FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2002022970> ?p ?o ?g. }

• W2002022970 endingPage "7102" @default.
• W2002022970 startingPage "7095" @default.
• W2002022970 abstract "The p38 mitogen-activated protein kinases (MAPK) play a crucial role in stress and inflammatory responses and are also involved in activation of the human immunodeficiency virus gene expression. We have isolated the murine cDNA clones encoding p38-δ MAPK, and we have localized the p38-δ gene to mouse chromosome 17A3-B and human chromosome 6p21.3. By using Northern and in situ hybridization, we have examined the expression of p38-δ in the mouse adult tissues and embryos. p38-δ was expressed primarily in the lung, testis, kidney, and gut epithelium in the adult tissues. Although p38-δ was expressed predominantly in the developing gut and the septum transversum in the mouse embryo at 9.5 days, its expression began to be expanded to many specific tissues in the 12.5-day embryo. At 15.5 days, p38-δ was expressed virtually in most developing epithelia in embryos, suggesting that p38-δ is a developmentally regulated MAPK. Interestingly, p38-δ and p38-α were similar serine/threonine kinases but differed in substrate specificity. Overall, p38-δ resembles p38-γ, whereas p38-β resembles p38-α. Moreover, p38-δ is activated by environmental stress, extracellular stimulants, and MAPK kinase-3, -4, -6, and -7, suggesting that p38-δ is a unique stress-responsive protein kinase. The p38 mitogen-activated protein kinases (MAPK) play a crucial role in stress and inflammatory responses and are also involved in activation of the human immunodeficiency virus gene expression. We have isolated the murine cDNA clones encoding p38-δ MAPK, and we have localized the p38-δ gene to mouse chromosome 17A3-B and human chromosome 6p21.3. By using Northern and in situ hybridization, we have examined the expression of p38-δ in the mouse adult tissues and embryos. p38-δ was expressed primarily in the lung, testis, kidney, and gut epithelium in the adult tissues. Although p38-δ was expressed predominantly in the developing gut and the septum transversum in the mouse embryo at 9.5 days, its expression began to be expanded to many specific tissues in the 12.5-day embryo. At 15.5 days, p38-δ was expressed virtually in most developing epithelia in embryos, suggesting that p38-δ is a developmentally regulated MAPK. Interestingly, p38-δ and p38-α were similar serine/threonine kinases but differed in substrate specificity. Overall, p38-δ resembles p38-γ, whereas p38-β resembles p38-α. Moreover, p38-δ is activated by environmental stress, extracellular stimulants, and MAPK kinase-3, -4, -6, and -7, suggesting that p38-δ is a unique stress-responsive protein kinase. The mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; MAP, mitogen-activated protein; ERK, extracellular signal-regulated protein kinase; JNK, c-Jun amino-terminal kinase; SAPK, stress-activated protein kinase; MKK, MAPK kinase; EST, expressed sequence tag; PCR, polymerase chain reaction; GST, glutathione S-transferase; mAb, monoclonal antibody; MBP, myelin basic protein; TNF, tumor necrosis factor; IL, interleukin; EGF, epidermal growth factor; PAGE, polyacrylamide gel electrophoresis; ISH, in situ hybridization; FISH, fluorescence in situ hybridization; DAPI, 4′,6′-diamidino-2-phenylindole; bp, base pair(s); Mops, 4-morpholinepropanesulfonic acid cascade is a major signaling system by which cells transduce extracellular stimuli into intracellular signals to control the expression of genes essential for cellular processes such as cell proliferation, differentiation, and stress responses (1Hunter T. Cell. 1995; 80: 225-236Abstract Full Text PDF PubMed Scopus (2608) Google Scholar, 2Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1663) Google Scholar). In mammalian cells, these kinases include extracellular signal-regulated protein kinases (ERKs) (3Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar), the c-Jun amino-terminal kinases (JNK)/stress-activated protein kinases (SAPKs) (4Derijard B. Hibi M. Wu I.-H. Barret T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2957) Google Scholar, 5Kyriakis J. Banerjee P. Nikolakaki E. Dai T. Rubie E. Ahmad M. Avruch J. Woodgett J. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2415) Google Scholar, 6Gupta S. Barrett T. Whitmarsh A.J. Cavanagh J. Sluss H.K. Derijard B. Davis R.J. EMBO J. 1996; 15: 2760-2770Crossref PubMed Scopus (1183) Google Scholar), and p38 MAPKs (7Han J. Lee J.-D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2420) Google Scholar, 8Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3147) Google Scholar, 9Rouse J. Cohen P. Trigon S. Morange M. Alonso-Llamazares A. Zammanillo D. Hunt T. Nebreda A.R. Cell. 1994; 78: 1027-1037Abstract Full Text PDF PubMed Scopus (1507) Google Scholar, 10Wang X.S. Diener K. Manthey C.L. Wang S. Rosenzweig B. Bray J. Delaney J. Cole C.N. Chan-Hui P.Y. Mantlo N. Lichenstein H.S. Zukowski M. Yao Z. J. Biol. Chem. 1997; 272: 23668-23674Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Whereas ERKs are activated rapidly in response to the binding of growth factors to growth factor receptors (11Boulton T.G. Yancopoulos G.D. Gregory J.S. Slaughter C. Moomaw C. Hsu J. Cobb M.H. Science. 1990; 249: 64-67Crossref PubMed Scopus (483) Google Scholar, 12Boulton T.G. Nye S.H. Robbins D.J. Ip N.Y. Radziejewska E. Morgenbesser S.D. DePinho R.A. Panayotatos N. Cobb M.H. Yancopoulos G.D. Cell. 1991; 65: 663-675Abstract Full Text PDF PubMed Scopus (1490) Google Scholar), JNKs/SAPKs and p38 MAPKs are stimulated by environmental stress (i.e. osmotic shock, ultraviolet irradiation, cytotoxic chemicals, etc.) and proinflammatory cytokines (i.e. interleukin-1 (IL-1) and tumor necrosis factor (TNF)) (4Derijard B. Hibi M. Wu I.-H. Barret T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2957) Google Scholar, 5Kyriakis J. Banerjee P. Nikolakaki E. Dai T. Rubie E. Ahmad M. Avruch J. Woodgett J. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2415) Google Scholar, 7Han J. Lee J.-D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2420) Google Scholar, 8Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3147) Google Scholar, 13Hibi M. Lin A. Smeal T. Minden A. Karin M. Genes Dev. 1993; 7: 2135-2148Crossref PubMed Scopus (1710) Google Scholar, 14Minden A. Lin A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1012) Google Scholar, 15Kallunki T. Su B. Tsigelny I. Sluss H.K. Derijard B. Moore G. Davis R.J. Karin M. Genes Dev. 1994; 8: 2996-3007Crossref PubMed Scopus (595) Google Scholar, 16Galcheva-Gargova Z. Derijard B. Wu I.-H. Davis R.J. Science. 1994; 265: 806-808Crossref PubMed Scopus (531) Google Scholar, 17Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2046) Google Scholar). Although the physiological function of the ERKs and JNKs/SAPKs in signal transduction pathways has been extensively studied (1Hunter T. Cell. 1995; 80: 225-236Abstract Full Text PDF PubMed Scopus (2608) Google Scholar, 3Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar, 18Davis R.J. Trends Biochem. Sci. 1994; 19: 470-473Abstract Full Text PDF PubMed Scopus (918) Google Scholar, 19Robbins D.J. Zhen E. Cheng M. Xu S. Ebert D. Cobb M.H. Adv. Cancer Res. 1994; 63: 93-116Crossref PubMed Google Scholar, 20Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2258) Google Scholar, 21Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4243) Google Scholar), the functional role of the p38 MAPK signaling pathway is relatively less understood (7Han J. Lee J.-D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2420) Google Scholar, 8Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3147) Google Scholar, 9Rouse J. Cohen P. Trigon S. Morange M. Alonso-Llamazares A. Zammanillo D. Hunt T. Nebreda A.R. Cell. 1994; 78: 1027-1037Abstract Full Text PDF PubMed Scopus (1507) Google Scholar, 17Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2046) Google Scholar, 22Freshney N.W. Rawlinson L. Guesdon F. Jones E. Cowley S. Hsuan J. Saklatvala J. Cell. 1994; 78: 1039-1049Abstract Full Text PDF PubMed Scopus (778) Google Scholar). Nevertheless, p38 MAPKs play an important role in stress and inflammatory responses and are also involved in activation of the human immunodeficiency virus type 1 promoter (23Kumar S. Orsini M.J. Lee J.C. McDonnell P.C. Debouck C. Young P.R. J. Biol. Chem. 1996; 271: 30864-30869Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). These MAP kinases have the unique feature of being activated by phosphorylation on threonine (Thr) and tyrosine (Tyr) residues by upstream dual-specificity kinases, i.e. MAP kinase kinases (MKKs or MEKs) (18Davis R.J. Trends Biochem. Sci. 1994; 19: 470-473Abstract Full Text PDF PubMed Scopus (918) Google Scholar, 20Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2258) Google Scholar). This dual phosphorylation Thr-X-Tyr motif is located within the kinase subdomain VIII where ERK is Thr-Glu-Tyr; JNK/SAPK is Thr-Pro-Tyr; and p38 MAPK is Thr-Gly-Tyr. MKK-1 and MKK-2 phosphorylate and activate ERK-1 and ERK-2 (24Crews C.M. Alessandrini A.A. Erickson R.L. Science. 1992; 258: 478-480Crossref PubMed Scopus (740) Google Scholar, 25Zheng C.-F. Guan K.-L. J. Biol. Chem. 1993; 268: 11435-11439Abstract Full Text PDF PubMed Google Scholar), whereas MKK-3 and MKK-6 activate p38 MAPK specifically (26Derijard B. Raingeaud J. Barrett T. Wu I.H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1415) Google Scholar, 27Lin A. Minden A. Martinetto H. Claret F.X. Langer-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (714) Google Scholar, 28Han J. Lee J.-D. Jiang Y. Li Z. Feng L. Ulevitch R.J. J. Biol. Chem. 1996; 271: 2886-2891Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar, 29Raingeaud J. Whitmarsh A.J. Barrett T. Derijard B. Davis R.J. Mol. Cell. Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1149) Google Scholar, 30Stein B. Brady H. Yang M.X. Young D.B. Barbosa M.S. J. Biol. Chem. 1996; 271: 11427-11433Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 31Moriguchi T. Kuroyanagi N. Yamaguchi K. Gotoh Y. Irie K. Kano T. Shirakabe K. Muro Y. Shibuya H. Matsumoto K. Nishida E. Hagiwara M. J. Biol. Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 32Moriguchi T. Toyoshima F. Gotoh Y. Iwamatsu A. Irie K. Mori E. Kuroyanagi N. Hagiwara M. Matsumoto K. Nishida E. J. Biol. Chem. 1996; 271: 26981-26988Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Although MKK-4 (SEK-1) stimulates JNK/SAPK and p38 MAPK (26Derijard B. Raingeaud J. Barrett T. Wu I.H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1415) Google Scholar, 27Lin A. Minden A. Martinetto H. Claret F.X. Langer-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (714) Google Scholar, 33Sanchez I. Hughes R.T. Mayer B.J. Yee K. Woodgett J.R. Avruch J. Kyriakis J.M. Zon L.I. Nature. 1994; 372: 794-798Crossref PubMed Scopus (917) Google Scholar), MKK-5 phosphorylates and activates ERK-5 (34English J.M. Vanderbilt C.A. Xu S. Marcus S. Cobb M.H. J. Biol. Chem. 1995; 270: 28897-28902Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 35Zhou G. Bao Z.Q. Dixon J.E. J. Biol. Chem. 1995; 270: 12665-12669Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar). Recently, MKK-7 has been shown to activate JNKs/SAPKs specifically but not p38 MAPKs and ERKs (36Yao Z. Diener K. Wang X.S. Zukowski M. Matsumoto G. Zhou G. Mo R. Sasaki T. Nishina H. Hui C.C. Tan T.-H. Woodgett J.P. Penninger J.M. J. Biol. Chem. 1997; 272: 32378-32383Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Each MAP kinase group has a unique substrate specificity and is regulated by a distinct signal transduction pathway (18Davis R.J. Trends Biochem. Sci. 1994; 19: 470-473Abstract Full Text PDF PubMed Scopus (918) Google Scholar, 20Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2258) Google Scholar, 21Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4243) Google Scholar). For instance, ERK-1 and ERK-2 phosphorylate and activate the transcription factor Elk-1 (37Gille H. Sharrocks A.D. Shaw P.E. Nature. 1992; 358: 414-417Crossref PubMed Scopus (816) Google Scholar, 38Marais R. Wynne J. Treisman R. Cell. 1993; 73: 381-393Abstract Full Text PDF PubMed Scopus (1108) Google Scholar). JNKs/SAPKs phosphorylate and activate the transcription factors c-Jun (4Derijard B. Hibi M. Wu I.-H. Barret T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2957) Google Scholar, 5Kyriakis J. Banerjee P. Nikolakaki E. Dai T. Rubie E. Ahmad M. Avruch J. Woodgett J. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2415) Google Scholar), ATF-2 (39Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1339) Google Scholar), and Elk-1 (40Whitmarsh A.J. Shore P. Sharrocks A.D. Davis R.J. Science. 1995; 269: 403-407Crossref PubMed Scopus (882) Google Scholar). The p38 MAPKs phosphorylate and activate the transcription factors ATF-2 (17Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2046) Google Scholar, 26Derijard B. Raingeaud J. Barrett T. Wu I.H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1415) Google Scholar) and Elk-1 (29Raingeaud J. Whitmarsh A.J. Barrett T. Derijard B. Davis R.J. Mol. Cell. Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1149) Google Scholar). The first p38 MAPK (hereafter designated as p38-α) was identified initially in lipopolysaccharide-stimulated macrophages and was found later to share significant homology with the yeast HOG1 kinase (7Han J. Lee J.-D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2420) Google Scholar, 41Brewster J.L. de Valoir T. Dyer N.D. Winter E. Gustin M.C. Science. 1993; 259: 1760-1763Crossref PubMed Scopus (1034) Google Scholar). Subsequently, the human p38-α homologues (CSBPs) were isolated by using radiolabeled and radiophotoaffinity labeled pyridinyl imidazole compounds, which block inflammatory cytokine biosynthesis by monocytes stimulated with lipopolysaccharide (8Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3147) Google Scholar). Another member (p38-β) of the p38 MAPK family was identified and cloned, which is very homologous (with 75% amino acid identity) to p38-α (42Jiang Y. Chen C. Li Z. Guo W. Gegner J.A. Lin S. Han J. J. Biol. Chem. 1996; 271: 17920-17926Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar). The third member (p38-γ) of the p38 MAPK family was recently isolated as ERK-6 (43Lechner C. Zahalka M.A. Giot J.-F. Moller N.P.H. Ullrich A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4355-4359Crossref PubMed Scopus (278) Google Scholar) and SAPK3 (44Cuenda A. Cohen P. Buee-Scherrer V. Goedert M. EMBO J. 1997; 16: 295-305Crossref PubMed Scopus (317) Google Scholar), which share significant homology (63% amino acid identity) with p38-α. All these p38 MAPK members contain a characteristic Thr-Gly-Tyr motif within the kinase subdomain VIII. Here, we present a murine p38 MAPK family member, p38-δ, whose sequence is significantly homologous to p38-α (63% amino acid identity). We showed that expression of p38-δ mRNA was regulated in different developmental stages, suggesting that p38-δ is a developmentally regulated MAPK. We characterized p38-δ by determining its chromosomal location, stimulation by extracellular stimuli, and activation by upstream kinases (MKKs). Moreover, we compared substrate specificity and inhibitor sensitivity between p38-δ and p38-α, and we showed that they are discrete. Our results indicate that p38-δ is a unique stress-responsive protein kinase. A rat 1.3-kilobase pair expressed sequence tag (EST) cDNA clone with ∼62% homology to mouse p38-β (GenBankTM accession number D83073) cDNA was used as a probe to screen a rat lung cDNA library in λgt11 phage vector (CLONTECHLaboratories). For hybridization, replicate filters were prehybridized for 1 h at 68 °C in Express hybridization buffer (CLONTECH Laboratories) and hybridized 12 h at 68 °C in the same solution with the [32P]dCTP-labeled probe. After hybridization, the filters were washed several times at high stringency, at 65 °C in 0.1% SDS, 0.2× SSC (1× SSC, 150 mm NaCl and 15 mm sodium citrate), and subjected to autoradiography. Several positive clones were picked and purified after screening 4 × 106 phages. The cDNA inserts of these positive phage clones were subsequently subcloned into pCR3.1 plasmid vector (Invitrogen). After analysis of the inserts, the longest cDNA clone was sequenced on both strands, using a PCR procedure employing fluorescent dideoxynucleotides and a model 373A automated sequencer (Applied Biosystems). Similarly, for human p38-δ cDNA cloning, the same EST cDNA probe was used to screen a human lung cDNA library in λTripEx phage vector (CLONTECH Laboratories). Several positive clones were obtained, and the cDNA inserts of these phage clones were converted in vivo into pTripEx plasmid vector, according to the manufacturer's instructions. A candidate full-length cDNA clone was sequenced on both strands as described above. Sequence comparisons were aligned with the Bestfit program of the GCG sequence analysis software package (Wisconsin Package version 9.0). The Flag-tagged p38-δ expression plasmid was constructed from the murine p38-δ cDNA by the PCR technique using oligonucleotides 5′-AAGCTTGTCGACGCCACCATGGATTATAAAGATGATGATGATAAAAGCCTCATTCGGAAAAGGGGCTTC-3′ and 5′-TATTGCGGCCGCTTATCACTGCAGCTTCATCCCACTTCG-3′ as primers to incorporate a Flag epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) at the amino terminus of p38-δ. The PCR-generated product was cloned into the expression vector pCR3.1 plasmid vector (Invitrogen) and designated pFlag-p38-δ. The Flag-tagged p38-δ(AGF) mutant expression plasmid was generated from pFlag-p38-δ plasmid by the PCR technique using oligonucleotides 5′-GATGCGGAGATGGCTGGCTTTGTGGTGACCCGC-3′ and 5′-GCGGGTCACCACAAAGCCAGCCATCTCCGCATC-3′ as primers to replace the Thr180-Gly-Tyr182 motif with Ala180-Gly-Phe182, using a QuickChangeTM site-directed mutagenesis kit (Stratagene). The sequences of these cDNA constructs were confirmed by DNA sequencing on both strands as described. The pVA1 (containing the adenovirus VA1 RNA gene) plasmid was obtained as described previously (45Hu M.C.-T. Qiu W.R. Wang X. Meyer C.F. Tan T.-H. Genes Dev. 1996; 10: 2251-2264Crossref PubMed Scopus (195) Google Scholar). The Flag-tagged p38-α, MKK-3, MKK-4, MKK-6, MKK-7, and MEK kinase-5 (ASK-1) expression plasmids were kindly provided by Dr. R. Geronsin (Amgen Inc.). ATF-2-(1–96) and GST-MAPKAP kinase-2 were purchased from Santa Cruz Biotechnology and Upstate Biotechnology Inc., respectively. PHAS-1 and p38-α inhibitor were purchased from Stratagene. Myelin basic protein (MBP), anisomycin, and Na3VO4 were purchased from Sigma. GST-c-Jun was prepared as described previously (45Hu M.C.-T. Qiu W.R. Wang X. Meyer C.F. Tan T.-H. Genes Dev. 1996; 10: 2251-2264Crossref PubMed Scopus (195) Google Scholar), and anti-Flag M2 mAb was purchased from Kodak Scientific Imaging Systems. Human TNF-α, IL-1α, and epidermal growth factor (EGF) were purchased from R & D Systems. Poly(A)+ RNAs from various mouse tissues were obtained from CLONTECHLaboratories. Each sample (2 μg) was denatured and electrophoresed on a 1.2% agarose gel containing formaldehyde and then transferred to a Hybond-N membrane (Amersham Pharmacia Biotech) in 20× SSC as described (46Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Murine p38-δ or human β-actin cDNA was labeled with [32P]dCTP to a specific activity of approximately 108 dpm/μg. Membranes were hybridized with either the p38-δ or β-actin cDNA probe, then washed at high stringency, at 65 °C in 0.2× SSC, 0.1% SDS, and subjected to autoradiography. Probes were removed in 0.5% SDS at 95–100 °C. ISH was performed as described (47Lyons G.E. Schiaffino S. Sassoon D. Barton P. Buckingham M. J. Cell Biol. 1990; 111: 2427-2436Crossref PubMed Scopus (326) Google Scholar). Briefly, fetuses and tissues were fixed in 4% paraformaldehyde in phosphate-buffered saline overnight, dehydrated, and infiltrated with paraffin. Serial sections at thickness of 5–7 μm were mounted on gelatin-coated slides, deparaffinized in xylene, rehydrated, and post-fixed. The tissue sections were digested with proteinase K, post-fixed, treated with triethanolamine/acetic anhydride, washed, and dehydrated. The cRNA transcripts were synthesized from linearized cDNA templates to generate antisense and sense probes, according to manufacturer's conditions (Ambion) and labeled with35S-UTP (>1000 Ci/mmol; Amersham Pharmacia Biotech). cRNA transcripts larger than 200 nucleotides were subjected to alkali hydrolysis to give a mean size of 70 nucleotides. The tissue slides were hybridized overnight at 52 °C in 50% deionized formamide, 0.3m NaCl, 20 mm Tris-HCl, pH 7.4, 10 mm NaPO4, 5 mm EDTA, 10% dextran sulfate, 1× Denhardt's, 50 μg/ml total yeast RNA, and 5–7.5 × 104 cpm/μl 35S-labeled cRNA probe. The tissue slides were subjected to stringent washing at 65 °C in 50% formamide, 2× SSC, 10 mm dithiothreitol, and washed in phosphate-buffered saline before treatment with 20 μg/ml RNase A at 37 °C for 30 min. Following washes in 2× SSC and 0.1× SSC at 37 °C for 10 min, the slides were dehydrated and dipped in Kodak NTB-2 nuclear track emulsion and exposed for 2–3 weeks in light-tight boxes with desiccant at 4 °C. Photographic development was carried out in Kodak D-19. The tissue slides were counterstained lightly with toluidine blue and analyzed using both light and dark field optics of a microscope. Sense control cRNA probes indicate the background levels of the hybridization signal. 293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Life Technologies, Inc.). Cells to be transfected were plated the day before transfection at a density of 2 × 106 cells per 100-mm dish. 293T cells were co-transfected with expression plasmids (10 μg each plasmid per dish) as indicated with pVA1 (10 μg per dish) to enhance transient protein expression, using the calcium phosphate precipitation protocol (Specialty Media, Inc.). The transfected 293T cells were harvested 48 h after transfection. For cell stimulation, 293T cells were treated with human TNF-α (20 ng/ml) for 10 min before harvest. Cells were lysed in WCE lysis buffer (20 mm HEPES, pH 7.4, 2 mm EGTA, 50 mm β-glycerophosphate, 1% Triton X-100, 10% glycerol, 1 mm dithiothreitol, 2 μg/ml leupeptin, 5 μg/ml aprotinin, 1 mm Pefabloc (Boehringer Mannheim) or phenylmethylsulfonyl fluoride, 1 mm sodium orthovanadate). Soluble lysates were prepared by centrifugation at 10,000 × g for 30 min at 4 °C. The lysates were precleared using Pansorbin cells (Calbiochem) and then incubated with specific antibodies. After 16 h of incubation, immunocomplexes were recovered with the aid of Gamma-Bind Sepharose beads (Amersham Pharmacia Biotech) and then washed four times with lysis buffer. Subsequently, immunoprecipitates were analyzed by Western blotting after SDS-PAGE (10%), electroblotted onto polyvinylidene difluoride membranes (Novex, Inc.), and probed with the corresponding rabbit antiserum or mouse monoclonal antibody. Immunocomplexes were visualized by enhanced chemiluminescence (ECL) detection (Amersham Pharmacia Biotech) using goat anti-rabbit or anti-mouse antisera conjugated to horseradish peroxidase as a secondary antibody (Pierce). Immunocomplex kinase assays were carried out as described previously (48Hu M.C.-T. Qiu W.R. Wang Y.-P. Oncogene. 1997; 15: 2277-2287Crossref PubMed Scopus (110) Google Scholar). Specifically, cellular p38-δ or p38-α proteins were immunoprecipitated by incubation with anti-Flag mAb and protein A-agarose beads (Bio-Rad) in WCE lysis buffer. After 3 h of incubation at 4 °C, the immunoprecipitates were collected and washed twice with WCE lysis buffer, twice with LiCl buffer (500 mm LiCl, 100 mm Tris-Cl, pH 7.6, and 0.1% Triton X-100), and twice with kinase buffer (20 mm Mops, pH 7.6, 2 mm EGTA, 10 mmMgCl2, 1 mm dithiothreitol, 0.1% Triton X-100, and 1 mm Na3VO4). Pellets were then mixed with 5 μg of substrate, 20 μCi of [γ-32P]ATP, and 15 μm unlabeled ATP in 30 μl of kinase buffer. The substrates included MBP, GST-c-Jun, GST-MAPKAP kinase-2, PHAS-1, and ATF-2-(1–96) in the absence or presence of p38-α inhibitor (SB203580). The kinase reaction was performed for 30 min at 30 °C and terminated by boiling in an equal volume of Laemmli sample buffer, and the products were resolved by SDS-PAGE (10%). The gel was dried and subjected to autoradiography. The phosphorylated proteins obtained from immunocomplex kinase assays were transferred electrophoretically to polyvinylidene difluoride membranes. The spots containing phosphoproteins on the membranes were excised according to the bands on autoradiograms and then hydrolyzed in 50 μl of 6n HCl for 1 h at 110 °C. The supernatant was lyophilized and dissolved in 6 μl of pH 1.9 buffer (2.2% formic acid and 7.8% acetic acid) containing cold phosphoamino acids as markers. The phosphoamino acids were resolved electrophoretically in two dimensions using a thin layer cellulose (TLC) plate with two pH systems as described (49Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1276) Google Scholar). The markers were visualized by staining with 0.2% ninhydrin in acetone, and the 32P-labeled residues were detected by autoradiography. Lymphocytes isolated from human blood were cultured in α-minimal essential medium supplemented with 10% fetal bovine serum and phytohemagglutinin at 37 °C for 68–72 h. The lymphocyte cultures were treated with bromodeoxyuridine (0.18 mg/ml, Sigma) to synchronize the cell population. The synchronized cells were washed three times with serum-free medium to release the block and recultured at 37 °C for 6 h in α-minimal essential medium with thymidine (2.5 μg/ml, Sigma). The cells were harvested, and the cell slides were prepared by using standard procedures including hypotonic treatment, fixation, and air-drying. The procedure for FISH detection was performed as described previously (50Heng H.H.Q. Squire J. Tsui L.-C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9509-9513Crossref PubMed Scopus (521) Google Scholar, 51Heng H.H.Q. Tsui L.-C. Chromosoma (Berl .). 1993; 102: 325-332Crossref PubMed Scopus (431) Google Scholar). Briefly, the cell slides were baked at 55 °C for 1 h. After RNase treatment, the slides were denatured in 70% formamide in 2× SSC for 2 min at 70 °C followed by dehydration with ethanol. DNA probes were labeled with biotinylated dATP at 15 °C for 1 h, using the Life Technologies, Inc., BioNick labeling kit (Life Technologies, Inc.). Probes were denatured at 75 °C for 5 min in a hybridization buffer containing 50% formamide and 10% dextran sulfate and loaded onto the denatured chromosomal slides. After 16–20 h hybridization, the slides were washed and incubated with fluorescein isothiocyanate-conjugated avidin (Vector Laboratories), and the signal was amplified as described (51Heng H.H.Q. Tsui L.-C. Chromosoma (Berl .). 1993; 102: 325-332Crossref PubMed Scopus (431) Google Scholar). FISH signals and the 4′,6′-diamidino-2-phenylindole (DAPI) banding patterns were recorded separately by taking photographs, and the assignment of the FISH mapping data with chromosomal bands was achieved by superimposing FISH signals with the DAPI-banded chromosomes (52McDonnell P.C. DiLella A.G. Lee J.C. Young P.R. Genomics. 1995; 29: 301-302Crossref PubMed Scopus (13) Google Scholar). A 1328-bp partial cDNA sequence with high homology (∼62% amino acid identity) to the kinase domain of mouse p38-β cDNA was identified from the Amgen EST data base of a rat colon cDNA library. Initially, we termed this cDNA an IKK-like kinase. By using this rat cDNA as a probe, we have isolated a putative full-length cDNA clone from a rat" @default.
• W2002022970 created "2016-06-24" @default.
• W2002022970 creator A5030516198 @default.
• W2002022970 creator A5041338628 @default.
• W2002022970 creator A5061480578 @default.
• W2002022970 creator A5085429091 @default.
• W2002022970 creator A5087039695 @default.
• W2002022970 date "1999-03-01" @default.
• W2002022970 modified "2023-10-14" @default.
• W2002022970 title "Murine p38-δ Mitogen-activated Protein Kinase, a Developmentally Regulated Protein Kinase That Is Activated by Stress and Proinflammatory Cytokines" @default.
• W2002022970 cites W1014698510 @default.
• W2002022970 cites W1528493806 @default.
• W2002022970 cites W1572174728 @default.
• W2002022970 cites W1572416479 @default.
• W2002022970 cites W1573821584 @default.
• W2002022970 cites W1964100540 @default.
• W2002022970 cites W1964504843 @default.
• W2002022970 cites W1968590447 @default.
• W2002022970 cites W1971887536 @default.
• W2002022970 cites W1972145919 @default.
• W2002022970 cites W1973766879 @default.
• W2002022970 cites W1976462601 @default.
• W2002022970 cites W1977472414 @default.
• W2002022970 cites W1983917564 @default.
• W2002022970 cites W1984665850 @default.
• W2002022970 cites W1984903682 @default.
• W2002022970 cites W1992494265 @default.
• W2002022970 cites W1995819749 @default.
• W2002022970 cites W1998487776 @default.
• W2002022970 cites W1999157780 @default.
• W2002022970 cites W2000082154 @default.
• W2002022970 cites W2001399809 @default.
• W2002022970 cites W2002282120 @default.
• W2002022970 cites W2003867300 @default.
• W2002022970 cites W2005900169 @default.
• W2002022970 cites W2007986629 @default.
• W2002022970 cites W2019599792 @default.
• W2002022970 cites W2020442337 @default.
• W2002022970 cites W2023189063 @default.
• W2002022970 cites W2025548430 @default.
• W2002022970 cites W2026951979 @default.
• W2002022970 cites W2027880172 @default.
• W2002022970 cites W2028886214 @default.
• W2002022970 cites W2029055264 @default.
• W2002022970 cites W2030016364 @default.
• W2002022970 cites W2030900078 @default.
• W2002022970 cites W2032731042 @default.
• W2002022970 cites W2036985660 @default.
• W2002022970 cites W2038087154 @default.
• W2002022970 cites W2053449050 @default.
• W2002022970 cites W2068502127 @default.
• W2002022970 cites W2070144920 @default.
• W2002022970 cites W2072061824 @default.
• W2002022970 cites W2072410510 @default.
• W2002022970 cites W2077880395 @default.
• W2002022970 cites W2079588007 @default.
• W2002022970 cites W2089686093 @default.
• W2002022970 cites W2118897660 @default.
• W2002022970 cites W2127195256 @default.
• W2002022970 cites W2134468110 @default.
• W2002022970 cites W2147678437 @default.
• W2002022970 cites W2149333426 @default.
• W2002022970 cites W2150792239 @default.
• W2002022970 cites W2155868854 @default.
• W2002022970 cites W9176561 @default.
• W2002022970 doi "https://doi.org/10.1074/jbc.274.11.7095" @default.
• W2002022970 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10066767" @default.
• W2002022970 hasPublicationYear "1999" @default.
• W2002022970 type Work @default.
• W2002022970 sameAs 2002022970 @default.
• W2002022970 citedByCount "94" @default.
• W2002022970 countsByYear W20020229702012 @default.
• W2002022970 countsByYear W20020229702013 @default.
• W2002022970 countsByYear W20020229702014 @default.
• W2002022970 countsByYear W20020229702015 @default.
• W2002022970 countsByYear W20020229702017 @default.
• W2002022970 countsByYear W20020229702018 @default.
• W2002022970 countsByYear W20020229702020 @default.
• W2002022970 countsByYear W20020229702022 @default.
• W2002022970 crossrefType "journal-article" @default.
• W2002022970 hasAuthorship W2002022970A5030516198 @default.
• W2002022970 hasAuthorship W2002022970A5041338628 @default.
• W2002022970 hasAuthorship W2002022970A5061480578 @default.
• W2002022970 hasAuthorship W2002022970A5085429091 @default.
• W2002022970 hasAuthorship W2002022970A5087039695 @default.
• W2002022970 hasBestOaLocation W20020229701 @default.
• W2002022970 hasConcept C124160383 @default.
• W2002022970 hasConcept C132149769 @default.
• W2002022970 hasConcept C137361374 @default.
• W2002022970 hasConcept C164027704 @default.
• W2002022970 hasConcept C184235292 @default.
• W2002022970 hasConcept C185592680 @default.
• W2002022970 hasConcept C203014093 @default.
• W2002022970 hasConcept C2776914184 @default.
• W2002022970 hasConcept C51551487 @default.
• W2002022970 hasConcept C86803240 @default.
• W2002022970 hasConcept C90934575 @default.
• W2002022970 hasConcept C95444343 @default.

• next