 269: 31443-31449Abstract Full Text PDF PubMed Google Scholar, 10Bhat G.J. Thekkumkara T.J. Thomas W.G. Conrad K.M. Baker K.M. J. Biol. Chem. 1995; 270: 19059-19065Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) have independently demonstrated that angiotensin II, acting through the AT1receptor, also stimulates the Jak/STAT pathway. In rat aortic smooth muscle (RASM) 1The abbreviations used are: RASM, rat aortic smooth muscle; DMEM, Dulbecco's modified Eagle's medium; GST, glutathione S-transferase. 1The abbreviations used are: RASM, rat aortic smooth muscle; DMEM, Dulbecco's modified Eagle's medium; GST, glutathione S-transferase. cells, angiotensin II leads to the rapid tyrosine phosphorylation and activation of Jak2 (5Marrero M.B. Schieffer B. Paxton W.G. Heerdt L. Berk B.C. Delafontaine P. Bernstein K.E. Nature. 1995; 375: 247-250Crossref PubMed Scopus (649) Google Scholar). Angiotensin II also induces the physical association of Jak2 with the AT1 receptor. The AT1 receptor contains no intrinsic kinase activity. However, it is now known that ligand occupancy of this receptor stimulates several different intracellular signaling cascades in which tyrosine phosphorylation plays an important role (11Bernstein K.E. Marrero M.B. Trends Cardiovasc. Med. 1996; 6: 179-187Crossref PubMed Scopus (27) Google Scholar). At present, the structural features of the AT1 receptor necessary for intracellular tyrosine kinase activation are not understood. In this study, we show that the carboxyl-terminal 54 amino acids of the rat AT1A receptor physically bind to Jak2 in an angiotensin II-dependent manner. Both in vitro and in vivo analyses show that this association is dependent on the AT1A receptor motif YIPP (amino acids 319–322). The binding of the AT1A receptor to the intracellular tyrosine kinase Jak2 supports the concept that the seven-transmembrane superfamily of receptors can physically associate with enzymatically active intracellular proteins, creating a signaling complex mechanistically similar to that observed with growth factor and cytokine receptors.RESULTS AND DISCUSSIONA GST fusion protein containing the carboxyl-terminal 54 amino acids of the rat AT1A receptor was purified to homogeneity. As a control, equally pure GST protein was made. Cultured RASM cells were treated with 100 nm angiotensin II for time points of 0.5–10 min. A cell lysate was prepared, and 1 mg of cell protein was mixed with 5 μg of the GST-AT1A fusion protein bound to glutathione-agarose beads. After a 2-h incubation, the beads were washed, and any remaining bound proteins were eluted by boiling. Jak2 was detected by Western blot analysis using a polyclonal anti-Jak2 antibody. As shown in Fig. 1 A, there was both a ligand- and a time-dependent binding of Jak2 to the GST-AT1A fusion protein. In the absence of angiotensin II (lane 1), Jak2 did not bind to the fusion protein, an observation that was confirmed in >10 separate experiments. Maximal Jak2 binding was observed 3 min after the addition of angiotensin II (lane 4). Treating RASM cells with angiotensin II for longer periods of time (lanes 5 and6) resulted in less Jak2 binding to the GST-AT1Afusion protein. An equivalent experiment was performed using GST (lanes 7 and 8); no Jak2 binding to GST was detected.We have also used an identical protocol to investigate the binding of Jak1 and Tyk2. In contrast to Jak2, angiotensin II induced no detectable complex formation of Jak1 or Tyk2 with the GST-AT1A fusion protein (data not shown).To investigate the specificity of the binding assay, we covalently linked the GST-AT1A fusion protein to an agarose matrix. This preparation allowed us to compete for the binding of Jak2 to the GST fusion protein matrix with increasing concentrations of free GST-AT1A fusion protein, GST, or an irrelevant protein such as albumin (Fig. 1 B). Lane 1 shows that in the absence of a competitor, there was abundant binding of Jak2 to the GST-AT1A fusion protein matrix. Increasing concentrations of free GST-AT1A fusion protein competed with this binding (lanes 2–4). In contrast, free GST or bovine serum albumin showed no competition with the binding of Jak2 to the fusion protein matrix (lanes 5–8).To identify specific regions within the AT1A receptor carboxyl tail important for the association with Jak2, we created a series of GST fusion proteins containing overlapping portions of the AT1A receptor tail (Fig. 2,upper panel). Each of these proteins was purified and individually tested for the ability to bind to Jak2 kinase. As before, Jak2 association was quantitated by Western blot analysis. We observed that fusion proteins containing the motif YIPP (Tyr-Ile-Pro-Pro, amino acids 319–322) bound Jak2 in a fashion similar to the full-length construct (Fig. 2, lower panel, lanes 1–4,7, and 10). Receptor fusion proteins lacking this motif showed either markedly reduced or no binding of Jak2 (lanes 5, 6, 8, 9, and11).The carboxyl-terminal 54 amino acids of the AT1A receptor contain tyrosine residues at positions 319 and 339. While Jak2 association with cytokine receptors is not thought to be dependent on tyrosines, the presence of a tyrosine in the 319YIPP motif convinced us to make GST-AT1A fusion proteins in which these individual tyrosine residues were converted to phenylalanine. When the conversion at position 319 was made, the GST fusion protein bound less Jak2 than the parent protein (Fig. 3, lower panel, lane 2). This was consistently observed in five separate experiments. In contrast, the Y339F change had no effect on Jak2 binding (lane 3).To further investigate the specificity of the YIPP motif, we created an 8-amino acid peptide (KYIPPKAK) corresponding to positions 318–325 of the AT1A receptor. As controls, we also synthesized this peptide in a scrambled configuration (IKKPAPYK) and a peptide corresponding to positions 311–318 of the AT1A receptor (KYFLQLLK). Increasing amounts of the synthetic peptides were used to compete for the angiotensin II-dependent binding of Jak2 to a GST-AT1A fusion protein containing the carboxyl-terminal 54 amino acids of the receptor (Fig. 4 A). Peptide P1 demonstrated a dose-dependent inhibition of Jak2 binding to the AT1A receptor fusion protein (lanes 2–4). No inhibition was observed using either the scrambled peptide (P2) or the peptide encompassing amino acids 311–318 (P3) (lanes 5–10). Even when 50 μg of competitor peptide P2 or P3 was used, no competition was observed (data not shown). On a molar basis, peptide P1 is a less effective inhibitor than the entire GST-AT1Afusion protein (Fig. 1 B), a finding probably related to the small size of the peptide.Figure 4Importance of the YIPP motif. A,lane 1 shows the binding of Jak2 to a GST-AT1Areceptor fusion protein containing the carboxyl-terminal 54 amino acids of the receptor. The addition of 1, 5, or 10 μg of peptide P1 (KYIPPKAK), encoding amino acids 318–325 of the AT1Areceptor (lanes 2–4), inhibited the binding of Jak2 in a dose-dependent fashion. Peptide P2 (IKKPAPYK) contains the same amino acids as peptide P1, but in a scrambled configuration. This peptide showed no inhibition of Jak2 binding (lanes 5–7). Peptide P3 (KYFLQLLK), containing amino acids 311–318 of the AT1A receptor, also did not inhibit the binding of Jak2 (lanes 8–10). All experiments were performed with a RASM cell lysate prepared from cells treated with angiotensin II for 3 min.B, inhibition studies similar to those described inA were performed with 10 μg of peptides P1 and P4–P8. Peptides P4–P6 contain single alanine substitutions for the sequence IPP (amino acids 320–322), but were unable to inhibit the binding of Jak2 to the GST-AT1A receptor fusion protein. Peptide P8, containing phenylalanine in place of tyrosine 319, has a reduced capacity to inhibit the binding of Jak2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The specificity of individual amino acids within the YIPP motif was tested by synthesizing peptides P4–P8 and testing the ability of each peptide to inhibit Jak2 binding to the GST-AT1A fusion protein (Fig. 4 B). Competition assays were performed using 10 μg of competitor peptide. These data demonstrate that peptides containing a single change in the IPP portion of peptide P1 were no longer able to compete (Peptides P4–P6). Peptide P7, containing a 3-amino acid substitution, was also unable to compete. Peptide P8, containing FIPP in place of YIPP, showed a partial ability to inhibit Jak2 binding to the GST-AT1A fusion protein. These results are consistent with data in Fig. 3, showing that a GST-AT1Afusion protein with a Y319F mutation bound Jak2, but with reduced efficiency.To investigate the behavior of the AT1A receptor carboxyl tail in vivo, RASM cells were electroporated with the GST-AT1A fusion protein; deletion construct D-35, D-55, or D-85; or GST (Fig. 5). We have previously shown that electroporation is an effective method of inserting proteins into RASM cells (15Marrero M.B. Schieffer B. Paxton W.G. Schieffer E. Bernstein K.E. J. Biol. Chem. 1995; 270: 15734-15738Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). After electroporation, the cells were incubated for 30 min at 37 °C and then washed extensively. Angiotensin II was added for 3 min, and the cells were lysed and incubated with glutathione-Sepharose beads at 4 °C. The beads were washed with buffer containing 1 m NaCl and eluted with SDS sample buffer. Jak2 was then assessed by Western blot analysis. In the absence of angiotensin II, no Jak2 associated with the GST-AT1Afusion protein (data not shown). However, after angiotensin II addition, both the GST-AT1A fusion protein and deletion construct D-35 bound Jak2 (Fig. 5, lanes 1 and2). In contrast, deletion constructs D-55 and D-85, both of which lack the YIPP motif, bound very much less Jak2 (lanes 3and 4). No binding of Jak2 was observed with GST (lane 5). These data recapitulate the in vitrostudies and indicate the importance of the YIPP sequence.Figure 5Insertion of fusion proteins into cells.RASM cells were electroporated with the indicated GST fusion proteins (defined in Fig. 2) using a protocol that has previously been shown to be effective in inserting proteins into these adherent cells (15Marrero M.B. Schieffer B. Paxton W.G. Schieffer E. Bernstein K.E. J. Biol. Chem. 1995; 270: 15734-15738Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). The cells were then cultured for 30 min and washed. Angiotensin II was added for 3 min, and the cells were lysed. Fusion proteins were collected by affinity for glutathione and washed with buffer containing 1 m NaCl. Associated Jak2 was determined by Western blot analysis. Fusion proteins containing the AT1A receptor YIPP motif bound Jak2 (lanes 1 and 2), whereas proteins lacking this motif bound very little or no Jak2 (lanes 3–5). WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We have also used a cell transfection approach to evaluate the importance of the YIPP motif. These experiments used the mammalian expression vector pZeo containing either a full-length, wild-type AT1A receptor (called pZeo/WT) or a construct in which the YIPP motif was converted to FAAA (called pZeo/FAAA). Transient transfection was performed using COS-7 cells, which contain little endogenous AT1 receptor or Jak2 (data not shown). Initial experiments used standard procedures to measure the binding of [125I-Sar1,Ile8]angiotensin II to the wild-type receptor or the FAAA mutant expressed in these cells. Scatchard analysis indicated K d values of 0.172 nm for the wild-type receptor and 0.225 nm for the FAAA mutant. Thus, the conversion of the YIPP motif to FAAA does not markedly affect ligand affinity.To measure the physical association of the AT1A receptor with Jak2, constructs encoding both these proteins were transfected into COS-7 cells (Fig. 6). Two days after transfection, the cells were treated with angiotensin II for 0, 3, 6, or 15 min. Cells were lysed, and proteins were immunoprecipitated using a rabbit polyclonal anti-rat AT1A receptor antibody and Protein A/G. The precipitated proteins were washed, and associated Jak2 was measured by Western blot analysis using polyclonal anti-Jak2 antibody. Controls for this protocol included transfection of the vector pZeo lacking any angiotensin II receptor insert (lanes 1–4). When pZeo/WT was cotransfected with a plasmid encoding Jak2 (lanes 5–8), treatment of cells with angiotensin II for 3 min induced the physical association of the AT1A receptor with Jak2 (lane 6). This experiment was performed four times, and association was typically strongest after 3 min of ligand treatment. However, other repetitions of the experiment indicated association above the background levels at the 6-min point (data not shown). Equivalent cotransfection experiments were also performed with pZeo/FAAA (lanes 9–12). In contrast to the wild type, an AT1A receptor bearing FAAA in place of the YIPP motif never showed ligand-dependent association of the receptor with Jak2.Figure 6Cotransfection of the AT1Areceptor and Jak2. Using Lipofectin, COS-7 cells were cotransfected with plasmid pBOSwtJk2 encoding murine Jak2 (16Zhuang 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) and the expression vector pZeo (lanes 1–4), pZeo encoding the wild-type rat AT1A receptor (pZeo/WT) (lanes 5–8), or pZeo containing an AT1A construct in which the YIPP motif was converted to FAAA (pZeo/FAAA) (lanes 9–12). Two days after transfection, the cells were treated with angiotensin II for 0, 3, 6, or 15 min. Cells were lysed, and proteins were precipitated with polyclonal anti-AT1A antisera. Associated Jak2 was measured by Western blot analysis. In response to angiotensin II, the wild-type AT1A receptor formed a complex with Jak2 that peaked at 3 min (lane 6) and often remained above background levels at 6 min. In four separate experiments, no such ligand-dependent complex was observed with the pZeo/FAAA construct.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In smooth muscle cells, angiotensin II stimulates the tyrosine phosphorylation of Jak2 (5Marrero M.B. Schieffer B. Paxton W.G. Heerdt L. Berk B.C. Delafontaine P. Bernstein K.E. Nature. 1995; 375: 247-250Crossref PubMed Scopus (649) Google Scholar). We have analyzed this using cotransfection of the AT1A receptor and Jak2 into COS-7 cells. Two days after transfection, cells were treated with angiotensin II, lysed, and immunoprecipitated with polyclonal anti-Jak2 antibody. Proteins were collected, washed, and analyzed by Western blotting using monoclonal anti-phosphotyrosine antibodies (Fig. 7 A). The blot was then stripped and reprobed with anti-Jak2 antibody to verify equivalent loading of protein (Fig. 7 B). In the absence of ligand, there was a basal level of Jak2 phosphorylation. In response to ligand, the wild-type receptor induced increased tyrosine phosphorylation of Jak2 (Fig. 7 A, lanes 5–8). In contrast, no such increase was ever seen with the AT1A receptor containing the FAAA mutation (lanes 9–12). In the absence of transfected Jak2 (lane 13), no endogenous Jak2 was identified with this protocol.Figure 7Tyrosine phosphorylation of Jak2. A, COS-7 cells were transfected with plasmid pBOSwtJk2 encoding murine Jak2 (16Zhuang 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) and pZeo (lanes 1–4), pZeo/WT (lanes 5–8), or pZeo/FAAA (lanes 9–13) as described in the legend to Fig. 6. After the addition of angiotensin II, the level of Jak2 tyrosine phosphorylation was measured by immunoprecipitating cellular proteins with anti-Jak2 antibody, followed by Western blot analysis of the precipitated proteins with anti-phosphotyrosine antibody. The wild-type AT1A receptor induced increased Jak2 tyrosine phosphorylation in response to angiotensin II (lanes 5–8). No such increase was observed with the AT1A receptor lacking the YIPP motif (lanes 9–12). In the absence of transfected Jak2 (lane 13), no Jak2 was identified using this protocol. B, the Western blots from A were stripped and reprobed with anti-Jak2 antibody to verify equivalent loading of protein within each group.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The major observation in this study is the binding of Jak2 to a GST fusion protein containing the carboxyl-terminal portion of the rat AT1A receptor. This association appears to be dependent on the receptor motif YIPP and is stable to washing in 1.5 mNaCl (Fig. 2, lower panel, lane 10). Transient cellular expression of either the wild-type AT1A receptor or the receptor containing a mutation of the YIPP motif confirmed the importance of this sequence in the association of Jak2 with the seven-transmembrane AT1A receptor. The YIPP motif also appears to be important in angiotensin II-dependent tyrosine phosphorylation of Jak2. These data are consistent with previously published observations showing the coprecipitation of the AT1A receptor and Jak2 from RASM cells (5Marrero M.B. Schieffer B. Paxton W.G. Heerdt L. Berk B.C. Delafontaine P. Bernstein K.E. Nature. 1995; 375: 247-250Crossref PubMed Scopus (649) Google Scholar). In that study, the association of Jak2 with the AT1A receptor was dependent on the addition of angiotensin II to the RASM cells. This dependence on ligand for binding Jak2 was recapitulated in both thein vitro and in vivo studies reported here. At present, the precise changes induced by angiotensin II are not known. Presumably, ligand binding to the AT1A receptors within cells changes the chemistry of these cells such that the Jak2 present in a cell lysate now binds to the GST-receptor fusion proteins. Whether our protocol is stripping activated Jak2 from endogenous receptors or whether activated Jak2 is naturally shuttling onto and off of AT1 receptors is not known. Indeed, it is not known whether the association of Jak2 with the GST-AT1A fusion protein is a bimolecular event or whether additional linker molecules participate. Finally, we are not certain if the Jak2-AT1A association that occurs within cells is a precedent or a consequence of Jak2 activation.We have considered the possibility that the interaction of Jak2 with the GST-AT1A fusion protein is the result of the YIPP sequence acting as a substrate-binding site for Jak2. This seems unlikely given that Jak2 still shows affinity for a receptor fusion protein lacking Tyr319 (Fig. 3). A peptide lacking this same tyrosine also showed some ability to inhibit the interaction of Jak2 with the GST-AT1A fusion protein (Fig. 4 B). To directly address this issue, we measured Jak2 binding to the GST-AT1A fusion protein in the presence of increasing concentrations of AG-490, a selective inhibitor of Jak2 kinase activity (Fig. 8) (17Meydan N. Grunberger T. Dadi H. Shahar M. Arpaia E. Lapidot Z. Leeder J.S. Freedman M. Cohen A. Gazit A. Levitzki A. Roifman C.M. Nature. 1995; 379: 645-648Crossref Scopus (847) Google Scholar). This compound was added to a RASM cell lysate before the addition of the GST-AT1Afusion protein. Even in the presence of 50 μm AG-490, no reduction in Jak2 binding to the GST-AT1A fusion protein was observed.Figure 8Effect of the Jak2 inhibitor AG-490.RASM cells were treated with angiotensin II for 3 min, and a cell lysate was prepared. AG-490 was added to a final concentration of 0, 10, or 50 μm, with all samples receiving an equivalent amount of the vehicle Me2SO. The lysate was incubated for 30 min at room temperature, and Jak2 binding to the GST-AT1A fusion protein was then measured as described under "Experimental Procedures." AG-490 had no effect on the association of Jak2 with the GST-AT1A fusion protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Previous studies of Jak2 binding to cytokine receptors have demonstrated the importance of the "box 1" motif (18Murakami M. Narazaki M. Hibi M. Yawata H. Yazukawa K. Hamagu-chi M. Taga T. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11349-11353Crossref PubMed Scopus (486) Google Scholar, 19Lebrun J.-J. Ali S. Ullrich A. Kelly P.A. J. Biol. Chem. 1995; 270: 10664-10670Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 20Colamonici O. Yan H. Domanski P. Handa R. Smalley D. Mullersman J. Witte M. Krishnan K. Krolewski J. Mol. Cell. Biol. 1994; 14: 8133-8142Crossref PubMed Google Scholar, 21Dinerstein H. Lago F. Goujon L. Ferrag F. Esposito N. Finidori J. Kelly P.A. Postel-Vinay M.-C. Mol. Endocrinol. 1995; 9: 1701-1707Crossref PubMed Google Scholar, 22Tanner J.W. Chen W. Young R.L. Longmore G.D. Shaw A.S. J. Biol. Chem. 1995; 270: 6523-6530Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). This is a region of 8–11 amino acids containing the consensus sequence PXXPXP, but such a sequence is not found in the AT1A receptor. To our knowledge, the YIPP motif has not been associated with Jak2 binding to cytokine or other receptor types. Whether this sequence acts alone to define the Jak2-binding site or acts in combination with other regions of the AT1 receptor awaits further analysis. In a sense, it is not surprising that the319YIPP motif is functionally important. It is the only region of the AT1A receptor in which 2 prolines are adjacent or separated by 1 amino acid. Previously, we (23Marrero M.B. Schieffer B. Paxton W.G. Duff J.L. Berk B.C. Bernstein K.E. Cardiovasc. Res. 1995; 30: 530-536Crossref PubMed Scopus (35) Google Scholar) and others (24Berk B.C. Corson M.A. Circ. Res. 1997; 80: 607-616Crossref PubMed Scopus (282) Google Scholar) have noted that this region is analogous to the motif YIIP found within the platelet-derived growth factor receptor and the motif YLPP found within the epidermal growth factor receptor. In these growth factor receptors, the sequences have been shown to be SH2 target sequences when the tyrosine is phosphorylated (25Pascal S.M. Singer A.U. Gish G. Yamazaki T. Shoelson S.E. Pawson T. Kay L.E. Forman K.-J.D. Cell. 1994; 77: 461-472Abstract Full Text PDF PubMed Scopus (229) Google Scholar). For instance, phospholipase C-γ1 contains an SH2 domain that interacts with the indicated SH2 target sequences of the platelet-derived growth factor and epidermal growth factor receptors (26Ohyama K. Yamano Y. Chaki S. Kondo T. Inagami T. Biochem. Biophys. Res. Commun. 1992; 189: 677-683Crossref PubMed Scopus (128) Google Scholar, 27Fantl W.J. Johnson D.E. Williams L.T. Annu. Rev. Biochem. 1993; 62: 453-481Crossref PubMed Scopus (927) Google Scholar). In contrast to phospholipase C, the study of cytokine receptors has suggested that Jak2 binding is not dependent on tyrosines and is consistent with the lack of an SH2 domain in these kinases (2Briscoe J. Kohlhuber F. Muller M. Trends Cell Biol. 1996; 6: 336-340Abstract Full Text PDF PubMed Scopus (73) Google Scholar, 3Ihle J.N. Philos. Trans. R S
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