C-terminal Elements Control Location, Activation Threshold, and p38 Docking of Ribosomal S6 Kinase B (RSKB)
2001; Elsevier BV; Volume: 276; Issue: 8 Linguagem: Inglês
10.1074/jbc.m005822200
ISSN1083-351X
AutoresMar Tomás-Zuber, Jean‐Luc Mary, François Lamour, Daniel Bur, Werner Lesslauer,
Tópico(s)Enzyme Structure and Function
ResumoRSKB, a p90 ribosomal S6 protein kinase with two catalytic domains, is activated by p38- and extracellular signal-regulated kinase mitogen-activated protein kinase pathways. The sequences between the two catalytic domains and of the C-terminal extension contain elements that control RSKB activity. The C-terminal extension of RSKB presents a putative bipartite713KRX 14KRRKQKLRS737nuclear location signal. The distinct cytoplasmic and nuclear locations of various C-terminal truncation mutants supported the hypothesis that the nuclear location signal was essential to direct RSKB to the nuclear compartment. The725APLAKRRKQKLRS737 sequence also was essential for the intermolecular association of RSKB with p38. The activation of RSKB through p38 could be dissociated from p38 docking, because RSKB truncated at Ser681 strongly responded to p38 pathway activity. Interestingly, Δ725–772-RSKB was nearly nonresponsive to p38. Sequence alignment with the autoinhibitory C-terminal extension of Ca+2/calmodulin-dependent protein kinase I predicted a conserved regulatory 708AFN710motif. Alanine mutation of the key Phe709 residue resulted in strongly elevated basal level RSKB activity. A regulatory role also was assigned to Thr687, which is located in a mitogen-activated protein kinase phosphorylation consensus site. These findings support that the RSKB C-terminal extension contains elements that control activation threshold, subcellular location, and p38 docking. RSKB, a p90 ribosomal S6 protein kinase with two catalytic domains, is activated by p38- and extracellular signal-regulated kinase mitogen-activated protein kinase pathways. The sequences between the two catalytic domains and of the C-terminal extension contain elements that control RSKB activity. The C-terminal extension of RSKB presents a putative bipartite713KRX 14KRRKQKLRS737nuclear location signal. The distinct cytoplasmic and nuclear locations of various C-terminal truncation mutants supported the hypothesis that the nuclear location signal was essential to direct RSKB to the nuclear compartment. The725APLAKRRKQKLRS737 sequence also was essential for the intermolecular association of RSKB with p38. The activation of RSKB through p38 could be dissociated from p38 docking, because RSKB truncated at Ser681 strongly responded to p38 pathway activity. Interestingly, Δ725–772-RSKB was nearly nonresponsive to p38. Sequence alignment with the autoinhibitory C-terminal extension of Ca+2/calmodulin-dependent protein kinase I predicted a conserved regulatory 708AFN710motif. Alanine mutation of the key Phe709 residue resulted in strongly elevated basal level RSKB activity. A regulatory role also was assigned to Thr687, which is located in a mitogen-activated protein kinase phosphorylation consensus site. These findings support that the RSKB C-terminal extension contains elements that control activation threshold, subcellular location, and p38 docking. family of 90 kDa ribosomal S6 protein kinases mitogen-activated protein kinase stress-activated 38kDa MAPK extracellular signal-regulated kinase ribosomal S6 protein kinase B Ca+2/calmodulin-dependent protein kinase p38 MAPK kinase constitutively active mutant of MKK6 MAPK-activated protein kinase type 2 nuclear location signal cAMP response element-binding protein wild-type polyacrylamide gel electrophoresis The p90 ribosomal S6 protein kinases (RSKs)1 are a family of Ser/Thr protein kinases composed of two catalytic domains, each with canonical ATP-binding site and activation loop sequences. In addition, RSKs contain a regulatory linker sequence connecting the two kinase domains and an extended C-terminal tail. RSKs comprise RSK1–RSK3, which are stimulated through the ERK pathway (1Blenis J. Proc. Natl. Acad. Sci. U. S. 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Biol. 1998; 18: 1978-1984Crossref PubMed Scopus (313) Google Scholar). Deficient mutants of RSK2 in man are linked to Coffin-Lowry syndrome, characterized by mental retardation and malformations (19Merienne K. Jacquot S. Pannetier S. Zeniou M. Bankier A. Gecz J. Mandel J.L. Mulley J. Sassone-Corsi P. Hanauer A. Nat. Genet. 1999; 22: 13-14Crossref PubMed Scopus (131) Google Scholar). Deletion of RSK4 is common in patients with X-linked mental retardation (10Yntema H.G. van den Helm B. Kissing J. van Duijnhoven G. Poppelaars F. Chelly J. Moraine C. Fryns J.P. Hamel B.C. Heilbronner H. Pander H.J. Brunner H.G. Ropers H.H. Cremers F.P. van Bokhoven H. Genomics. 1999; 62: 332-343Crossref PubMed Scopus (93) Google Scholar). Interestingly, RSKB maps to theBBS1 locus (20Zhu S. Gerhard D.S. Hum. Genet. 1998; 103: 674-680Crossref PubMed Scopus (9) Google Scholar), which is associated with Bardet-Biedl syndrome with manifestations reminiscent of Coffin-Lowry syndrome (21Bruford E.A. Riise R. Teague P.W. Porter K. Thomson K.L. Moore A.T. Jay M. Warburg M. Schinzel A. Tommerup N. Tornqvist K. Rosenberg T. Patton M. Mansfield D.C. Wright A.F. Genomics. 1997; 41: 93-99Crossref PubMed Scopus (108) Google Scholar), and may be a candidate BBS gene. Many Ser/Thr-kinases present a resting state characterized by an autoinhibitory conformation of the C-terminal extension. Phosphorylations, or interactions with other proteins, that relax autoinhibition, are required for the activation of these enzymes. For example, the activation of MAPKAPK2, in addition to phosphorylation of Thr205 in the catalytic site, correlated with phosphorylation of a threonine in a PXTP318 motif in the C-terminal tail, which contains an autoinhibitory domain with homology to the amphiphilic A-helix of other kinases (22Engel K. Schultz H. Martin F. Kotlyarov A. Plath K. Hahn M. Heinemann U. Gaestel M. J. Biol. Chem. 1995; 270: 21-27213Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 23Stokoe D. Campbell D.G. Nakielny S. Hidaka H. Leevers S.J. Marshall C. Cohen P. EMBO J. 1992; 11: 3985-3994Crossref PubMed Scopus (392) Google Scholar, 24Veron M. Radzio-Andzelm E. Tsigelny I. Ten Eyck L.F. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10618-10622Crossref PubMed Scopus (68) Google Scholar). In CaMKII, a C-terminal segment blocks the catalytic site in the absence of calmodulin; upon calmodulin binding, a threonine within that segment is phosphorylated, disrupting autoinhibition and leading to a calcium-independent active state of the enzyme (25Yang E. Schulman H. J. Biol. Chem. 1999; 274: 26199-26208Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 26Miller S.G. Patton B.L. Kennedy M.B. Neuron. 1988; 1: 593-604Abstract Full Text PDF PubMed Scopus (228) Google Scholar). When compared with these single-domain enzymes, RSKs with two kinase domains and regulatory sites in linker and C-terminal tail present more complex regulation. Commonly, the N-terminal kinase of RSKs phosphorylates substrates, whereas the C-terminal kinase has a role in regulating RSKs activity. For example, the stepwise activation of RSK1 involved phosphorylations of a threonine in the C-terminal activation loop and of a serine in the linker through ERK, and further phosphorylations of linker and N-terminal activation loop sites through autophosphorylation (27Dalby N.K. Morrice N. Caudwell F.B. Avruch J. Cohen P. J. Biol. Chem. 1998; 273: 1496-1505Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). RSK2 is activated through integrating signals from two independent upstream kinase pathways, ERK and 3-phosphoinositide-dependent protein kinase 1, targeting the C-terminal and N-terminal domains, respectively (28Jensen C.J. Buch M.B. Krag T.O. Hemmings B.A. Gammeltoft S. Frodin M. J. Biol. Chem. 1999; 274: 27168-27176Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Specific docking sites in the C-terminal tail of RSKs, facilitating interaction with upstream MAPKs, in some instances were found to be essential for activation (8Pierrat B. da Silva Correia J. Mary J.L. Tomás-Zuber M. Lesslauer W. J. Biol. Chem. 1998; 273: 29661-29671Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 29Smith J.A. Poteet-Smith C.E. Malarkey K. Sturgill T.W. J. Biol. Chem. 1999; 274: 2893-2898Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 30Gavin A. Nebreda A. Curr. Biol. 1999; 9: 281-284Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The profound control exerted by C-terminal tail elements was further demonstrated by the constitutive activity generated by truncation or mutation in the conserved putative autoinhibitory C-terminal helix of RSK2 (31Poteet-Smith C.E. Smith J.A. Lannigan D.A. Freed T.A. Sturgill T.W. J. Biol. Chem. 1999; 274: 22135-22138Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). RSK1–RSK3 interact with ERK independent of activation state and locate both to the cytoplasm and nucleus under resting conditions; upon activation, the complex translocates to the nucleus (32Zhao Y. Bjorbaek C. Moller E.M. J. Biol. Chem. 1996; 271: 29773-29779Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Sequence comparison suggested that elements directing RSKs in general to the nuclear compartment reside in the C-terminal tail (29Smith J.A. Poteet-Smith C.E. Malarkey K. Sturgill T.W. J. Biol. Chem. 1999; 274: 2893-2898Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Thus, the control of subcellular location, autoinhibition, and protein-protein recognition in the association with upstream MAPKs all appear to be functions of the C-terminal sequences of RSKs. Here, we present a study of regulatory sites in the C-terminal tail of RSKB. Sequential truncations of the C-terminal tail revealed elements mediating nuclear location and p38 association. The C-terminal kinase of RSKB has sequence similarities with CaMKs. The structure of rat CaMKI (33Goldberg J. Nairn A.C. Kuriyan J. Cell. 1996; 84: 875-887Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar) was of particular interest, because it presented an autoinhibited conformation with a key AFN motif adjoining a helical stretch of the C-terminal extension (26Miller S.G. Patton B.L. Kennedy M.B. Neuron. 1988; 1: 593-604Abstract Full Text PDF PubMed Scopus (228) Google Scholar, 34Rich R.C. Schulman H. J. Biol. Chem. 1998; 273: 28424-28429Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar); the phenylalanine of this motif played a crucial role in the binding of the C-terminal extension to the body of CaMKI and maintaining the resting state (33Goldberg J. Nairn A.C. Kuriyan J. Cell. 1996; 84: 875-887Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). This AFN motif is conserved in RSKB. Sequence alignment and structure prediction suggested a study of the role of Phe709 within that motif in the control of RSKB function. Standard reagents were from various sources as reported (8Pierrat B. da Silva Correia J. Mary J.L. Tomás-Zuber M. Lesslauer W. J. Biol. Chem. 1998; 273: 29661-29671Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 35Da Silva J. Pierrat B. Mary J.L. Lesslauer W. J. Biol. Chem. 1997; 272: 28373-28380Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 36Tomás-Zuber M. Mary J.L. Lesslauer W. J. Biol. Chem. 2000; 275: 23549-23558Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Transfectam procedure was from Promega (Madison, WI). CREBtide (123KRREILSRRPSYRK136) was purchased from Genosys Biotechnologies (Lake Front Circle, TX). Antibodies to p38 (C-20) and epitope tag FLAG (antibody M2) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Sigma, respectively. SB202190 (4-(4-fluorophenyl-2-2-(4-hydroxyphenyl)-5-(4-pyridyl)-imidazole) was obtained from Dr. Wyss (Hoffmann-La Roche, Switzerland). Phorbol myristate acetate was from Sigma. PD98059 was purchased from Calbiochem. Expression constructs for MAPKs and wt-RSKB were generated as described (8Pierrat B. da Silva Correia J. Mary J.L. Tomás-Zuber M. Lesslauer W. J. Biol. Chem. 1998; 273: 29661-29671Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Mutated MAPK/ERK kinase was obtained from Stratagene (La Jolla, CA). RSKB point mutants were generated by site-directed mutagenesis using the Altered Sites in vitromutagenesis system (Promega) according to the recommendations of the manufacturer. Expression plasmids of truncated RSKB were constructed by replacing the NotI-SalI insert of Flag-wt-RSKB/pALTER (8Pierrat B. da Silva Correia J. Mary J.L. Tomás-Zuber M. Lesslauer W. J. Biol. Chem. 1998; 273: 29661-29671Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) by the NotI-SalI fragment of the polymerase chain reaction product generated using the forward primer 5′-GAAAATCATCGACTTCGGG and one of the following reverse primers, (i) 5′-ACTAGGAGCTCGCGCTGCCGT (Δ682–772-RSKB), (ii) 5′-ATTAGAGCTCATTCTCCACGCTCTT (Δ725–772-RSKB), and (iii) 5′-ATTAGGAGCTCCGCAGCTTCTGCTT (Δ738–772-RSKB). One day after transfection with the indicated plasmids, HEK293 cells were harvested, and 20,000 cells/cm2 were seeded on poly-d-lysine-coated coverslips and cultivated for 1 additional day in minimal essential medium containing 0.3% fetal calf serum. The slides were washed in phosphate-buffered saline, fixed for 5 min in 4% formaldehyde in phosphate-buffered saline, and processed for immunohistochemistry as previously reported (8Pierrat B. da Silva Correia J. Mary J.L. Tomás-Zuber M. Lesslauer W. J. Biol. Chem. 1998; 273: 29661-29671Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). For p38 detection, C-20 antibody (10 μg/ml) was used as primary reagent, with Texas red-labeled goat anti-rabbit immunoglobulin from Jackson ImmunoResearch (West Grove, PA) as secondary antibody. For FLAG-tagged truncated and wt-RSKB detection, M2 antibody (10 μg/ml) was used as primary reagent, with fluorescein isothiocyanate-labeled goat anti-mouse antibody from Dako (Glostrup, Denmark) as secondary antibody. Cells were analyzed on a Leica confocal fluorescence microscope. Subcellular RSKB localization was scored for each mutant by counting the first 100 successive, positively transfected cells using a Leica DRMB fluorescence microscope and assigning a nuclear, mixed (i.e.strong nuclear combined with weak cytoplasmic), or homogeneous cytoplasmic staining pattern. Immunoblots were incubated with specific primary and secondary horseradish-conjugated antibodies and revealed by chemiluminescence (ECL, Amersham Pharmacia Biotech) and Storm PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA). HEK293 (ATCC CRL 1573) cells were cultured in humidified air with 5% CO2 at 37 °C. Cells were cultured in minimal essential medium supplemented with 10% fetal calf serum, 2 mml-glutamine, 100 units/ml penicillin, 10 μg/ml streptomycin, pH 7.4. Transfections were done by the Transfectam procedure as recommended by manufacturer. In parallel transfection experiments, total amount of DNA was normalized with empty vector. In studies of cells stimulated by the cotransfected mMKK6/p38 upstream kinase pathway, a constitutively active mutant of MKK6 (mMKK6) was used (8Pierrat B. da Silva Correia J. Mary J.L. Tomás-Zuber M. Lesslauer W. J. Biol. Chem. 1998; 273: 29661-29671Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Transfected cells were cultured for 2 days, including serum starvation for the last 16 h in 0.3% fetal calf serum-containing medium. The p38 kinase inhibitor SB202190 (10 μm) was added together with the starvation medium; a second dose of SB202190 (10 μm) was added 1 h before harvesting the cells. After stimulation, cells were washed with ice-cold phosphate-buffered saline, and extracts were prepared with lysis buffer (50 mmTris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 10% glycerol, 500 μmdithiothreitol, 1% Triton X-100, 5 mm NaPPi, 1 mm Na3VO4, 50 mm NaF, CompleteTM protease inhibitor mixture (Roche Molecular Biochemicals)). Cell lysates were cleared at 14,000 rpm for 10 min at 4 °C. Protein concentration was determined using the BCA reagents (Pierce). wt-RSKB and truncated RSKB genes were in vitrotranscribed with T7 RNA polymerase and translated at 30 °C for 90 min in the presence of [35S]methionine using the TNT coupled rabbit reticulocyte lysate system as specified by Promega. Aliquots of reticulocyte lysate containing wt-RSKB or the various truncated RSKBs were analyzed by SDS-PAGE and autoradiography to verify expression of the in vitro translated proteins. 5–10 μl of the reticulocyte lysate were used in the p38 binding assay performed in 2 mm Tris, 30 mm HEPES, 10 mmMgCl2, 1 mm dithiothreitol (pH 7.4) in a final volume of 25 μl. Active recombinant Flag-tagged human p38α was purified by affinity chromatography from Escherichia coliexpression. 1 μg of unlabeled p38α was used in the binding assay. After incubation for 30 min at 30 °C, 5 μl of SDS-free loading buffer was added to the reaction that was then electrophoresed on a 7.5% native polyacrylamide gel at 7 mA overnight using standard SDS-PAGE running buffer (acrylamide/N,N′-methylene-bisacrylamide weight ratio of 29:1). The gels were fixed for 15 min (10% acetic acid, 40% ethanol) and amplified for 30 min in NAM-P 100 amplifier solution (Amersham Pharmacia Biotech). Gels were dried and autoradiographed with Kodak Scientific X-OMAT AR film overnight. Cell extracts were subjected to M2 immunoprecipitation followed by kinase assay using CREBtide as a substrate. Cell extracts normalized to total protein content (200 μg unless specified otherwise) were precleared twice with 30 μl of protein G-Sepharose slurry for 20 min at 4 °C under constant agitation. Antibody was added to precleared lysates and incubated overnight at 4 °C. 30 μl of protein G-Sepharose slurry were added. After 1 h of incubation, immune complexes were pelleted and washed twice with {min}-kinase buffer (50 mm Tris-HCl, pH 7.5, 0.1 mm EGTA, 140 mm KCl) supplemented with 5 mm NaPPi. Forin vitro kinase assays, beads were resuspended in 25 μl of {plus}-kinase buffer (33 μm CREBtide, 30 μm ATP, 10 mm MgCl2, 0.02 μCi/μl [γ-33P]ATP, in {min}-kinase buffer) and incubated at 22 °C for 30 min under constant agitation. Similar to wt-RSKB (36Tomás-Zuber M. Mary J.L. Lesslauer W. J. Biol. Chem. 2000; 275: 23549-23558Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), SB202190 (10 μm) added directly to the kinase reactions had no effect on the elevated basal activities of F709A- and T687E-RSKB. Furthermore, 10 μm SB202190 had no effect on stimulated F709A-RSKB activity when added to the in vitro kinase reaction with precipitates from cells activated by mMKK6/p38 cotransfection and cultured without SB202190. A minor reduction of stimulated activity (<15%) of T687E-RSKB was seen when 10 μm SB202190 was added to kinase assays with precipitates from cells activated by mMKK6/p38 cotransfection. A possible effect of SB202190 on T687E-RSKB depends on relative affinities and binding kinetics of ATP versus SB202190 for the C-terminal ATP binding site; ATP concentrations in the in vitro kinase assay and in vivo are 30 μmand in the mm range, respectively. The assay may also be influenced by coprecipitated p38, and spontaneous decay kinetics of activity of distinct RSKB mutants. Thus, the assay format may explain the weak reduction of stimulated T687E-RSKB activity by SB202190 independently of a hypothetical direct effect of the inhibitor. Reactions were stopped by addition of 100 μl of 0.75% phosphoric acid, and 100 μl of the mix reaction were filtered in a 96-well phosphocellulose filter plate (Millipore, Bedford, MA), washed (five times) with 100 μl of 0.75% phosphoric acid, washed once with ethanol, and air-dried. Bound radioactivity was measured in a Packard top counter, using 100 μl of microscintillation mixture (Packard). All alignments were obtained with the help of the in-house program Xsae 2C. Broger, unpublished data. using a modified version of CLUSTAL V (37Higgins D.G. Bleasby A.J. Fuchs R. Comput. Appl. Biosci. 1992; 8: 189-191PubMed Google Scholar). Sequences were accessed through the National Center for Biotechnology Information and Swiss-Prot data bases under the following accession numbers: AJ010119 (RSKB), L07597 (RSK1), P51812(RSK2), Q15349 (RSK3), Q14012 (human CaMKI, kcc1_human), and Q63450(rat CaMKI, kcc1_rat). All modeling calculations were made on a Silicon Graphics Octane with a single R12000 processor using in-house modeling package Moloc (38Gerber P.R. Mueller K. J. Comp. Aided Mol. Design. 1995; 9: 251-268Crossref PubMed Scopus (519) Google Scholar, 39Gerber P.R. J. Comp. Aided Mol. Design. 1998; 12: 37-51Crossref PubMed Scopus (103) Google Scholar). Model building was performed in a three-step procedure. An initial C-α model of the C-terminal domain of RSKB was built by fitting the aligned sequence on the C-α template of rat CaMK-I (33Goldberg J. Nairn A.C. Kuriyan J. Cell. 1996; 84: 875-887Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar) (Protein Data Bank accession number 1A06 (40Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27938) Google Scholar)). This model was subsequently improved by searching a Moloc internal data base of loops obtained from highly resolved protein structures. A loop selection was made on the basis of minimal steric interactions with the rest of the model. Subsequently newly introduced loops were optimized with the Moloc C-α force field. In a next step, a full atom model was generated. φ and ψ angles were obtained for aligned amino acids from the x-ray structure. χ angles were also adopted from the template where possible, and in cases of nonidentical amino acids generated by using the most probable value, applying the method of Ponder and Richards (41Ponder J.W. Richards F.M. J. Mol. Biol. 1987; 193: 775-791Crossref PubMed Scopus (1349) Google Scholar). An energy calculation of the initial full peptide structure revealed regions with bad van der Waals contacts of amino acid side chains that were subsequently improved by manually adjusting the relevant χ angles. Newly inserted loop regions were then optimized individually, with the rest of the protein kept stationary. Repulsive van der Waals interactions were removed manually where necessary. Refinement of the peptide model was performed using Moloc. Neither water nor other molecules were added. In the first step, only amino acid side chains were allowed to move while all backbone atoms were kept in fixed positions. This step greatly removed repulsive interactions between side chains, further improved χ angles of nonconserved amino acids, and revealed regions with unfavorable interactions. In further optimization, only C-α atoms were kept in a fixed position, and all other atoms were allowed to move. In a third round of optimization, no atoms were kept stationary, but positional constraints were applied to C-α atoms. The quality of the model was then checked with (i) Moloc internal programs, (ii) a program by Luthyet al. (42Luthy R. Bowie J.U. Eisenberg D. Nature. 1992; 356: 83-85Crossref PubMed Scopus (2616) Google Scholar), and (iii) PROCHECK (43Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Earlier immunostainings of transfected HEK293 cells had shown that RSKB and its isolated C-terminal domain located to the nucleus, whereas the N-terminal domain distributed in the cytoplasm (8Pierrat B. da Silva Correia J. Mary J.L. Tomás-Zuber M. Lesslauer W. J. Biol. Chem. 1998; 273: 29661-29671Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The C-terminal tail sequence of RSKB contains a putative bipartite713KRX 14KRRKQKLRS nuclear location signal (NLS) (Fig. 1). To investigate its role, mutants were generated with successive truncations of the C-terminal tail, Δ682–772-RSKB, Δ725–772-RSKB, and Δ738–772-RSKB (Fig.1), and their subcellular location was determined in HEK293 cell transfectants by immunostaining for the fused Flag epitope. wt-RSKB and Δ738–772-RSKB presented clearly dominant nuclear staining, but some cells showed cytoplasmic and mixed nuclear/cytoplasmic staining; to quantify, the first 100 successive transfected cells were counted up in the fluorescence microscope with each mutant and assigned to one of the three staining patterns as defined under "Experimental Procedures" (TableI and Fig. 2). In contrast to wt-RSKB and Δ738–772-RSKB, Δ725–772-RSKB and Δ682–772-RSKB, both of which lack the NLS, were predominantly found in the cytoplasm (Table I and Fig. 2). This indicates that elements of the sequence between Ala725 and Ser737 were essential to direct RSKB to the nuclear compartment.Table IPercentages of transfected cells with nuclear, cytoplasmic, and mixed nuclear/cytoplasmic location of Δ682–772-RSKB, Δ725–772-RSKB, Δ738–772-RSKB, and wt-RSKBNuclearCytoplasmicMixedΔ682–772-RSKB177112Δ725–772-RSKB19765Δ738–772-RSKB65278wt-RSKB76169 Open table in a new tab Figure 2RSKB nuclear location is determined by the NLS element. HEK293 cells were transfected with deletion mutants Δ682–772-RSKB, Δ725–772-RSKB, and Δ738–772-RSKB and wt-RSKB, as indicated. All RSKB constructs were tagged with Flag epitope. After transfection cells were cultured for 1 day, seeded on poly-d-lysine-coated coverslips, and cultured for an additional day. For immunodetection, the cells were simultaneously stained with M2 and fluorescein isothiocyanate-conjugated secondary antibodies and with C-20 anti-p38 and Texas red-labeled secondary antibodies. Confocal microscopy showing two panels at higher and lower magnification are shown for each mutant, as indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) RSKB was first identified as a partial cDNA encoding the sequence downstream of Leu60
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