FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2034303876> ?p ?o ?g. }

• W2034303876 endingPage "22752" @default.
• W2034303876 startingPage "22745" @default.
• W2034303876 abstract "Control over the nuclear import of transcription factors (TFs) represents a level of gene regulation integral to cellular processes such as differentiation and transformation. TheDrosophila TF Dorsal shares with other rel TF family members the fact that it contains a phosphorylation site for the cAMP-dependent protein kinase (PKA) 22 amino acids N-terminal to the nuclear localization signal (NLS) at amino acids 335–340. This study examines for the first time the nuclear import kinetics of Dorsal fusion proteins in rat hepatoma cells in vivo and in vitro. Nuclear uptake was found to be not only NLS-dependent, but also strongly dependent on the PKA site, whereby substitution of Ser312 by either Ala or Glu using site-directed mutagenesis severely reduced nuclear accumulation. Exogenous cAMP or PKA catalytic subunit significantly enhanced the nuclear import of wild-type proteins both in vivo andin vitro. Using a direct binding assay, the molecular basis of PKA site enhancement of Dorsal fusion protein nuclear import was determined to be PKA site-mediated modulation of NLS recognition by the importin 58/97 complex. The physiological relevance of these results is supported by the observation that Drosophila embryos expressing PKA site Dorsal mutant variants were impaired in development. We conclude that the Dorsal NLS and PKA site constitute a phosphorylation-regulated NLS essential to Dorsal function and able to function in heterologous mammalian cell systems, where phosphorylation modulates the affinity of NLS recognition by importin. Control over the nuclear import of transcription factors (TFs) represents a level of gene regulation integral to cellular processes such as differentiation and transformation. TheDrosophila TF Dorsal shares with other rel TF family members the fact that it contains a phosphorylation site for the cAMP-dependent protein kinase (PKA) 22 amino acids N-terminal to the nuclear localization signal (NLS) at amino acids 335–340. This study examines for the first time the nuclear import kinetics of Dorsal fusion proteins in rat hepatoma cells in vivo and in vitro. Nuclear uptake was found to be not only NLS-dependent, but also strongly dependent on the PKA site, whereby substitution of Ser312 by either Ala or Glu using site-directed mutagenesis severely reduced nuclear accumulation. Exogenous cAMP or PKA catalytic subunit significantly enhanced the nuclear import of wild-type proteins both in vivo andin vitro. Using a direct binding assay, the molecular basis of PKA site enhancement of Dorsal fusion protein nuclear import was determined to be PKA site-mediated modulation of NLS recognition by the importin 58/97 complex. The physiological relevance of these results is supported by the observation that Drosophila embryos expressing PKA site Dorsal mutant variants were impaired in development. We conclude that the Dorsal NLS and PKA site constitute a phosphorylation-regulated NLS essential to Dorsal function and able to function in heterologous mammalian cell systems, where phosphorylation modulates the affinity of NLS recognition by importin. Precisely scheduled nuclear import of transcription factors (TFs) 1The abbreviations used are: TFtranscription factorNLSnuclear localization sequenceprNLSphosphorylation-regulated NLSPKAcAMP-dependent protein kinaseT-agSV40 large tumor-antigenCK2protein kinase CK2 (casein kinase II)CLSMconfocal laser scanning microscopyELISAenzyme-linked immunosorbent assayGTPγSguanosine 5′-O-(thiotriphosphate). is a key factor in eukaryotic cell function (1Schmitz M.L. Henkel T. Baeuerle P.A. Trends Cell Biol. 1991; 1: 130-137Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 2Jans D.A. Biochem. J. 1995; 311: 705-716Crossref PubMed Scopus (179) Google Scholar, 3Jans D.A. Hübner S. Physiol. Rev. 1996; 76: 651-685Crossref PubMed Scopus (388) Google Scholar). While proteins such as histones appear to be constitutively targeted to the nucleus, TFs such as those of therel family (1Schmitz M.L. Henkel T. Baeuerle P.A. Trends Cell Biol. 1991; 1: 130-137Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 4Govind S. Steward R. Trends Genet. 1991; 7: 119-125Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 5Shirakawa F. Mizel S.B. Mol. Cell. Biol. 1989; 9: 2424-2430Crossref PubMed Scopus (302) Google Scholar, 6Lenardo M.J. Baltimore D. Cell. 1989; 58: 227-229Abstract Full Text PDF PubMed Scopus (1258) Google Scholar, 7Mosialos G. Hamer P. Capobianco A.J. Laursen R.A. Gilmore T.D. Mol. Cell. Biol. 1991; 11: 5867-5877Crossref PubMed Scopus (95) Google Scholar), the nuclear factor of activated T-cells (8Nolan G.P. Cell. 1994; 77: 795-798Abstract Full Text PDF PubMed Scopus (97) Google Scholar), SWI5 from yeast (9Moll T. Tebb G. Surana U. Robitsch H. Nasymth K. Cell. 1991; 66: 743-758Abstract Full Text PDF PubMed Scopus (414) Google Scholar, 10Jans D.A. Moll T. Nasmyth K. Jans P. J. Biol. Chem. 1995; 270: 17064-17067Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), and the cytokine responsive signal transducers and activators of transcription (STATs) (11Shuai K. Schindler C. Prezioso V.R. Darnell Jr., J.E. Science. 1992; 258: 1808-1812Crossref PubMed Scopus (657) Google Scholar, 12Schindler C. Shuai K. Prezioso V.R. Darnell Jr., J.E. Science. 1992; 257: 809-813Crossref PubMed Scopus (723) Google Scholar) are translocated to the nucleus only under specific conditions, being otherwise cytoplasmic and thereby directly accessible to cytoplasmic signal-transducing systems (1Schmitz M.L. Henkel T. Baeuerle P.A. Trends Cell Biol. 1991; 1: 130-137Abstract Full Text PDF PubMed Scopus (68) Google Scholar). The fact that nuclear translocation of many TFs and oncogene products accompanies changes in the differentiation or metabolic state of eukaryotic cells underlines the fact that nuclear protein import is a key control point in the regulation of gene expression (2Jans D.A. Biochem. J. 1995; 311: 705-716Crossref PubMed Scopus (179) Google Scholar, 3Jans D.A. Hübner S. Physiol. Rev. 1996; 76: 651-685Crossref PubMed Scopus (388) Google Scholar). transcription factor nuclear localization sequence phosphorylation-regulated NLS cAMP-dependent protein kinase SV40 large tumor-antigen protein kinase CK2 (casein kinase II) confocal laser scanning microscopy enzyme-linked immunosorbent assay guanosine 5′-O-(thiotriphosphate). Proteins larger than 45 kDa require a nuclear localization sequence (NLS) (2Jans D.A. Biochem. J. 1995; 311: 705-716Crossref PubMed Scopus (179) Google Scholar, 3Jans D.A. Hübner S. Physiol. Rev. 1996; 76: 651-685Crossref PubMed Scopus (388) Google Scholar) in order to be targeted to the nucleus. We and others have shown that phosphorylation in the vicinity of NLSs plays a central role in regulating NLS-dependent nuclear protein import (7Mosialos G. Hamer P. Capobianco A.J. Laursen R.A. Gilmore T.D. Mol. Cell. Biol. 1991; 11: 5867-5877Crossref PubMed Scopus (95) Google Scholar, 8Nolan G.P. Cell. 1994; 77: 795-798Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 9Moll T. Tebb G. Surana U. Robitsch H. Nasymth K. Cell. 1991; 66: 743-758Abstract Full Text PDF PubMed Scopus (414) Google Scholar, 10Jans D.A. Moll T. Nasmyth K. Jans P. J. Biol. Chem. 1995; 270: 17064-17067Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 11Shuai K. Schindler C. Prezioso V.R. Darnell Jr., J.E. Science. 1992; 258: 1808-1812Crossref PubMed Scopus (657) Google Scholar, 12Schindler C. Shuai K. Prezioso V.R. Darnell Jr., J.E. Science. 1992; 257: 809-813Crossref PubMed Scopus (723) Google Scholar, 13Jans D.A. Ackermann M. Bischoff J.R. Beach D.H. Peters R. J. Cell Biol. 1991; 115: 1203-1212Crossref PubMed Scopus (183) Google Scholar, 14Rihs H.-P. Jans D.A. Fan H. Peters R. EMBO J. 1991; 10: 633-639Crossref PubMed Scopus (302) Google Scholar, 15Jans D.A. Jans P. Oncogene. 1994; 9: 2961-2968PubMed Google Scholar, 16Beg A.A. Ruben S.M. Scheinman R.I. Haskill S. Rosen C.A. Baldwin Jr., A.S. Genes Dev. 1992; 6: 1899-1913Crossref PubMed Scopus (612) Google Scholar, 17Henkel T. Zabel U. van Zee K. Müller J.M. Fanning E. Baeuerle P.A. Cell. 1992; 68: 1121-1133Abstract Full Text PDF PubMed Scopus (304) Google Scholar, 18Zabel U. Henkel T. dos Santos Silva M. Baeuerle P.A. EMBO J. 1993; 12: 201-211Crossref PubMed Scopus (267) Google Scholar, 19Xiao C-Y. Hübner S. Elliot R.M. Caon A. Jans D.A. J. Biol. Chem. 1996; 271: 6451-6457Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 20Vancurova I. Paine T.M. Lou W. Paine P.L. J. Cell Sci. 1995; 108: 779-787Crossref PubMed Google Scholar, 21Hennekes H. Peter M. Weber K. Nigg E.A. J. Cell Biol. 1993; 120: 1293-1304Crossref PubMed Scopus (102) Google Scholar). The modular sequence motifs able to confer regulated nuclear protein import on heterologous proteins, called phosphorylation-regulated NLSs (prNLSs) (2Jans D.A. Biochem. J. 1995; 311: 705-716Crossref PubMed Scopus (179) Google Scholar, 3Jans D.A. Hübner S. Physiol. Rev. 1996; 76: 651-685Crossref PubMed Scopus (388) Google Scholar), have been identified for a number of proteins, including the CcN motif of the simian virus SV40 large tumor antigen (T-ag) where transport is regulated by dual phosphorylation by protein kinase CK2 (CK2) and the cyclin-dependent kinasecdc2 (13Jans D.A. Ackermann M. Bischoff J.R. Beach D.H. Peters R. J. Cell Biol. 1991; 115: 1203-1212Crossref PubMed Scopus (183) Google Scholar, 14Rihs H.-P. Jans D.A. Fan H. Peters R. EMBO J. 1991; 10: 633-639Crossref PubMed Scopus (302) Google Scholar, 15Jans D.A. Jans P. Oncogene. 1994; 9: 2961-2968PubMed Google Scholar), and the cell cycle-dependent NLS of the yeast TF SWI5 (9Moll T. Tebb G. Surana U. Robitsch H. Nasymth K. Cell. 1991; 66: 743-758Abstract Full Text PDF PubMed Scopus (414) Google Scholar, 10Jans D.A. Moll T. Nasmyth K. Jans P. J. Biol. Chem. 1995; 270: 17064-17067Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Significantly in the case of SWI5, cyclin-dependent kinase site-mediated inhibition of SWI5 nuclear transport functions in higher eukaryotes (10Jans D.A. Moll T. Nasmyth K. Jans P. J. Biol. Chem. 1995; 270: 17064-17067Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). A variety of kinases are known to modulate nuclear protein import in response to specific hormonal or other cellular signals regulating their activity (2Jans D.A. Biochem. J. 1995; 311: 705-716Crossref PubMed Scopus (179) Google Scholar, 3Jans D.A. Hübner S. Physiol. Rev. 1996; 76: 651-685Crossref PubMed Scopus (388) Google Scholar). In the case of the T-ag CcN motif, we have shown that substitution of one kinase site by a consensus site for another can alter the cellular signals able to regulate the nuclear import of proteins carrying the modified prNLS (19Xiao C-Y. Hübner S. Elliot R.M. Caon A. Jans D.A. J. Biol. Chem. 1996; 271: 6451-6457Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Although a number of prNLSs have been defined, the mechanistic basis of prNLS-mediated regulation of nuclear transport is largely unclear. Our recent work with respect to CK2 enhancement of T-ag nuclear import indicates that phosphorylation facilitates recognition of the T-ag NLS by the NLS-binding importin subunits (22Hübner S. Xiao C-Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). We were interested in measuring the kinetics of nuclear accumulation of the relfamily member Dorsal, the Drosophila melanogaster morphogen whose graded nuclear translocation is integral in determining dorsal-ventral polarity during embryogenesis (4Govind S. Steward R. Trends Genet. 1991; 7: 119-125Abstract Full Text PDF PubMed Scopus (120) Google Scholar). Modifying genes that regulate Dorsal nuclear accumulation include the transmembrane receptor protein Toll, the Raf family kinase Pelle, the protein Tube, which may have a chaperone function with respect to Dorsal or Pelle, and the cytoplasmic retention factor Cactus (23Norris J.L. Manley J.L. Genes Dev. 1992; 6: 1654-1667Crossref PubMed Scopus (67) Google Scholar, 24Norris J.L. Manley J.L. Genes Dev. 1995; 9: 358-369Crossref PubMed Scopus (26) Google Scholar, 25Kidd S. Cell. 1992; 71: 632-635Abstract Full Text PDF Scopus (184) Google Scholar, 26Govind S. Drier E. Huang L.H. Steward R. Mol. Cell. Biol. 1996; 16: 1103-1114Crossref PubMed Scopus (34) Google Scholar, 27Lehming N. McGuire S. Brickman J.M. Ptashne M. Proc. Natl. Acad. Sci. U. S. A. 1996; 92: 10242-10246Crossref Scopus (42) Google Scholar), which binds Dorsal and prevents its nuclear localization although not through direct binding to the Dorsal NLS (27Lehming N. McGuire S. Brickman J.M. Ptashne M. Proc. Natl. Acad. Sci. U. S. A. 1996; 92: 10242-10246Crossref Scopus (42) Google Scholar). Results using a number of experimental systems have suggested the involvement of phosphorylation in regulating Dorsal nuclear localization (23Norris J.L. Manley J.L. Genes Dev. 1992; 6: 1654-1667Crossref PubMed Scopus (67) Google Scholar, 28Whalen A.M. Steward R. J. Cell Biol. 1993; 123: 523-534Crossref PubMed Scopus (110) Google Scholar, 29Gillespie S.K.H. Wasserman S.A. Mol. Cell. Biol. 1994; 14: 3559-3568Crossref PubMed Scopus (83) Google Scholar, 30Kubota K. Keith F.J. Gay N.J. Biochem. J. 1993; 296: 497-503Crossref PubMed Scopus (20) Google Scholar, 31Reach M. Galindo R.L. Towb P. Allen J.L. Karin M. Wasserman S.A. Dev. Biol. 1996; 180: 353-364Crossref PubMed Scopus (101) Google Scholar, 32Belvin M.P. Anderson K.V. Annu. Rev. Cell. Dev. Biol. 1996; 12: 393-416Crossref PubMed Scopus (685) Google Scholar). In identical fashion to otherrel family members, Dorsal possesses a consensus site for PKA 22 amino acids N-terminal to a 6- amino acid NLS within the ∼300-amino acid rel homology domain (see Fig. 1), where the enhancing role of PKA in terms of nuclear localization has been qualitatively described, using transfection systems for Dorsal (23Norris J.L. Manley J.L. Genes Dev. 1992; 6: 1654-1667Crossref PubMed Scopus (67) Google Scholar) and c-rel (7Mosialos G. Hamer P. Capobianco A.J. Laursen R.A. Gilmore T.D. Mol. Cell. Biol. 1991; 11: 5867-5877Crossref PubMed Scopus (95) Google Scholar), and implicated for NF-κB p65 (5Shirakawa F. Mizel S.B. Mol. Cell. Biol. 1989; 9: 2424-2430Crossref PubMed Scopus (302) Google Scholar, 6Lenardo M.J. Baltimore D. Cell. 1989; 58: 227-229Abstract Full Text PDF PubMed Scopus (1258) Google Scholar, 23Norris J.L. Manley J.L. Genes Dev. 1992; 6: 1654-1667Crossref PubMed Scopus (67) Google Scholar). In this study we define the Dorsal prNLS kinetically by quantitating the nuclear uptake of β-galactosidase fusion proteins at the single cell level both in vivo and in vitro in the HTC rat hepatoma line using confocal laser scanning microscopy (CLSM) (10Jans D.A. Moll T. Nasmyth K. Jans P. J. Biol. Chem. 1995; 270: 17064-17067Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar,13Jans D.A. Ackermann M. Bischoff J.R. Beach D.H. Peters R. J. Cell Biol. 1991; 115: 1203-1212Crossref PubMed Scopus (183) Google Scholar, 14Rihs H.-P. Jans D.A. Fan H. Peters R. EMBO J. 1991; 10: 633-639Crossref PubMed Scopus (302) Google Scholar, 15Jans D.A. Jans P. Oncogene. 1994; 9: 2961-2968PubMed Google Scholar, 19Xiao C-Y. Hübner S. Elliot R.M. Caon A. Jans D.A. J. Biol. Chem. 1996; 271: 6451-6457Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). While Dorsal fusion protein nuclear uptake is NLS-dependent, it is also strongly dependent on the PKA site, whereby substitution of Ser312 by either Ala or Glu using site-directed mutagenesis essentially abolishes nuclear accumulation. Exogenous addition of cAMP or PKA catalytic (C-) subunit enhances nuclear import of the wild-type Dorsal proteins. Results using an ELISA-based binding assay indicate that the mechanistic basis of the PKA site modulation of Dorsal fusion protein nuclear import is PKA-site enhancement of the binding interaction between the NLS-binding importin 58/97 complex and the Dorsal NLS. The fact that Drosophilaembryos expressing PKA site and NLS Dorsal mutant variants show impaired development and fail to hatch supports the physiological relevance of the results. We conclude that the Dorsal NLS and PKA site constitute a prNLS essential to Dorsal function, and able to function in heterologous mammalian cell systems, where phosphorylation regulates interaction with importin 58/97. PKA (EC 2.7.1.37) catalytic (C-) subunit (bovine heart) was from Sigma; other reagents were from the sources previously described (10Jans D.A. Moll T. Nasmyth K. Jans P. J. Biol. Chem. 1995; 270: 17064-17067Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 15Jans D.A. Jans P. Oncogene. 1994; 9: 2961-2968PubMed Google Scholar, 19Xiao C-Y. Hübner S. Elliot R.M. Caon A. Jans D.A. J. Biol. Chem. 1996; 271: 6451-6457Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 22Hübner S. Xiao C-Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Cells of the HTC rat hepatoma tissue culture (a derivative of Morris hepatoma 7288C) line were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum as described previously (10Jans D.A. Moll T. Nasmyth K. Jans P. J. Biol. Chem. 1995; 270: 17064-17067Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 15Jans D.A. Jans P. Oncogene. 1994; 9: 2961-2968PubMed Google Scholar, 19Xiao C-Y. Hübner S. Elliot R.M. Caon A. Jans D.A. J. Biol. Chem. 1996; 271: 6451-6457Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Plasmids expressing the Dorsal β-galactosidase fusion proteins were derived by standard recombinant DNA technology. Inserts encoding Dorsal amino acids 1–678 (D1 construct, full-length Dorsal), 1–346 (D2 construct, encoding therel homology domain), and 297–346 (D3 construct, encoding the PKA site at Ser312 together with the NLS at amino acids 335–340), were derived using a polymerase chain reaction and ligated into the NcoI site of a derivative of plasmid vector pPR2 (14Rihs H.-P. Jans D.A. Fan H. Peters R. EMBO J. 1991; 10: 633-639Crossref PubMed Scopus (302) Google Scholar) in which the oligonucleotide 5′-GCCATGGTGTTA-3′ was inserted into the SmaI site. The D3 construct containing the NLS-deficient mutant (Thr337 Glu339), as well as plasmids encoding the PKA site Ser312 substitutions by Ala or Glu (see Fig. 1) were derived by oligonucleotide site-directed mutagenesis of the wild-type Dorsal β-galactosidase fusion protein-expressing constructs using the Amersham Pharmacia Biotech U.S.E. mutagenesis kit. The T-ag-CcN-β-galactosidase fusion protein used as a control contains T-ag amino acids 111–135, encompassing the CcN motif (including CK2 and cyclin-dependent kinase phosphorylation sites and NLS) fused N-terminal to the Escherichia coliβ-galactosidase enzyme sequence (amino acids 9–1023) (13Jans D.A. Ackermann M. Bischoff J.R. Beach D.H. Peters R. J. Cell Biol. 1991; 115: 1203-1212Crossref PubMed Scopus (183) Google Scholar, 14Rihs H.-P. Jans D.A. Fan H. Peters R. EMBO J. 1991; 10: 633-639Crossref PubMed Scopus (302) Google Scholar). 1 mm isopropyl-1-thio-β-d-galactopyranoside was used to induce expression of β-galactosidase fusion proteins inE. coli, which were then purified by affinity chromatography and labeled with 5-(iodacetamido)-fluorescein as described previously (13Jans D.A. Ackermann M. Bischoff J.R. Beach D.H. Peters R. J. Cell Biol. 1991; 115: 1203-1212Crossref PubMed Scopus (183) Google Scholar, 14Rihs H.-P. Jans D.A. Fan H. Peters R. EMBO J. 1991; 10: 633-639Crossref PubMed Scopus (302) Google Scholar). Protein concentrations were measured using the dye binding assay of Bradford (33Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216261) Google Scholar). Analysis of nuclear import kinetics at the single cell level in microinjected HTC cells using CLSM (Bio-Rad MRC-500) was as described previously (10Jans D.A. Moll T. Nasmyth K. Jans P. J. Biol. Chem. 1995; 270: 17064-17067Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 19Xiao C-Y. Hübner S. Elliot R.M. Caon A. Jans D.A. J. Biol. Chem. 1996; 271: 6451-6457Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 22Hübner S. Xiao C-Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). HTC cells were fused with polyethylene glycol about 1 h prior to microinjection to produce polykaryons (13Jans D.A. Ackermann M. Bischoff J.R. Beach D.H. Peters R. J. Cell Biol. 1991; 115: 1203-1212Crossref PubMed Scopus (183) Google Scholar, 14Rihs H.-P. Jans D.A. Fan H. Peters R. EMBO J. 1991; 10: 633-639Crossref PubMed Scopus (302) Google Scholar, 15Jans D.A. Jans P. Oncogene. 1994; 9: 2961-2968PubMed Google Scholar, 19Xiao C-Y. Hübner S. Elliot R.M. Caon A. Jans D.A. J. Biol. Chem. 1996; 271: 6451-6457Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Image analysis of CLSM files, using the MacIntosh NIH Image 1.49 public domain software, and curve fitting was performed as described elsewhere (19Xiao C-Y. Hübner S. Elliot R.M. Caon A. Jans D.A. J. Biol. Chem. 1996; 271: 6451-6457Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 22Hübner S. Xiao C-Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Analysis of nuclear import kinetics at the single cell level using mechanically perforated HTC cells in conjunction with CLSM was as described previously (13Jans D.A. Ackermann M. Bischoff J.R. Beach D.H. Peters R. J. Cell Biol. 1991; 115: 1203-1212Crossref PubMed Scopus (183) Google Scholar, 19Xiao C-Y. Hübner S. Elliot R.M. Caon A. Jans D.A. J. Biol. Chem. 1996; 271: 6451-6457Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar,34Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). NLS-dependent nuclear protein import can be reconstituted in this system through the exogenous addition of cytosolic extract (untreated reticulocyte lysate, Promega catalog no. L415A), an ATP-regenerating system (0.125 mg/ml creatine phosphokinase, 30 mm creatine phosphate, 2 mm ATP), and transport substrate (0.2 mg/ml 5-(iodacetamido)-fluorescein-labeled fusion protein). Image analysis and curve-fitting were performed as forin vivo assays. Where indicated, proteins were either coinjected with cAMP (250 μm in the pipette) in the case of the in vivoexperiments, or 25 μm cAMP, 2.5 μm PKA C-subunit inhibitor peptide PKI 5–24, and 400 picomolar units PKA C-subunit/μl included in the cytosol in the case of in vitro experiments (19Xiao C-Y. Hübner S. Elliot R.M. Caon A. Jans D.A. J. Biol. Chem. 1996; 271: 6451-6457Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In experiments where the dependence of transport on the GTP-binding protein Ran/TC4 was tested, cytosolic extract was treated with 850 μm GTPγS (nonhydrolyzable GTP analog) for 5 min at room temperature, prior to use in the in vitro assay (34Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The lectin wheat germ agglutinin, which impairs nuclear pore complex function, was used at 240 μg/ml. In vitrophosphorylation of fusion proteins by PKA C-subunit was analyzed quantitatively by determination of the stoichiometry of phosphorylation as described previously (13Jans D.A. Ackermann M. Bischoff J.R. Beach D.H. Peters R. J. Cell Biol. 1991; 115: 1203-1212Crossref PubMed Scopus (183) Google Scholar, 19Xiao C-Y. Hübner S. Elliot R.M. Caon A. Jans D.A. J. Biol. Chem. 1996; 271: 6451-6457Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 22Hübner S. Xiao C-Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Mouse importin 58- and 97-glutathioneS-transferase fusion proteins were expressed and purified as described previously (22Hübner S. Xiao C-Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar), with glutathioneS-transferase-free mouse importin 58 prepared by subsequent thrombin cleavage (22Hübner S. Xiao C-Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Binding of importin subunits to β-galactosidase fusion proteins was quantitated using an ELISA as described previously (22Hübner S. Xiao C-Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 34Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Briefly, fusion proteins (0.5 μg/well) were coated overnight at 4 °C in 96-well microtiterplates (Nunc). After blocking, appropriate dilutions of mouse importin 58-glutathione S-transferase or precomplexed importin 58/97-glutathione S-transferase complex were then added to the wells and incubated for 16 h at 4 °C. Bound importin was detected using rabbit anti-glutathioneS-transferase and goat anti-rabbit IgG alkaline phosphatase-conjugated antibodies (Sigma) and the colorimetric substrate p-nitrophenyl phosphate. A405 was followed with time using a plate reader (Molecular Devices), with values corrected by subtracting both the absorbance at 0 min and the absorbance of wells incubated without importin 58- or 58/97-glutathione S-transferase complexes. Correction was made for differences in coating efficiency as described previously using a parallel β-galactosidase ELISA (22Hübner S. Xiao C-Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 34Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Plasmid pBP-dorsal is a derivative of pSP64 (35Melton D.A. Krieg P.A. Rebagliati M.R. Maniatis T. Zinn K. Green M.R. Nucleic Acids Res. 1984; 12: 7035-7056Crossref PubMed Scopus (4057) Google Scholar) that carries the wild-type dorsal cDNA (36Steward R. Science. 1987; 238: 692-694Crossref PubMed Scopus (344) Google Scholar) cloned immediately downstream of the Xenopus globin gene leader, which is known to direct efficient translational initiation in Drosophila embryos (37Driever W. Siegel V. Nüsslein-Volhard C. Development. 1990; 109: 811-820PubMed Google Scholar). Site-directed mutagenesis (see above) was used to change the PKA site Ser312 to Ala and to Glu, and to create the NLS-deficient double mutant (Lys337 to Thr, Gln339 to Glu) in pBP-dorsal to generatedorsal mutants exactly comparable to those described above for the bacterially expressed Dorsal fusion proteins. For eachdorsal construct, a cassette comprising the globin translation signals and modified dorsal cDNA was excised and introduced in the appropriate orientation into plasmid pCasperbcdBglII, a P-element-derived vector (37Driever W. Siegel V. Nüsslein-Volhard C. Development. 1990; 109: 811-820PubMed Google Scholar) carrying the promoter region of the bcd gene (38Berleth T. Burri M. Thoma G. Richstein S. Frigerio G. Noll M. Nüsslein-Volhard C. EMBO J. 1988; 7: 1749-1756Crossref PubMed Scopus (577) Google Scholar), which directs transcription in the female germ line. As a wild-type control, a dorsalcDNA carrying the Met-His6 N-terminal epitope tag, cloned in pBP4-dorsal, was similarly introduced into pCasperbcdBglII. The epitope tag has no effect on Dorsal protein function. The P-element vectors carrying the dorsalcassettes were then introduced into the Drosophila genome through conventional transformation methods (39Rubin G.M. Spradling A.C. Science. 1982; 218: 348-353Crossref PubMed Scopus (2336) Google Scholar). To determine their ability to substitute for the wild-typedorsal gene in defining embryonic dorsal-ventral pattern, the mutant transgenes were crossed into females of the genotypew/w; dl 1 /dl 1 . The presence of thew + eye marker allowed identification of females carrying the transgenes by their colored eyes. Genetically, thedl 1 allele behaves like a null mutation; Dorsal protein is not present in embryos produced bydl 1 /dl 1 females (see also Fig.7 B). dorsal mutant females carrying the variousdorsal mutant transgenes were placed on yeast and allowed to lay eggs. Cuticular preparations of the embryos produced by these females were made 72 h after egg deposition (40van der Meer J.M. Drosophila Inform. Serv. 1977; 52: 160Google Scholar). Multiple transgenic lines were obtained and analyzed for each construct; significant differences in expression are associated with different chromosomal sites of insertion. Although the level of phenotypic rescue was relatively constant for each insert identified for a particular dorsal transgene from line to line, there were differences in the level of rescue, and analysis was restricted to those insertions that provided the highest level of rescue of thedorsal mutant phenotype. About a third of transgenic inserts obtained for a particular construct were found to provide the highest level of observable rescue (see “Results”); two of six stocks carrying p(w +, bcd-dorsal, NLS mutant) led to the formation of embryos producing Filzkörper material, three of 10 stocks carrying either p(w +, bcd-dorsal, Ala312) or p(w +, bcd-dorsal, Glu312) led to the formation of embryos producing ventral denticles as well as Filzkörper material, and one third of transgenic lines carrying the wild-type dorsal gene under the transcriptional control of the bcd promoter produced hatching embryos when pre" @default.
• W2034303876 created "2016-06-24" @default.
• W2034303876 creator A5006386713 @default.
• W2034303876 creator A5020712448 @default.
• W2034303876 creator A5038954081 @default.
• W2034303876 creator A5054290176 @default.
• W2034303876 creator A5070182251 @default.
• W2034303876 creator A5080160147 @default.
• W2034303876 creator A5087863949 @default.
• W2034303876 date "1998-08-01" @default.
• W2034303876 modified "2023-09-29" @default.
• W2034303876 title "The cAMP-dependent Protein Kinase Site (Ser312) Enhances Dorsal Nuclear Import through Facilitating Nuclear Localization Sequence/Importin Interaction" @default.
• W2034303876 cites W1498224103 @default.
• W2034303876 cites W1571632059 @default.
• W2034303876 cites W1826494689 @default.
• W2034303876 cites W1892741576 @default.
• W2034303876 cites W1917049701 @default.
• W2034303876 cites W1951130496 @default.
• W2034303876 cites W1963289746 @default.
• W2034303876 cites W1965398762 @default.
• W2034303876 cites W1968863167 @default.
• W2034303876 cites W1975437758 @default.
• W2034303876 cites W1976656176 @default.
• W2034303876 cites W1991153512 @default.
• W2034303876 cites W1991484745 @default.
• W2034303876 cites W1992908236 @default.
• W2034303876 cites W1994528729 @default.
• W2034303876 cites W2005484362 @default.
• W2034303876 cites W2014619010 @default.
• W2034303876 cites W2017467565 @default.
• W2034303876 cites W2019894624 @default.
• W2034303876 cites W2037928479 @default.
• W2034303876 cites W2057120350 @default.
• W2034303876 cites W2057381460 @default.
• W2034303876 cites W2067626539 @default.
• W2034303876 cites W2069355785 @default.
• W2034303876 cites W2076854746 @default.
• W2034303876 cites W2078119356 @default.
• W2034303876 cites W2079544213 @default.
• W2034303876 cites W2093489683 @default.
• W2034303876 cites W2097239500 @default.
• W2034303876 cites W2098336582 @default.
• W2034303876 cites W2109550723 @default.
• W2034303876 cites W2119691744 @default.
• W2034303876 cites W2128584338 @default.
• W2034303876 cites W2139005210 @default.
• W2034303876 cites W2139412961 @default.
• W2034303876 cites W2145488825 @default.
• W2034303876 cites W2149093922 @default.
• W2034303876 cites W2154627422 @default.
• W2034303876 cites W2157244412 @default.
• W2034303876 cites W4293247451 @default.
• W2034303876 cites W45974969 @default.
• W2034303876 cites W645989409 @default.
• W2034303876 doi "https://doi.org/10.1074/jbc.273.35.22745" @default.
• W2034303876 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9712906" @default.
• W2034303876 hasPublicationYear "1998" @default.
• W2034303876 type Work @default.
• W2034303876 sameAs 2034303876 @default.
• W2034303876 citedByCount "88" @default.
• W2034303876 countsByYear W20343038762012 @default.
• W2034303876 countsByYear W20343038762013 @default.
• W2034303876 countsByYear W20343038762014 @default.
• W2034303876 countsByYear W20343038762015 @default.
• W2034303876 countsByYear W20343038762018 @default.
• W2034303876 crossrefType "journal-article" @default.
• W2034303876 hasAuthorship W2034303876A5006386713 @default.
• W2034303876 hasAuthorship W2034303876A5020712448 @default.
• W2034303876 hasAuthorship W2034303876A5038954081 @default.
• W2034303876 hasAuthorship W2034303876A5054290176 @default.
• W2034303876 hasAuthorship W2034303876A5070182251 @default.
• W2034303876 hasAuthorship W2034303876A5080160147 @default.
• W2034303876 hasAuthorship W2034303876A5087863949 @default.
• W2034303876 hasBestOaLocation W20343038761 @default.
• W2034303876 hasConcept C104317684 @default.
• W2034303876 hasConcept C105702510 @default.
• W2034303876 hasConcept C133882971 @default.
• W2034303876 hasConcept C140530291 @default.
• W2034303876 hasConcept C184235292 @default.
• W2034303876 hasConcept C185592680 @default.
• W2034303876 hasConcept C2780114586 @default.
• W2034303876 hasConcept C2780723820 @default.
• W2034303876 hasConcept C29512474 @default.
• W2034303876 hasConcept C49805395 @default.
• W2034303876 hasConcept C55493867 @default.
• W2034303876 hasConcept C59157529 @default.
• W2034303876 hasConcept C86339819 @default.
• W2034303876 hasConcept C86803240 @default.
• W2034303876 hasConcept C95444343 @default.
• W2034303876 hasConcept C97029542 @default.
• W2034303876 hasConceptScore W2034303876C104317684 @default.
• W2034303876 hasConceptScore W2034303876C105702510 @default.
• W2034303876 hasConceptScore W2034303876C133882971 @default.
• W2034303876 hasConceptScore W2034303876C140530291 @default.
• W2034303876 hasConceptScore W2034303876C184235292 @default.
• W2034303876 hasConceptScore W2034303876C185592680 @default.
• W2034303876 hasConceptScore W2034303876C2780114586 @default.
• W2034303876 hasConceptScore W2034303876C2780723820 @default.

• next