FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2072563654> ?p ?o ?g. }

• W2072563654 endingPage "31198" @default.
• W2072563654 startingPage "31191" @default.
• W2072563654 abstract "Pituitary tumor-transforming gene (PTTG) is a recently characterized oncogene that can act as a transcriptional activator. In this study, we have characterized the transactivation domain of PTTG. Transient transfection of fusion constructs containing GAL4 DNA-binding domain and different parts of PTTG indicated the transactivation domain of PTTG is located between amino acids 119 and 164. Mitogen-activated protein (MAP) kinase cascade is important in the regulation of cell growth, apoptosis, and differentiation. Therefore, we have explored the possibility that this kinase cascade plays a role in regulating PTTG transactivation function. Activation of the MAP kinase cascade by epidermal growth factor or an expression vector for a constitutively active form of the MAP kinase kinase (MEK1) led to stimulation of PTTG transactivation activity. We showed that PTTG is phosphorylated in vitro on Ser162 by MAP kinase and that this phosphorylation site plays an essential role in PTTG transactivation function. We demonstrated that PTTG interacts directly with MEK1 through a putative SH3 domain-binding site located between amino acids 51 and 54 and that this interaction is crucial for PTTG transactivation function. In addition, we showed that activation of MAP kinase phosphorylation cascade resulted in nuclear translocation of PTTG. Together, our data establish that a growth factor-stimulated MAP kinase plays an important role in modulating PTTG function. Pituitary tumor-transforming gene (PTTG) is a recently characterized oncogene that can act as a transcriptional activator. In this study, we have characterized the transactivation domain of PTTG. Transient transfection of fusion constructs containing GAL4 DNA-binding domain and different parts of PTTG indicated the transactivation domain of PTTG is located between amino acids 119 and 164. Mitogen-activated protein (MAP) kinase cascade is important in the regulation of cell growth, apoptosis, and differentiation. Therefore, we have explored the possibility that this kinase cascade plays a role in regulating PTTG transactivation function. Activation of the MAP kinase cascade by epidermal growth factor or an expression vector for a constitutively active form of the MAP kinase kinase (MEK1) led to stimulation of PTTG transactivation activity. We showed that PTTG is phosphorylated in vitro on Ser162 by MAP kinase and that this phosphorylation site plays an essential role in PTTG transactivation function. We demonstrated that PTTG interacts directly with MEK1 through a putative SH3 domain-binding site located between amino acids 51 and 54 and that this interaction is crucial for PTTG transactivation function. In addition, we showed that activation of MAP kinase phosphorylation cascade resulted in nuclear translocation of PTTG. Together, our data establish that a growth factor-stimulated MAP kinase plays an important role in modulating PTTG function. pituitary tumor-transforming gene mitogen-activated protein mitogen-activated protein kinase extracellular signal-regulated kinase MAP/ERK kinase hemagglutinin polyacrylamide gel electrophoresis epidermal growth factor green fluorescent protein glutathione S-transferase signal transducers and activators of transcription phosphate-buffered saline basic fibroblast growth factor PTTG1 was originally isolated by its differential expression in pituitary tumor cells (1Pei L. Melmed S. Mol. Endocrinol. 1997; 11: 433-441Crossref PubMed Scopus (443) Google Scholar). Overexpression of PTTG protein in 3T3 fibroblasts resulted in cell transformation in vitro, and injection of transfected 3T3 cells into nude mice resulted in tumor formation, indicating PTTG is a transforming gene (1Pei L. Melmed S. Mol. Endocrinol. 1997; 11: 433-441Crossref PubMed Scopus (443) Google Scholar, 2Zhang X. Horwitz G.A. Prezant T.R. Valentini A. Nakashoma M. Bronstein M.D. Melmed S. Mol. Endocrinol. 1999; 13: 156-166Crossref PubMed Scopus (278) Google Scholar). In addition to pituitary tumors, PTTG mRNA is also expressed in a variety of primary tumors and tumor cell lines including carcinomas of lung, breast, melanoma, leukemia, and lymphoma (3Zhang X. Horwitz G.A. Heaney A.P. Nakashoma M. Prezant T.R. Bronstein M.D. Melmed S. J. Clin. Endocrinol. & Metab. 1999; 84: 761-767Crossref PubMed Scopus (0) Google Scholar, 4Saez C. Japon M.A. Ramos-Morales F. Romero F. Rios R.M. Dreyfus F. Tortolero M. Pintor-Toro J.A. Oncogene. 1999; 18: 5473-5476Crossref PubMed Scopus (129) Google Scholar), suggesting that PTTG may be involved in tumorigenesis of other tissues in addition to the pituitary. However, among normal adult tissues, PTTG mRNA is only expressed to high levels in the testis, with lower expression detected in thymus and intestines (1Pei L. Melmed S. Mol. Endocrinol. 1997; 11: 433-441Crossref PubMed Scopus (443) Google Scholar, 2Zhang X. Horwitz G.A. Prezant T.R. Valentini A. Nakashoma M. Bronstein M.D. Melmed S. Mol. Endocrinol. 1999; 13: 156-166Crossref PubMed Scopus (278) Google Scholar, 5Dominguez A. Ramos-Morales F. Romero F. Segura D.I. Tortolero M. Pintor-Toro J.A. Oncogene. 1998; 17: 2187-2193Crossref PubMed Scopus (166) Google Scholar). PTTG mRNA is expressed in spermatocytes and spermatids in a stage-specific manner during the rat spermatogenic cycle (6Pei L. J. Biol. Chem. 1999; 274: 3151-3158Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), and an enhancer element important for PTTG transcriptional activation in germ cells has been identified (7Pei L. J. Biol. Chem. 1998; 273: 5219-5225Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Yeast two-hybrid screening identified ribosomal protein S10 and a novel DnaJ homologue HSJ2 as binding partners to PTTG in testicular germ cells (7Pei L. J. Biol. Chem. 1998; 273: 5219-5225Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). These findings suggest that PTTG may be involved in regulating male germ cell differentiation. In a recent study, PTTG was identified as a vertebrate sister-chromatid separation inhibitor, and degradation of PTTG was required for sister-chromatid separation (8Zou H. McGarry T.J. Bernal T. Kirschner M.W. Science. 1999; 285: 418-422Crossref PubMed Scopus (672) Google Scholar). The expression level of PTTG was found to be up-regulated in rapidly proliferating cells and was regulated in a cell cycle-dependent manner, peaking in mitosis (9Ramos-Morales F. Dominguez A. Romero F. Luna R. Multon M.-C. Tortolero M. Pintor-Toro J.A. Oncogene. 2000; 19: 403-409Crossref PubMed Scopus (96) Google Scholar), suggesting that PTTG may play a role in regulatory pathways involved in controlling cell proliferation. Transcriptional regulation is an essential control point for diverse cellular functions such as cell proliferation, differentiation, and transformation. The involvement of PTTG in transcriptional control was first demonstrated by the finding that the C-terminal region of PTTG could act as a transactivation domain when fused to GAL4 DNA-binding domain (5Dominguez A. Ramos-Morales F. Romero F. Segura D.I. Tortolero M. Pintor-Toro J.A. Oncogene. 1998; 17: 2187-2193Crossref PubMed Scopus (166) Google Scholar). One of the target genes for PTTG transactivation function is basic fibroblast growth factor (bFGF). Overexpression of PTTG in transfected cells stimulated expression of bFGF, and point mutations of the putative SH3-binding sites within the C-terminal region of PTTG abrogated the increase in bFGF expression (2Zhang X. Horwitz G.A. Prezant T.R. Valentini A. Nakashoma M. Bronstein M.D. Melmed S. Mol. Endocrinol. 1999; 13: 156-166Crossref PubMed Scopus (278) Google Scholar). Recently, we demonstrated that PTTG was able to transactivate the bFGF transcription in the presence of a novel PTTG-binding factor (10Chien W. Pei L. J. Biol. Chem. 2000; 275: 19422-19427Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). This evidence suggests that transcriptional activation is one of the important mechanisms for PTTG actions. However, the mechanism of PTTG transactivation function and the amino acid sequences involved in this function have yet to be characterized. In this study we have performed detailed deletion and point mutation analysis to localize precisely PTTG transactivation domain. The stimulation of cellular proliferation and differentiation involves the activation of signaling pathways that are initiated by specific receptors at the cell surface. Many of these signaling pathways converge on the mitogen-activated protein (MAP) kinase cascade, a module consisting of MAP kinase kinase (MEK1 and -2, also known as ERK kinase or as MKK), MAP kinase (MAPK 1 and 2, also known as extracellular signal-regulated kinase or ERK), and its downstream targets. These kinases form three successive tiers of a cascade in which MEK phosphorylates and activates MAPK and mitogen-activated protein kinase phosphorylates and activates its downstream target (11Sturgill T.W. Ray L.B. Erikson E. Maller J.L. Nature. 1988; 334: 715-718Crossref PubMed Scopus (751) Google Scholar, 12Gregory J.S. Boulton T.G. Sang B.C. Cobb M.H. J. Biol. Chem. 1989; 264: 18397-18401Abstract Full Text PDF PubMed Google Scholar, 13Ahn N.G. Weiel J.E. Chan C.P. Krebs E.G. J. Biol. Chem. 1990; 265: 11487-11494Abstract Full Text PDF PubMed Google Scholar, 14Ahn N.G. Seger R. Bratlien R.L. Diltz C.D. Tonks N.K. Krebs E.G. J. Biol. Chem. 1991; 266: 4220-4227Abstract Full Text PDF PubMed Google Scholar, 15Matsuda S. Kosoda H. Takenaka K. Moriyama K. Sakai H. Akiyama T. Gotoh Y. Nishida E. EMBO J. 1992; 11: 973-982Crossref PubMed Scopus (160) Google Scholar, 16Gomez N. Cohen P. Nature. 1991; 353: 170-173Crossref PubMed Scopus (417) Google Scholar). An important target of MAP kinase cascade is the regulation of gene expression in the nucleus. MAP kinase was shown to phosphorylate and activate several nuclear transcription factors. The proto-oncogene product c-Myc is phosphorylated on Ser62 by MAP kinase (17Alvarez E. Northwood I.C. Gonzales F.A. Latour D.A. Seth A. Abate C. Curran T. Davis R.J. J. Biol. Chem. 1991; 266: 15277-15285Abstract Full Text PDF PubMed Google Scholar, 18Gupta S. Seth A. Davis R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3216-3220Crossref PubMed Scopus (137) Google Scholar). Phosphorylation at this site is associated with enhanced transactivation function of c-Myc (18Gupta S. Seth A. Davis R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3216-3220Crossref PubMed Scopus (137) Google Scholar, 19Seth A. Alvarez E. Gupta S. Davis R.J. J. Biol. Chem. 1991; 266: 23521-23524Abstract Full Text PDF PubMed Google Scholar, 20Seth A. Gonzales F.A. Gupta S. Raden D.L. Davis R.J. J. Biol. Chem. 1991; 267: 24796-24804Abstract Full Text PDF Google Scholar). Similarly, phosphorylation of NF-IL6 at Thr235 by MAP kinase caused an increase in transactivation of gene expression by NF-IL6 (21Nagajima T. Kinoshita S. Sasagawa T. Sasaki K. Naruto M. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2207-2211Crossref PubMed Scopus (513) Google Scholar). MAP kinase phosphorylation of p62TCF/Elk-1, a component of the ternary complex bound to the serum response element, was shown to be responsible for mitogen-stimulated function of serum response element (22Gille H. Sharrocks A.D. Shaw P.E. Nature. 1992; 358: 414-417Crossref PubMed Scopus (811) Google Scholar, 23Marais R. Wynne J. Treisman R. Cell. 1993; 73: 381-393Abstract Full Text PDF PubMed Scopus (1102) Google Scholar). The possible involvement of PTTG in regulating cellular proliferation as well as the presence of a consensus MAP kinase phosphorylation site (Pro-X-Ser/Thr-Pro) (17Alvarez E. Northwood I.C. Gonzales F.A. Latour D.A. Seth A. Abate C. Curran T. Davis R.J. J. Biol. Chem. 1991; 266: 15277-15285Abstract Full Text PDF PubMed Google Scholar) within the transactivation domain of PTTG led us to investigate whether activation of MAP kinase signal transduction pathway affects PTTG transactivation function. We show here that transactivation function of PTTG is enhanced by activation of MAP kinase cascade. We demonstrate that PTTG is phosphorylated by MAP kinase at Ser162 in vitroand that this site plays a critical role in PTTG transactivation function. We explored the possibility that PTTG directly interacts with one or more components of the MAP kinase cascade. We show here that PTTG interacts with MEK1 through an SH3 domain-binding motif located at the N terminus of PTTG and that this interaction is required to mediate the effect of MAPK cascade activation on PTTG transactivation function. Furthermore, we provide evidence that activation of MAPK phosphorylation cascade leads to PTTG nuclear translocation. Together our data establish that MAP kinase phosphorylation cascade has an important functional role in regulating PTTG transactivation activity. The reporter plasmid (5×GAL4-E1B-LUC) containing five GAL4-binding sites inserted in front of E1B minimal promoter linked to luciferase (24Sun P. Enslen H. Myung P.S. Maurer R.A. Genes Dev. 1994; 8: 2527-2539Crossref PubMed Scopus (635) Google Scholar) was kindly provided by Dr. R. A. Maurer (Oregon Health Science University, Portland, OR). The construction of expression vector containing GAL4 DNA-binding domain (pGAL4DBD) and the GAL4DBD-VP16 fusion constructs were described previously (25Baniahmad A. Kohne A.C. Renkawitz R. EMBO J. 1992; 11: 1015-1023Crossref PubMed Scopus (238) Google Scholar) and were obtained from Dr. Bariahmad (Justus-Liebig-Universitat, Germany). The bFGF-luciferase fusion containing 1 kilobase pair of bFGF promoter linked to luciferase was described previously (10Chien W. Pei L. J. Biol. Chem. 2000; 275: 19422-19427Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The GAL4DBD-PTTG fusion constructs were constructed as follows. The wild type PTTG and the N-terminal 60-amino acid deletion mutants were constructed by digesting pAS2PTTG and pAS2PTTGm2 (6Pei L. J. Biol. Chem. 1999; 274: 3151-3158Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) with NcoI andBamHI. After filling the ends with DNA polymerase I, the blunt ended insert was clone at SmaI site of GAL4DBD to generate GAL-PTTG-(1–199) and GAL-PTTG-(61–199). To generate N-terminal 118-amino acid deletion mutant, GST-PTTG (6Pei L. J. Biol. Chem. 1999; 274: 3151-3158Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) was cut withXbaI and XhoI; the ends were repaired by DNA polymerase I, and the blunt ended insert was cloned at the PuvII site of GAL4DBD, resulting in plasmid GAL-PTTG-(119–199). To generate the deletion mutant containing only the C-terminal 35 amino acids, GST-PTTG (6Pei L. J. Biol. Chem. 1999; 274: 3151-3158Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) was digested with PstI and XhoI. The 5′ overhang and the 3′ overhang were repaired by DNA polymerase I and T4 DNA polymerase, respectively. The blunt-ended insert was cloned intoSmaI site of GAL4DBD, resulting in plasmid GAL-PTTG-(164–199). Deletion mutant GAL-PTTG-(119–164) was generated using ExSite polymerase chain reaction-based mutagenesis kit following the manufacturer's instructions (Stratagene). To generate C-terminal 81-amino acid deletion mutant, GAL-PTTG-(1–199) was digested withXbaI. After filling the end with DNA polymerase I, the plasmid was digested with SmaI. The religation of the larger fragment resulted in GAL-PTTG-(1–118). Point mutations of PTTG were generated using QickChange site-directed mutagenesis kit following the manufacturer's instructions (Stratagene). All mutations were verified by DNA sequencing. Fusion protein between PTTG and green fluorescent protein (GFP) was obtained by subcloning the coding region of PTTG into theBglII and HindIII sites of pEGFP-C1. Point mutations of PTTG were generated as described above. Histidine-tagged PTTG was generated by inserting the coding region of PTTG atEcoRI site of pcDNA/His expression vector (Invitrogen). GST-PTTG fusion construct was described previously (6Pei L. J. Biol. Chem. 1999; 274: 3151-3158Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Deletions and point mutations of the fusion protein were made similar to GAL-PTTG fusion plasmids. HA-tagged, constitutively active and inactive mutants of MEK1 were provided by Dr. N. G. Ahn (University of Colorado, Boulder, CO). Expression plasmids for MAPK (wild type and kinase defective mutants) were obtained from Dr. M. Cobb (University of Texas, Dallas, TX). COS-7 and NIH-3T3 cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. EGF and PD098059 were purchased from Calbiochem. Transfections were performed using calcium phosphate precipitation as described previously (7Pei L. J. Biol. Chem. 1998; 273: 5219-5225Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). All transfections were performed in triplicate, and each DNA construct was tested in at least three independent experiments. Forty eight hours post-transfection cells were lysed in 0.25 m Tris, pH 7.8, with three freeze and thaw cycles. Cell lysates (50 μg/assay) were assayed for luciferase activity as described previously (7Pei L. J. Biol. Chem. 1998; 273: 5219-5225Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Transfected cells were lysed in the lysis buffer (20 mm Tris, pH 7.4, 140 mm NaCl, 1% Nonidet P-40, 10 mm NaF, 1 mm Na3VaO4, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, and 5 μg/ml leupeptin). For immunoprecipitation, 0.5 mg of the cell lysate was incubated with 2 μg of anti-Express for 2 h at 4 °C. The protein A/G-agarose was then added and was incubated for 1 h at 4 °C. The immunocomplexes were washed 5 time with the lysis buffer, eluted in loading buffer, and fractionated on 10% SDS-PAGE. After electron transfer, the membrane was probed using anti-HA monoclonal antibody (Covance Research Product Inc.), diluted 1:2000. For Western blot analysis using anti-GAL4DBD antibody (Santa Cruz Biotechnology), 20 μg of cell lysate was fractionated on 10% SDS-PAGE, transferred to nylon membrane, and incubated with the antibody diluted 1:500. Detection was performed using ECL system. COS-7 cells were transfected with HA-MEK1 or HA-MPK expression vectors. MEK1 and MAPK were immunoprecipitated from 100 μg of lysate using 2 μg of anti-HA antibody as described above. The immunoprecipitates were washed 3 times with the lysis buffer and 3 times with PBS. They were then incubated in a 40-μl reaction mixture containing 25 mm HEPES, pH 7.2, 10 mm MgCl2, 1 mm dithiothreitol, [γ-32P]ATP (NEN Life Science Products, 10 μCi per assay), and GST-PTTG expressed in and purified fromEscherichia coli (10 μg/reaction) as substrate at 30 °C for 30 min. Reactions were terminated by addition of sample buffer and analyzed by SDS-PAGE. The phosphorylated products were visualized by autoradiography. Expression of GST fusion proteins was induced with 0.5 mm isopropyl-β-thiogalactopyranoside at 37 °C for 90 min. Cells were centrifuged, and the resulting pellet was resuspended in a lysis buffer containing 20 mm Tris, pH 7.4, 140 mm NaCl, 1% Nonidet P-40, 10 mm NaF, 1 mm Na3VaO4, 1 mmEDTA, 1 mm phenylmethylsulfonyl fluoride, and 5 μg/ml leupeptin. Cells were lysed by three freeze-thaw cycles. Cell debris was removed by centrifugation, and the supernatant was added to Sepharose 4B beads (Amersham Pharmacia Biotech) and incubated at room temperature for 30 min. After three washes with the lysis buffer, the fusion proteins immobilized on the beads were incubated with 300 μg of cell lysate from cells transfected with HA-tagged MEK1 at 4 °C for 2 h. After 5 washes in the lysis buffer, the bound proteins were eluted in SDS sample buffer, separated on 12% SDS-PAGE, and blotted onto nylon membrane. The membrane was probed with anti-HA antibody (Covance Research Product Inc.), diluted 1:2000. COS-7 cells were transfected with GFP-PTTG (wild type or mutants) and HA-MEK1, either alone or in combination. Cells were fixed 24 h post-transfection with 2% neutral buffered formaldehyde (2% formaldehyde, 20 mmNaPO4, pH 7.4) for 15 min at 37 °C, washed 3 times with PBS, and blocked with 1% fetal calf serum in PBS. Cells were then incubated at 37 °C with 1:100 anti-HA antibody (Covance Research Product Inc.) for 1 h and with 1:50 anti-mouse Ig rhodamine (Chemicon) for 1 h at 37 °C, with 3 PBS washes after each incubation. Slides were examined with fluorescence microscope. The fluorescence data are based on multiple transfection experiments. Previous studies showed that PTTG could function as a transcriptional activator (5Dominguez A. Ramos-Morales F. Romero F. Segura D.I. Tortolero M. Pintor-Toro J.A. Oncogene. 1998; 17: 2187-2193Crossref PubMed Scopus (166) Google Scholar) and activate transcription of bFGF gene (10Chien W. Pei L. J. Biol. Chem. 2000; 275: 19422-19427Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). However, the amino acid sequences responsible for this function have yet to be defined. To localize the transcriptional activation domain of PTTG, fusion constructs were made between various regions of PTTG and the GAL4 DNA-binding domain (GAL4-DBD, amino acids 1–147). The GAL4-DBD contains signals for dimerization (26Carey M.F. Kakidani H. Leatherwood J. Mostashari F. Ptashne M. J. Mol. Biol. 1989; 209: 423-432Crossref PubMed Scopus (234) Google Scholar) and nuclear translocation (27Silver P.A. Chiang A. Sadler I. Genes Dev. 1988; 2: 707-717Crossref PubMed Scopus (48) Google Scholar) in addition to its specific DNA binding activity and shows no transactivation function (26Carey M.F. Kakidani H. Leatherwood J. Mostashari F. Ptashne M. J. Mol. Biol. 1989; 209: 423-432Crossref PubMed Scopus (234) Google Scholar, 27Silver P.A. Chiang A. Sadler I. Genes Dev. 1988; 2: 707-717Crossref PubMed Scopus (48) Google Scholar). The expression plasmids coding for GAL4-DBD fused to various parts of PTTG were transfected into COS-7 cells together with a reporter plasmid containing five GAL4-binding sites in front of E1B minimal promoter linked to luciferase (24Sun P. Enslen H. Myung P.S. Maurer R.A. Genes Dev. 1994; 8: 2527-2539Crossref PubMed Scopus (635) Google Scholar). As shown in Fig.1 A, wild type PTTG activated reporter gene transcription about 82-fold (Fig. 1 A, construct wt). Deletion of up to 118 amino acids from the N terminus does not affect PTTG transactivation activity (Fig. 1 A, constructs 61–199 and 119–199). Additional deletion of the C-terminal 35 amino acids has little effect on PTTG transactivation function (Fig. 1 A, construct 119–164). However, deletion of either 164 amino acids from the N terminus or 81 amino acids from the C terminus resulted in complete loss of transcriptional activation (Fig.1 A, constructs 164–199 and 1–118). The expression of all the GAL4-PTTG fusion constructs in transfected cells was verified by Western blot analysis (Fig. 1 B). These results indicate that amino acids between positions 118 and 164 are responsible for PTTG transcriptional activation activity. Compared with the strong transcriptional activator VP16, PTTG is a relatively weak activator (Fig. 1 A). The stimulation of cellular proliferation and differentiation involves the activation of signaling pathways that are initiated by specific receptors at the cell surface. Many of these signaling pathways converge on MAP kinase cascade and its downstream targets. MAP kinase was shown to phosphorylate and activate several nuclear transcription factors (18Gupta S. Seth A. Davis R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3216-3220Crossref PubMed Scopus (137) Google Scholar, 19Seth A. Alvarez E. Gupta S. Davis R.J. J. Biol. Chem. 1991; 266: 23521-23524Abstract Full Text PDF PubMed Google Scholar, 20Seth A. Gonzales F.A. Gupta S. Raden D.L. Davis R.J. J. Biol. Chem. 1991; 267: 24796-24804Abstract Full Text PDF Google Scholar, 21Nagajima T. Kinoshita S. Sasagawa T. Sasaki K. Naruto M. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2207-2211Crossref PubMed Scopus (513) Google Scholar). The presence of a consensus phosphorylation site within the PTTG transactivation domain (amino acids 161–164 PPSP) (17Alvarez E. Northwood I.C. Gonzales F.A. Latour D.A. Seth A. Abate C. Curran T. Davis R.J. J. Biol. Chem. 1991; 266: 15277-15285Abstract Full Text PDF PubMed Google Scholar) prompted us to investigate whether activation of the MAP kinase signal transduction pathway affects PTTG transcriptional activation function. EGF is one of the growth factors that is known to activate MAPK signaling transduction pathway (28Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar, 29Blennis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5889-5892Crossref PubMed Scopus (1151) Google Scholar). We therefore tested the effect of EGF on transcriptional activation function of PTTG. As shown in Fig.2 A, treatment of transfected cells with EGF resulted in 6-fold increase in GAL-PTTG transactivation activity. A primary pathway for MAP kinase activation by EGF consists of sequential activation of guanine exchange factor SOS, the guanosine triphosphate-binding protein Ras, and the protein kinases Raf-1, MEK1 or -2, and ERKs (30Egan S.E. Giddings B.W. Brooks M.W. Buday L. Sizeland A.M. Weinberg R.A. Nature. 1993; 363: 45-51Crossref PubMed Scopus (1003) Google Scholar, 31Moodie S.A. Willumsen B.M. Weber M.J. Wolfman A. Science. 1993; 260: 1658-1661Crossref PubMed Scopus (771) Google Scholar, 32Warne P.H. Viciana P.R. Downward J. Nature. 1993; 364: 352-355Crossref PubMed Scopus (579) Google Scholar, 33Kyriakis J.M. App H. Zhang X.-F. Banerjee P. Brautigan D.L. Rapp U.R. Avruch J. Nature. 1992; 358: 417-421Crossref PubMed Scopus (964) Google Scholar). To test whether EGF exerts its effect on PTTG transactivation function through this pathway, we initially asked whether PD098059, a specific inhibitor of MEK1, would affect EGF-stimulated transactivation function of PTTG. Fig. 2 Ashows that in the presence of 50 μm PD098059, EGF-stimulated transactivation activity of PTTG was inhibited. We then tested the ability of a constitutively active form of MEK1 to activate the MAP kinase phosphorylation cascade. This mutant form of MEK1 was shown to have basal activity several hundred times greater than that of the unphosphorylated wild type kinase and was able to stimulate MAP kinase activity (34Mansour S.J. Matten W.T. Herman A.S. Candia J.M. Rong S. Fukusawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1253) Google Scholar). As shown in Fig. 2 B, expression of constitutively active MEK1 (R4F) resulted in 40-fold increase in transactivation activity of the wild type PTTG, whereas expression of inactive MEK1 mutant (8E) had no effect on PTTG activity. Although expression of constitutively active MEK1 could also increase the transactivation function of the PTTG N-terminal deletion mutants, the effects were much less compared with wild type PTTG (3-versus 40-fold, Fig. 2 B). These results indicate that activation of PTTG transactivation function by MAP kinase cascade requires not only PTTG transactivation domain but also residues within the N-terminal 60 amino acids. We then tested whether activation of MAP kinase cascade also affects the ability of PTTG to transactivate bFGF transcription. Fig.2 C shows that either EGF treatment or co-transfection of the constitutively active form of MEK1 resulted in 2- or 5-fold increase in luciferase activity of bFGF-LUC, respectively, whereas the inactive MEK1 mutant had no effect. The stimulatory effect of EGF and MEK1 on PTTG transactivation of bFGF was also attenuated by treatment of cells with PD098059 (Fig. 2 C). To confirm that enhanced PTTG transactivation activity by expression of the constitutively active MEK1 is mediated by MAP kinase, we then tested the ability of expression vector for kinase-defective mutants of MAPK to function as inhibitors of MEK1-activated reporter gene activity. Although these mutants MAPK can be phosphorylated on activating site, they possess less than 5% of the wild type activity (35Robbins D.J. Zhen E. Owaki H. Vanderbilt C.A. Ebert D. Geppert T.D. Cobb M.H. J. Biol. Chem. 1993; 268: 5016-5097Abstract Full Text PDF Google Scholar), and they appear to interfere with endogenous MAPK activity (36Frost J.A. Grppert T.D. Cobb M.H. Feramisco J.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3844-3848Crossref PubMed Scopus (202) Google Scholar,37Sontag E. Fedorov S. Kamibayashi C. Robbins D. Cobb M.H. Mumby M. Cell. 1993; 75: 887-897Abstract Full Text PDF PubMed Scopus (457) Google Scholar). As shown in Fig. 2 D, expression vector for kinase-defective MAPK was able to attenuate the ability of the constitutively active MEK1 to activate the GAL-PTTG fusion protein, consistent with role for MAPK in mediating activation signal from MEK1. To determine whether modulation of PTTG transactivation function by activation of MAP kinase signal transduction pathway is a result of direct phosphorylation of PTTG, we tested the ability of MEK1 and MAPK to phosphorylate PTTG in vitro. COS-7 cells were transfected with either constitutively active MEK1 or MAPK expression plasmid. MEK1 and MAPK were immunoprecipitated, and the immunocomplexes were incubated with PTTG expressed in and purified from E. coli in the presence of [γ-32P]ATP. As shown in Fig. 3, while MEK1 phosphorylated recombinant MAPK (Fig. 3, lane 2), it did not phosphorylate PTTG (Fig. 3, lane 1). MAPK, on the other hand, was able to phosphorylate PTTG (Fig. 3, lane 3). These results indicate that MAPK is kinase that phosphorylates PTTG. To determine whether the consensus MAPK phosphorylation site located between amino acids 160 and 163 (PPSP) was responsible for PTTG phosphorylation by MAP kinase, si" @default.
• W2072563654 created "2016-06-24" @default.
• W2072563654 creator A5024070604 @default.
• W2072563654 date "2000-10-01" @default.
• W2072563654 modified "2023-10-14" @default.
• W2072563654 title "Activation of Mitogen-activated Protein Kinase Cascade Regulates Pituitary Tumor-transforming Gene Transactivation Function" @default.
• W2072563654 cites W135983215 @default.
• W2072563654 cites W139618738 @default.
• W2072563654 cites W1499613870 @default.
• W2072563654 cites W1504158642 @default.
• W2072563654 cites W1507957953 @default.
• W2072563654 cites W1531194664 @default.
• W2072563654 cites W1532900970 @default.
• W2072563654 cites W1543158853 @default.
• W2072563654 cites W1555409791 @default.
• W2072563654 cites W1573821584 @default.
• W2072563654 cites W1854798937 @default.
• W2072563654 cites W1964201687 @default.
• W2072563654 cites W1964842789 @default.
• W2072563654 cites W1965398762 @default.
• W2072563654 cites W1971475788 @default.
• W2072563654 cites W1975957264 @default.
• W2072563654 cites W1979647722 @default.
• W2072563654 cites W1983475354 @default.
• W2072563654 cites W1991321447 @default.
• W2072563654 cites W1997218197 @default.
• W2072563654 cites W1997664222 @default.
• W2072563654 cites W1999157780 @default.
• W2072563654 cites W2000082154 @default.
• W2072563654 cites W2001521752 @default.
• W2072563654 cites W2010955929 @default.
• W2072563654 cites W2022658303 @default.
• W2072563654 cites W2038197766 @default.
• W2072563654 cites W2039076738 @default.
• W2072563654 cites W2041187882 @default.
• W2072563654 cites W2044959710 @default.
• W2072563654 cites W2052203027 @default.
• W2072563654 cites W2054937093 @default.
• W2072563654 cites W2057050252 @default.
• W2072563654 cites W2057126981 @default.
• W2072563654 cites W2058036485 @default.
• W2072563654 cites W2067626539 @default.
• W2072563654 cites W2070023258 @default.
• W2072563654 cites W2071198735 @default.
• W2072563654 cites W2071649852 @default.
• W2072563654 cites W2072243374 @default.
• W2072563654 cites W2078330502 @default.
• W2072563654 cites W2082190322 @default.
• W2072563654 cites W2094042805 @default.
• W2072563654 cites W2097585410 @default.
• W2072563654 cites W2140566131 @default.
• W2072563654 cites W2145330934 @default.
• W2072563654 cites W2146513771 @default.
• W2072563654 cites W2162322742 @default.
• W2072563654 cites W2328187042 @default.
• W2072563654 doi "https://doi.org/10.1074/jbc.m002451200" @default.
• W2072563654 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10906323" @default.
• W2072563654 hasPublicationYear "2000" @default.
• W2072563654 type Work @default.
• W2072563654 sameAs 2072563654 @default.
• W2072563654 citedByCount "73" @default.
• W2072563654 countsByYear W20725636542012 @default.
• W2072563654 countsByYear W20725636542013 @default.
• W2072563654 countsByYear W20725636542015 @default.
• W2072563654 countsByYear W20725636542016 @default.
• W2072563654 countsByYear W20725636542017 @default.
• W2072563654 countsByYear W20725636542018 @default.
• W2072563654 countsByYear W20725636542019 @default.
• W2072563654 countsByYear W20725636542020 @default.
• W2072563654 countsByYear W20725636542022 @default.
• W2072563654 crossrefType "journal-article" @default.
• W2072563654 hasAuthorship W2072563654A5024070604 @default.
• W2072563654 hasBestOaLocation W20725636541 @default.
• W2072563654 hasConcept C104317684 @default.
• W2072563654 hasConcept C1292079 @default.
• W2072563654 hasConcept C132149769 @default.
• W2072563654 hasConcept C137361374 @default.
• W2072563654 hasConcept C14036430 @default.
• W2072563654 hasConcept C150194340 @default.
• W2072563654 hasConcept C184235292 @default.
• W2072563654 hasConcept C185592680 @default.
• W2072563654 hasConcept C502942594 @default.
• W2072563654 hasConcept C54355233 @default.
• W2072563654 hasConcept C86803240 @default.
• W2072563654 hasConcept C95444343 @default.
• W2072563654 hasConcept C97029542 @default.
• W2072563654 hasConceptScore W2072563654C104317684 @default.
• W2072563654 hasConceptScore W2072563654C1292079 @default.
• W2072563654 hasConceptScore W2072563654C132149769 @default.
• W2072563654 hasConceptScore W2072563654C137361374 @default.
• W2072563654 hasConceptScore W2072563654C14036430 @default.
• W2072563654 hasConceptScore W2072563654C150194340 @default.
• W2072563654 hasConceptScore W2072563654C184235292 @default.
• W2072563654 hasConceptScore W2072563654C185592680 @default.
• W2072563654 hasConceptScore W2072563654C502942594 @default.
• W2072563654 hasConceptScore W2072563654C54355233 @default.
• W2072563654 hasConceptScore W2072563654C86803240 @default.
• W2072563654 hasConceptScore W2072563654C95444343 @default.

• next