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• W2060715138 abstract "The TOUSLED (TSL) gene is essential for the proper morphogenesis of leaves and flowers in Arabidopsis thaliana. Protein sequence analysis predicts TSL is composed of a carboxyl-terminal protein kinase catalytic domain and a large amino-terminal regulatory domain. TSL fusion proteins, expressed in and purified from yeast, were used to demonstrate TSL protein kinase activity in vitro. TSL trans-autophosphorylates on serine and threonine residues, and phosphorylates exogenous substrates. Using the yeast two-hybrid system, TSL was found to oligomerize via its NH2-terminal domain. A deletion series indicates that a region containing two α-helical segments predicted to participate in a coiled-coil structure is essential for oligomerization. TSL localizes to the nucleus in plant cells through an essential NH2-terminal nuclear localization signal; however, this signal is not necessary for protein kinase activity. Finally, deletion mutants demonstrate a strict correlation between catalytic activity and the ability to oligomerize, arguing that activation of the protein kinase requires interaction between TSL molecules. The TOUSLED (TSL) gene is essential for the proper morphogenesis of leaves and flowers in Arabidopsis thaliana. Protein sequence analysis predicts TSL is composed of a carboxyl-terminal protein kinase catalytic domain and a large amino-terminal regulatory domain. TSL fusion proteins, expressed in and purified from yeast, were used to demonstrate TSL protein kinase activity in vitro. TSL trans-autophosphorylates on serine and threonine residues, and phosphorylates exogenous substrates. Using the yeast two-hybrid system, TSL was found to oligomerize via its NH2-terminal domain. A deletion series indicates that a region containing two α-helical segments predicted to participate in a coiled-coil structure is essential for oligomerization. TSL localizes to the nucleus in plant cells through an essential NH2-terminal nuclear localization signal; however, this signal is not necessary for protein kinase activity. Finally, deletion mutants demonstrate a strict correlation between catalytic activity and the ability to oligomerize, arguing that activation of the protein kinase requires interaction between TSL molecules. INTRODUCTIONDuring development of many organisms, protein kinases function in signaling pathways important for proper morphogenesis and cell fate determination. Their activity can either be stimulatory, such as that of Sevenless receptor kinase (reviewed in Ref. 1Simon M.A. Dev. Biol. 1994; 166: 431-442Crossref PubMed Scopus (48) Google Scholar), or inhibitory, as exemplified by the inactivation of cAMP-dependent protein kinase through the Hedgehog cascade (reviewed in Ref. 2Perrimon N. Cell. 1995; 80: 517-520Abstract Full Text PDF PubMed Scopus (144) Google Scholar). Therefore, to understand the basic cellular processes involved during development requires knowledge of how the relevant kinase(s) is regulated, as well as how its target substrates are affected by phosphorylation.The TSL gene was identified by mutational analysis in Arabidopsis thaliana as a gene required for proper development of the flower and the margins of the leaf (3Roe J.L. Rivin C.J. Sessions R.A. Feldmann K.A. Zambryski P.C. Cell. 1993; 75: 939-950Abstract Full Text PDF PubMed Scopus (146) Google Scholar). tsl loss of function mutations are recessive and cause a phenotype characterized by two major floral defects. First, there is a stochastic decrease in the number of floral organs, implying that TSL functions at an early stage during flower formation, perhaps regulating the establishment of organ primordia by promoting specific cell divisions within the floral meristem. Second, specific regions of the ovule-housing organ, the gynoecium, fail to develop properly suggesting that TSL also may function to pattern developmental programs within an organ type (3Roe J.L. Rivin C.J. Sessions R.A. Feldmann K.A. Zambryski P.C. Cell. 1993; 75: 939-950Abstract Full Text PDF PubMed Scopus (146) Google Scholar). 1Roe, J. L., Nemhauser, J. L., and Zambryski, P. C. (1997) Plant Cell, in press.TSL encodes a 688-amino acid protein (TSL) which is a putative serine/threonine protein kinase with a COOH-terminal catalytic domain (amino acids 409-688) and an NH2-terminal domain (amino acids 1-408) of unknown function (3Roe J.L. Rivin C.J. Sessions R.A. Feldmann K.A. Zambryski P.C. Cell. 1993; 75: 939-950Abstract Full Text PDF PubMed Scopus (146) Google Scholar). Recent data base searches suggest that TSL is a member of an evolutionarily conserved protein kinase subfamily with closely-related homologs found in Caenorhabditis elegans, Caenorhabditis briggsae, humans, and maize. 2J. L. Roe, unpublished observation. The presence of TSL homologs in both plant and animal kingdoms implies the protein performs a fundamental function. Consistent with this hypothesis, TSL is expressed in all organs of the plant (3Roe J.L. Rivin C.J. Sessions R.A. Feldmann K.A. Zambryski P.C. Cell. 1993; 75: 939-950Abstract Full Text PDF PubMed Scopus (146) Google Scholar).The utilization of the same signaling components in multiple developmental pathways is emerging as a common theme in many organsims (1Simon M.A. Dev. Biol. 1994; 166: 431-442Crossref PubMed Scopus (48) Google Scholar, 4Siegfried E. Perrimon N. BioEssays. 1994; 16: 395-404Crossref PubMed Scopus (144) Google Scholar, 5Hammerschmidt M. Bitgood M.J. McMahon A.P. Genes Dev. 1996; 10: 647-658Crossref PubMed Scopus (319) Google Scholar, 6Sundaram M. Han M. BioEssays. 1996; 18: 473-480Crossref PubMed Scopus (56) Google Scholar, 7Duffy J.B. Perrimon N. Dev. Biol. 1994; 166: 380-395Crossref PubMed Scopus (97) Google Scholar). The requirement for TSL function at several stages of development suggests that this putative protein kinase is regulated in response to different unidentified developmental cues. The TSL NH2-terminal domain contains sequence motifs, such as a coiled-coil region and three consensus nuclear localization signal (NLS) 3The abbreviations used are:NLSnuclear localization signalGSTglutathione S-transferasePAGEpolyacrylamide gel electrophoresisPipes1,4-piperazinediethanesulfonic acidMBPmyelin basic proteinGUSβ-glucuronidaseDBDDNA-binding domain. sequences, which together could participate in modulating the activity of the COOH-terminal catalytic domain. The existence of multiple NLS sequences in the NH2-terminal domain may direct the subcellular localization of the protein, permitting access to potential regulatory factors and target substrates. The coiled-coil region, including a leucine-zipper motif, may participate in protein-protein interactions that affect kinase activity. Such interactions could include the formation of TSL oligomers. The ligand-binding induced dimerization of receptor protein kinases is known to be critical for kinase activation (8Heldin C.-H. Cell. 1995; 80: 213-223Abstract Full Text PDF PubMed Scopus (1427) Google Scholar, 9Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4581) Google Scholar). However, oligomerization has only recently been found to provide a possible means of regulation of non-receptor protein kinases (10Farrar M.A. Alberola-Ila J. Perlmutter R.M. Nature. 1996; 383: 178-181Crossref PubMed Scopus (266) Google Scholar, 11Luo Z. Tzivion G. Belshaw P.J. Vavvas D. Marshall M. Avruch J. Nature. 1996; 383: 181-185Crossref PubMed Scopus (201) Google Scholar).To begin to understand the basic properties of the TSL protein kinase and its regulation, we have isolated catalytically active TSL from yeast and characterized that activity in vitro. In this report, we show that TSL is a nuclear serine/threonine protein kinase and is capable of autophosphorylation in trans. The TSL NH2-terminal domain also is shown to mediate oligomerization in the two-hybrid system. Deletion mutants, analyzed for both their enzymatic activity in vitro and ability to oligomerize in the two-hybrid system, indicate that TSL requires self-association for protein kinase activity.EXPERIMENTAL PROCEDURESStrainsEscherichia coli DH5 (F−, recA1, endA1, hsdR17, supE44, thi1, gyrA, relA1) was the recipient for all plasmid transformations. For drug selections, LB media was supplemented with carbenicillin (100 μg/ml). The yeast strain Y153 (12Durfee T. Beecher K. Chen P.-L. Yeh S.-H. Yang Y. Kilburn A.E. Lee W.-H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1297) Google Scholar) (MATa leu2-3,112 ura3-52 trp1-901 his3-Δ200 ade2-101 gal4Δ gal80Δ URA3::GAL-lacZ LYS2::GAL-HIS3) was used for two-hybrid experiments. BJ5460 (MATa ura3-52 trp1 lys2-801 leu2Δ1 his3Δ200 pep4::HIS3 prb1Δ1.6R can1 GAL) was used for protein expression experiments. Yeast YPD and synthetic complete (SC) media was prepared as described (13Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics. A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar).PlasmidsTSL SubclonesPrecursor plasmids containing the full TSL coding sequence, and the coding sequence for the NH2-terminal domain, and NH2-terminal deletions were generated for subcloning into the expression vectors described below. Cloning details will be provided upon request.K438E MutationA polymerase chain reaction product containing the sequence for the catalytic domain with the codon for Lys-438 changed to a codon for Glu was generated by polymerase chain reaction-directed oligomutagenesis using the mutagenic primer 5′-GTGCGAGCTTCATGGTT-3′, and the T3 and T7 primers by the method of Bowman et al. (14Bowman S. Tischfield J.A. Stambrook P.J. Technique, J. Methods Cell Mol. Biol. 1990; 2: 254-260Google Scholar). The fragment was subcloned into pBluescriptSK+ at the EcoRI site to generate pTK2E. This plasmid was subsequently used to exchange the mutant fragment with wild-type fragments in the precursor plasmids where appropriate.GST Fusion PlasmidsYEpLG-GST 4C. Inouye and J. Thorner, unpublished. is a yeast shuttle vector containing a LEU2 and a β-lactamase gene for selection in yeast and E. coli, respectively, and a 2-μ and a ColE1 origin for replication in yeast and E. coli. It also contains the coding sequence for GST under GAL1 control followed by a polylinker. TSL sequences from precursor plasmids were subcloned into the polylinker region of YEpLG-GST such that the TSL coding sequences were in-frame with GST (details will be provided upon request).Two-hybrid ContructsTSL sequences from the precursor plasmids described above were subcloned into the two-hybrid vectors pAS1 (12Durfee T. Beecher K. Chen P.-L. Yeh S.-H. Yang Y. Kilburn A.E. Lee W.-H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1297) Google Scholar) and pACTII (15Bai C. Elledge S.J. Methods Enzymol. 1996; 273: 331-347Crossref PubMed Google Scholar) such that the TSL coding sequences were in-frame downstream of the Gal4 DBD or activation domain sequence, respectively (details will be provided upon request).Myc Epitope Tag Expression PlasmidsThe pA6M vector, containing a CaMV35S promoter upstream of the sequence encoding a Myc-epitope tag followed by a polylinker, was constructed by ligating the XhoI-XbaI restriction fragment from pJR1265, 5D. Gottschling, unpublished data. containing a translation start followed by a 6x Myc-epitope tag and polylinker, into the plant expression vector, pART7 (16Gleave A.P. Plant Mol. Biol. 1992; 20: 1203-1207Crossref PubMed Scopus (860) Google Scholar). The sequence for full-length TSL was introduced into pA6M to create pA6M/TSL.2 (for this subcloning only, the sequence at the translational start site of TSL was changed to introduce an NcoI site, resulting in a serine to alanine change at the second residue in TSL to create TSL.2). Deletion mutants were introduced into pA6M by excising appropriate restriction fragments from precursor plasmids and ligating to pA6M.GUS Expression PlasmidspRTL2GUS, an expression plasmid containing the GUS reporter gene under the control of the CaMV35S promoter has been described (17Restrepo M.A. Freed D.D. Carrington J.C. Plant Cell. 1990; 2: 987-998Crossref PubMed Scopus (299) Google Scholar). Sequences encoding amino acids 12-438 of TSL were inserted downstream as a BamHI-KpnI fragment replacing the BglII-KpnI Nia fragment from pRTL2SG-Nia/ΔB+K (17Restrepo M.A. Freed D.D. Carrington J.C. Plant Cell. 1990; 2: 987-998Crossref PubMed Scopus (299) Google Scholar) creating pGUS-NTΔN12.Expression and Purification of GST Fusion ProteinsYEpLG-GST constructs were introduced into BJ5460 by chemical transformation (18Schiestl R.H. Gietz R.D. Curr. Genet. 1989; 16: 339-346Crossref PubMed Scopus (1766) Google Scholar). For galactose inductions, transformants were grown overnight in 10 ml of SC media lacking leucine and containing 2% raffinose and 0.2% sucrose. 10 ml of fresh media was added and cells grown an additional 4 h. Galactose was added to 2% and cells grown another 4 h. Cells were pelleted by centrifugation and resuspended in lysis 250 buffer (50 mM Tris (pH 7.4), 250 mM NaCl, 5 mM EDTA, 0.1% Nonidet P-40, 50 mM sodium fluoride, supplemented with phenylmethylsulfonyl fluoride (100 μg/ml), aprotinin (2 μg/ml), leupeptin (2 μg/ml), pepstatin A (1 μg/ml), dithiothreitol (1 mM), and benzamidine (1 mM)) prior to freezing. Glass beads were added to thawed samples and cells lysed by vortexing. Extracts were cleared by centrifugation (14,000 × g for 20 min at 4°C) and protein concentration of supernatants estimated by OD280. Equal protein amounts were mixed with glutathione-agarose beads (Sigma) for 1 h at 4°C. Bound fractions were washed extensively with lysis 250 buffer. For all purifications, a ratio of 1 mg of protein extract/100 μl bed volume of glutathione-agarose beads was used.Western Blot AnalysisBound GST fusion proteins from equivalent aliquots of glutathione-agarose beads (10-μl bed volume) purified as above were eluted by boiling in Laemmli sample buffer and separated by SDS-PAGE (19Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206024) Google Scholar). For Western blots in Figs. 2A and 5A, the entire sample was loaded, and in Fig. 7A, one-half volume of eluted proteins was loaded. Gels were transferred to Immobilon-P and immunoblots were performed using rabbit anti-GST polyclonal antiserum, horseradish peroxidase-conjugated anti-rabbit IgG (Bio-Rad), and detection with enhanced chemiluminescence (Amersham). Blots were treated with Western blocking buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 0.05% Tween-20, 3% non-fat dry milk) for at least an hour, incubated overnight in primary antibody (1:1000) at 4°C, washed 3 times 20 min each at room temperature, incubated 1 h in secondary antibody (1:3000), washed 3 times 20 min each. All incubations and washes were performed in Western blocking buffer. After the final wash, blots were rinsed in Western blocking buffer without milk and subjected to chemiluminescent detection.Fig. 5Protein kinase activity of TSL deletion mutants correlates with their ability to oligomerize. A, Western blot analysis of purified TSL (lane 1) and NH2-terminal deletion GST fusion proteins (lanes 2-7) using anti-GST antiserum. Equivalent amounts of protein extract were purified for each sample. B, kinase assays of TSL (lane 1) and NH2-terminal deletion GST fusion proteins (lanes 2-7). 1 μg of MBP was added before addition of [γ-32P]ATP. After the reaction, Laemmli sample buffer was added, samples were boiled, and proteins were separated on a 12.5% SDS-polyacrylamide gel. The autoradiogram shows two different regions of the gel. The arrow indicates the position where MBP migrates. The results presented are representative of two independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 7Active TSL protein kinase can transphosphorylate an inactive TSL mutant and the two can form heterotypic complexes. A, Western blot analysis of purified GST fusion proteins from extracts of induced yeast cells expressing ΔN171 (lane 1), K438E (lane 2), and mixed extracts of ΔN171 and K438E (lane 3). Blot was probed with anti-GST antiserum. B, autoradiogram of kinase assays of purified GST fusion proteins (lanes as in A). The results in A and B are representative of two independent experiments. C, two-hybrid interaction assay. TSL deletions with or without the K438E mutation as indicated were inserted into the Gal4 DNA-binding domain and activation domain plasmids. Y153 transformants were analyzed for β-galactosidase activity (expressed as Miller units). Numbers represent the average of results from three independent transformants.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Kinase AssaysEquivalent aliquots of glutathione-agarose beads (10-μl bed volume) with bound GST fusion proteins purified as above were washed three times in kinase buffer (50 mM HEPES, pH 7.6, 150 mM NaCl, 10 mM MgCl2, 2 mM MnCl2) and resuspended in 37.5 μl of kinase buffer. 10 μCi of [γ-32P]ATP (Amersham, 3000 Ci/mmol) was added and the reaction was incubated at room temperature for 60 min. Laemmli sample buffer was added, samples were boiled for 3 min, centrifuged, and the supernatant was analyzed by SDS-PAGE. Kinase assays in Fig. 7B were washed in kinase buffer after the reaction prior to elution in Laemmli sample buffer. In kinase assays in Fig. 2, B and D, and Fig. 5B, the entire reaction was loaded, whereas in Fig. 7B, one-fourth volume of the reaction was loaded.Phosphoamino Acid AnalysisAliquots of purified GST-TSL were allowed to autophosphorylate as described above. Pellets were washed once in kinase buffer, samples were boiled after addition of Laemmli sample buffer and electrophoresed on an SDS-polyacrylamide gel. The portion of the gel containing the phosphorylated protein was transferred to Immobilon-P (Amersham) by electroblotting. The 32P-containing portion of the membrane was excised, and the sample was hydrolyzed by incubating the membrane in 6 N HCl for 1 h at 100°C (20Kamps M.P. Methods Enzymol. 1991; 201: 21-27Crossref PubMed Scopus (99) Google Scholar). The supernatant was dried, redried twice after resuspension in H2O, and analyzed by TLC and autoradiography by the method of Cooper et al. (21Cooper J.A. Sefton B.M. Hunter T. Methods Enzymol. 1983; 99: 387-402Crossref PubMed Scopus (703) Google Scholar).Quantitation of β-Galactosidase Activityβ-Galactosidase activity in yeast was quantified with chlorophenyl red-β-D-galactopyranoside (CPRG; Boehringer Mannheim). Y153 transformants were grown in 3 ml of SC media lacking tryptophan and leucine to OD600 1.0-1.5. Cells were then prepared and permeabilized as described (22Guarente L. Methods Enzymol. 1983; 101: 181-191Crossref PubMed Scopus (871) Google Scholar), except cell pellets were resuspended in 900 μl of H buffer (100 mM HEPES, 150 mM NaCl, 2 mM MgCl2, 1% bovine serum albumin, pH 7.0) and 100 μl of 50 mM chlorophenyl red-β-D-galactopyranoside was added following permeabilization. The amount of liberated chlorophenyl red was determined by OD574.Immunocytochemistry and GUS Fusion AssaysNicotiana tabacum (line XD) suspension cultures were grown as described (23McLean B.G. Zupan J. Zambryski P.C. The Plant Cell. 1995; 7: 2101-2114Crossref PubMed Scopus (266) Google Scholar). Protoplasts were prepared as described previously (24Howard E.A. Zupan J.R. Citovsky V. Zambryski P.C. Cell. 1992; 68: 109-118Abstract Full Text PDF PubMed Scopus (193) Google Scholar) and electroporated using a Bio-Rad electroporator according to McLean et al. (23McLean B.G. Zupan J. Zambryski P.C. The Plant Cell. 1995; 7: 2101-2114Crossref PubMed Scopus (266) Google Scholar). Cells were then grown at 22°C in suspension media (24Howard E.A. Zupan J.R. Citovsky V. Zambryski P.C. Cell. 1992; 68: 109-118Abstract Full Text PDF PubMed Scopus (193) Google Scholar) supplemented with 0.4% mannitol for 16-24 h. For immunofluoresence staining, cells were fixed with 3.7% (w/v) formaldehyde in fixation buffer (100 mM Pipes (pH 6.9), 10 mM EGTA, 5 mM MgSO4, 10% Me2SO, 100 μg/ml phenylmethylsulfonyl fluoride) for 45 min at room temperature. Fixed cells were permeabilized with 0.5% Triton X-100 in fixation buffer for 5 min. All antibody and washing steps were performed in blocking buffer (0.1 M Tris-HCl, pH 7.4, 0.15 M NaCl, 0.1% Tween 20, 5% (w/v) dry milk). Myc epitope-tagged proteins were detected using the monoclonal antibody, 9E10 (BAbCO), and a goat anti-mouse fluorescein isothiocyanate-conjugated secondary antibody (Calbiochem). Cells were resuspended in Citifluor (Ted Pella, Inc.) prior to visualization. Digital images of fluorescein isothiocyanate or 4,6-diamidino-2-phenylindole fluorescence, or of Normarski differential interference contrast, were obtained using a Zeiss Axiophot fluoresence microscope with a CCD camera. GUS staining was performed as described previously (24Howard E.A. Zupan J.R. Citovsky V. Zambryski P.C. Cell. 1992; 68: 109-118Abstract Full Text PDF PubMed Scopus (193) Google Scholar). When color developed, cells were photographed by bright-field illumination on a Zeiss Axiophot.DISCUSSIONThe loss of TSL gene function results in a mutant phenotype affecting both leaf and flower morphology, and TSL appears to function during both early and late stages of flower development (3Roe J.L. Rivin C.J. Sessions R.A. Feldmann K.A. Zambryski P.C. Cell. 1993; 75: 939-950Abstract Full Text PDF PubMed Scopus (146) Google Scholar). These results suggest that the TSL gene product participates in a commonly used developmental pathway. Sequence analysis predicted TSL to be a serine/threonine protein kinase composed of a carboxyl-terminal catalytic domain and a large amino-terminal regulatory domain. We have shown here that TSL is indeed a protein kinase which autophosphorylates on both serine and threonine residues. That a kinase-dead mutant can serve as a substrate for the wild-type protein demonstrates that some, if not all, sites are phosphorylated in trans. Furthermore, TSL can phosphorylate exogenous substrates such as MBP and casein in vitro.TSL function presumably is tightly controlled during Arabidopsis development. We have shown that the TSL NH2-terminal domain contributes at least two regulatory activities to the protein. First, this domain contains three NLS sequences, at least one of which appears to be functional in targeting TSL to the plant cell nucleus. Deletion of the first two NLS consensus sequences results in a cytoplasmically localized protein, suggesting that one or both of these NLS sequences are essential for nuclear localization. The third NLS is not sufficient for nuclear targeting. Catalytic activity is not required for the nuclear localizing function as the NH2-terminal domain alone efficiently targets a heterologous protein to the plant cell nucleus. Reciprocally, the deletion mutant ΔN171 is not nuclear-localized, but is catalytically active in vitro, suggesting that nuclear localization is not required for catalytic activity.The second regulatory function of the NH2-terminal domain is to mediate TSL oligomerization. Deletions studies indicate that at least the first α-helical segment of the coiled-coil region is critical for TSL oligomerization. The leucine-zipper motif found in the second α-helical segment is not sufficient for oligomerization, and its contribution to TSL function is not yet clear. Importantly, deletion mutants revealed a strict correlation between oligomerization in the two-hybrid system and the ability to both autophosphorylate and transphosphorylate exogenous substrates in vitro. That the catalytic domain of TSL alone is inactive indicates that the NH2-terminal domain must positively regulate the protein. Together, these data strongly suggest that oligomerization of TSL is required for activation of the catalytic domain. Although this interaction likely is due to direct binding of TSL molecules, it is possible that a cofactor present in the yeast cell mediates this apparent self-interaction.The kinase-dead K438E mutant can interact with itself and wild-type TSL, demonstrating that catalytic activity is not required for oligomerization. Based on values obtained for activation of the reporter gene in the two-hybrid assay, K438E apparently interacts with wild-type TSL more strongly than either protein interacts with itself. Perhaps this heterotypic combination represents an intermediate state where autophosphorylation of only one molecule in a complex has occurred and this intermediate is more stable. Functionally, it is not yet known whether a heterotypic complex is catalytically active. If not, the K438E mutant would be expected to exert a dominant negative effect when expressed in wild-type plants. In contrast, the NH2-terminal domain alone (NTΔN73) cannot interact with near full-length TSL containing the catalytic domain (ΔN73) and therefore, presumably will not function as a dominant-negative mutation. Experiments are underway to test these hypotheses.Ligand-mediated dimerization is generally required for activation of receptor protein kinases (reviewed in Refs. 8Heldin C.-H. Cell. 1995; 80: 213-223Abstract Full Text PDF PubMed Scopus (1427) Google Scholar and 9Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4581) Google Scholar). However, oligomerization is not a common mechanism of regulation for a non-receptor kinase, such as TSL. Recent results suggest that oligomerization plays a role in Raf-1 kinase activation (10Farrar M.A. Alberola-Ila J. Perlmutter R.M. Nature. 1996; 383: 178-181Crossref PubMed Scopus (266) Google Scholar, 11Luo Z. Tzivion G. Belshaw P.J. Vavvas D. Marshall M. Avruch J. Nature. 1996; 383: 181-185Crossref PubMed Scopus (201) Google Scholar). Several other cytoplasmic protein kinases, including double-stranded RNA-dependent protein kinase (37Langland J.O. Jacobs B.L. J. Biol. Chem. 1992; 267: 10729-10736Abstract Full Text PDF PubMed Google Scholar) and Type I and Type II cGMP-dependent protein kinases (38Gamm D.M. Francis S.H. Angelotti T.P. Corbin J.D. Uhler M.D. J. Biol. Chem. 1995; 270: 27380-27388Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 39Atkinson R.A. Saudek V. Huggins J.P. Pelton J.T. Biochemistry. 1991; 30: 9387-9395Crossref PubMed Scopus (60) Google Scholar), have been shown to exist as dimers, although the functional relevance of dimerization is not clear. Studies with double-stranded RNA-dependent protein kinase deletion mutants demonstrated that the catalytic domain alone, although unable to dimerize, was fully active in vivo (40Wu S. Kaufman R.J. J. Biol. Chem. 1996; 271: 1756-1763Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). In the case of the cGMP-dependent protein kinases isoforms, it is not known if dimerization is required for catalytic activity. Finally, the crystal structure of the regulatory domain of Lck, a Src family member, revealed that this domain dimerized, but again, it is unknown whether dimerization plays a role in regulating kinase activity (41Eck M.J. Atwell S.K. Shoelson S.E. Harrison S.C. Nature. 1994; 368: 764-769Crossref PubMed Scopus (240) Google Scholar).The apparent dependence of TSL catalytic activity on oligomerization argues that TSL self-association via the NH2-terminal domain transmits a conformational change to the catalytic domain, analogous to the effect of dimerization on receptor tyrosine kinases (reviewed in Ref. 9Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4581) Google Scholar). Dimerization per se is insufficient for activation, as GST can dimerize (42Walker J. Crowley P. Moreman A.D. Barrett J. Mol. Biochem. Parasitol. 1993; 61: 255-264Crossref PubMed Scopus (111) Google Scholar), and therefore, even the catalytically inactive GST-TSL deletion mutants may exist as dimers. TSL self-association mediated by its coiled-coil region, then, may not act merely to bring the catalytic domains into proximity with one another, but instead may cause the protein to adopt a highly specific structure. This conformational change may either allow the catalytic domain to take on a fully active structure, or permit an intermediate that allows one TSL molecule to activate another via trans-autophosphorylation. This is in contrast to other examples where activation of a protein kinase can be induced by fusion to a heterologous dimerization domain (Refs. 10Farrar M.A. Alberola-Ila J. Perlmutter R.M. Nature. 1996; 383: 178-181Crossref PubMed Scopus (266) Google Scholar and 11Luo Z. Tzivion G. Belshaw P.J. Vavvas D. Marshall M. Avruch J. Nature. 1996; 383: 181-185Crossref PubMed Scopus (201) Google Scholar, and reviewed in Ref. 8Heldin C.-H. Cell. 1995; 80: 213-223Abstract Full Text PDF PubMed Scopus (1427) Google Scholar), including fusion to GST (43Maru Y. Afar D.E. Witte O.N. Shibuya M. J. Biol. Chem. 1996; 271: 15353-15357Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). However, we cannot exclude the possibility that TSL activation requires complexes larger than dimers, and thus dimerization via GST might be insufficient for activation.How, then, might TSL oligomerization be controlled? Two obvious possibilities exist. First, the protein may spontaneously form oligomers with a subsequent modification(s) serving as the rate-limiting step for kinase activation. Second, the protein could normally adopt a structure that is unable to oligomerize. An activating event, such as ligand-binding or phosphorylation of a critical residue(s), would then occur to allow TSL self-ass" @default.
• W2060715138 created "2016-06-24" @default.
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• W2060715138 date "1997-02-01" @default.
• W2060715138 modified "2023-10-11" @default.
• W2060715138 title "TOUSLED Is a Nuclear Serine/Threonine Protein Kinase That Requires a Coiled-coil Region for Oligomerization and Catalytic Activity" @default.
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