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Nuclear Localization of Protein Kinase U-α Is Regulated by 14-3-3

1999; Elsevier BV; Volume: 274; Issue: 35 Linguagem: Inglês

10.1074/jbc.274.35.24865

1083-351X

Shaosong Zhang, Heming Xing, Anthony J. Muslin,

Fungal and yeast genetics research

14-3-3 proteins are intracellular, dimeric molecules that bind to and modify the activity of several signaling proteins. We used human 14-3-3ζ as a bait in the yeast two-hybrid system to screen a murine embryonic cDNA library. One interacting clone was found to encode the carboxyl terminus of a putative protein kinase. The coding sequence of the human form (protein kinase Uα, PKUα) of this protein kinase was found in GenBankTMon the basis of sequence homology. The two-hybrid clone was also highly homologous to TOUSLED, an Arabidopsis thaliana protein kinase that is required for normal flower and leaf development. PKUα has been found by coimmunoprecipitation to bind to 14-3-3ζ in vivo. Our confocal laser immunofluorescence microscopic experiments revealed that PKUα colocalizes with the cytoplasmic intermediate filament system of cultured fibroblasts in the G1 phase of the cell cycle. PKUα is found in the perinuclear area of S phase cells and in the nucleus of late G2 cells. Transfection of cells with a dominant negative form of 14-3-3η promotes the nuclear localization of PKUα. These results suggest that the subcellular localization of PKUα is regulated, at least in part, by its association with 14-3-3. 14-3-3 proteins are intracellular, dimeric molecules that bind to and modify the activity of several signaling proteins. We used human 14-3-3ζ as a bait in the yeast two-hybrid system to screen a murine embryonic cDNA library. One interacting clone was found to encode the carboxyl terminus of a putative protein kinase. The coding sequence of the human form (protein kinase Uα, PKUα) of this protein kinase was found in GenBankTMon the basis of sequence homology. The two-hybrid clone was also highly homologous to TOUSLED, an Arabidopsis thaliana protein kinase that is required for normal flower and leaf development. PKUα has been found by coimmunoprecipitation to bind to 14-3-3ζ in vivo. Our confocal laser immunofluorescence microscopic experiments revealed that PKUα colocalizes with the cytoplasmic intermediate filament system of cultured fibroblasts in the G1 phase of the cell cycle. PKUα is found in the perinuclear area of S phase cells and in the nucleus of late G2 cells. Transfection of cells with a dominant negative form of 14-3-3η promotes the nuclear localization of PKUα. These results suggest that the subcellular localization of PKUα is regulated, at least in part, by its association with 14-3-3. protein kinase U-α protein kinase U-β polyacrylamide gel electrophoresis glutathione S-transferase fluorescein isothiocyanate kilobase pairs dominant negative nuclear export signal 14-3-3 proteins are intracellular, acidic dimeric molecules that play a role in signal transduction pathways (1Aitken A. Trends Cell Biol. 1996; 6: 341-347Abstract Full Text PDF PubMed Scopus (348) Google Scholar, 2Aitken A. Jones D. Soneji Y. Howell S. Biochem. Soc. Trans. 1995; 23: 605-611Crossref PubMed Scopus (114) Google Scholar). They have been identified in many eukaryotic organisms, including plants and fungi, and are primarily found in the cytoplasmic compartment of eukaryotic cells. The biological function of 14-3-3 is best modeled in the budding yeast Saccharomyces cerevisiae. Certain yeast strains that lack both 14-3-3 homologues, BMH1 and BMH2, are inviable (3Gelperin D. Weigle J. Nelson K. Roseboom P. Irie K. Matsumoto K. Lemmon S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11539-11543Crossref PubMed Scopus (146) Google Scholar). Furthermore, strains that lack BMH1 and BMH2 can be partially “rescued” by overexpression of the Ras-stimulated kinase TPK1 or by overexpression of clathrin heavy chain. These results suggest that BMH proteins play a role in both the Ras pathway and the membrane sorting pathway. In Drosophila, 14-3-3 proteins positively regulate Ras signaling in R7 photoreceptor development (4Chang H.C. Rubin G.M. Genes Dev. 1997; 11: 1132-1139Crossref PubMed Scopus (123) Google Scholar, 5Kockel L. Vorbruggen G. Jackle H. Mlodzik M. Bohmann D. Genes Dev. 1997; 11: 1140-1147Crossref PubMed Scopus (80) Google Scholar). Genetic epistasis analyses in Drosophila suggest that 14-3-3 acts between Ras and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (4Chang H.C. Rubin G.M. Genes Dev. 1997; 11: 1132-1139Crossref PubMed Scopus (123) Google Scholar).In vertebrate organisms, 14-3-3 proteins regulate several facets of cell physiology, including binding to and promotion of the activation of tyrosine and tryptophan hydroxylases that are important in neurotransmitter synthetic pathways (6Ichimura T. Isobe T. Okuyama T. Takahashi N. Araki K. Kuwano R. Takahashi Y. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7084-7088Crossref PubMed Scopus (295) Google Scholar). 14-3-3 proteins bind to the protein kinases Raf-1 (7Fantl W.J. Muslin A.J. Kikuchi A. Martin J.A. MacNicol A.M. Gross R.W. Williams L.T. Nature. 1994; 371: 612-614Crossref PubMed Scopus (309) Google Scholar, 8Freed E. Symons M. Macdonald S.G. McCormick F. Ruggieri R. Science. 1994; 265: 1713-1716Crossref PubMed Scopus (352) Google Scholar, 9Irie K. Gotoh Y. Yashar B.M. Errede B. Nishida E. Matsumoto K. Science. 1994; 265: 1716-1719Crossref PubMed Scopus (255) Google Scholar, 10Fu H. Xia K. Pallas D.C. Cui C. Conroy K. Narsimhan R.P. Mamon H. Collier R.J. Roberts T.M. Science. 1994; 266: 126-129Crossref PubMed Scopus (242) Google Scholar), KSR-1 (11Xing H. Kornfeld K. Muslin A.J. Curr. Biol. 1997; 7: 294-300Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), BCR (12Reuther G.W. Fu H. Cripe L.D. Collier R.J. Pendergast A.M. Science. 1994; 266: 129-133Crossref PubMed Scopus (209) Google Scholar), and protein kinase C (13Aitken A. Howell S. Jones D. Madrazo J. Martin H. Patel Y. Robinson K. Mol. Cell. Biochem. 1995; 149/150: 41-49Crossref Scopus (60) Google Scholar) and are thought to modulate the activity of these kinases. In the case of protein kinase C, most data demonstrate that 14-3-3 binding inhibits its activity (13Aitken A. Howell S. Jones D. Madrazo J. Martin H. Patel Y. Robinson K. Mol. Cell. Biochem. 1995; 149/150: 41-49Crossref Scopus (60) Google Scholar). The interaction of 14-3-3 with Raf-1 is required for the Ras-dependent activation of Raf (14Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar, 15Thorson J.A., Yu, L.W.K. Hsu A.L. Shih N.Y. Graves P.R. Tanner J.W. Allen P.M. Shaw A.S. Mol. Cell. Biol. 1998; 18: 5229-5238Crossref PubMed Scopus (184) Google Scholar, 16Tzivion G. Luo Z. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (386) Google Scholar, 17Roy S. McPherson R.A. Apollini A. Yan J. Lane A. Clyde-Smith J. Hancock J.F. Mol. Cell. Biol. 1998; 18: 3947-3955Crossref PubMed Scopus (115) Google Scholar). 14-3-3 also interacts with the cell cycle protein phosphatase Cdc25c (18Peng C.-Y. Graves P.R. Thomas R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1178) Google Scholar) and the apoptosis-promoting protein BAD (19Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2241) Google Scholar). These interactions may play an important role in the regulation of apoptosis and the cell cycle.14-3-3 preferentially binds to serine-phosphorylated proteins (14Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar, 15Thorson J.A., Yu, L.W.K. Hsu A.L. Shih N.Y. Graves P.R. Tanner J.W. Allen P.M. Shaw A.S. Mol. Cell. Biol. 1998; 18: 5229-5238Crossref PubMed Scopus (184) Google Scholar,20Furukawa Y. Ikuta N. Omata S. Yamauchi T. Isobe T. Ichimura T. Biochem. Biophys. Res. Commun. 1993; 194: 144-149Crossref PubMed Scopus (87) Google Scholar, 21Michaud N.R. Fabian J.R. Mathes K.D. Morrison D.K. Mol. Cell. Biol. 1995; 15: 3390-3397Crossref PubMed Scopus (188) Google Scholar, 22Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar), but the biochemical significance of this is not clear, and there are several models of 14-3-3 “behavior” that are not mutually exclusive. In one, 14-3-3 binding alters the conformation of a target protein, altering its enzymatic activity. The ability of 14-3-3 to promote the activation of tyrosine and tryptophan hydroxylases in vitro supports this hypothesis (6Ichimura T. Isobe T. Okuyama T. Takahashi N. Araki K. Kuwano R. Takahashi Y. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7084-7088Crossref PubMed Scopus (295) Google Scholar). In another model, 14-3-3 functions as a “competitive inhibitor” that prevents the binding of other proteins to the target. This model is supported by data demonstrating that 14-3-3 binding to BAD inhibits the ability of BCL-XL to bind to BAD (19Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2241) Google Scholar). Another possibility is that 14-3-3 is a scaffolding protein that promotes the assembly of oligomeric signaling complexes. Indeed, Raf-1 and BCR can form a complex that is mediated by 14-3-3 protein (23Braselmann S. McCormick F. EMBO J. 1995; 14: 4839-4848Crossref PubMed Scopus (177) Google Scholar). A fourth possibility is that 14-3-3 is an attachable nuclear export signal that promotes the ability of binding partners to translocate out of the nucleus (24Lopes-Girona A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (501) Google Scholar).In an attempt to identify additional 14-3-3-binding partners, we performed a yeast two-hybrid screen with human 14-3-3ζ as a bait. One interacting clone was found to encode a serine/threonine kinase, named protein kinase U-α (PKUα).1 This protein kinase is homologous to a plant protein, TOUSLED, that is required for normal flower and leaf development (25Roe J.L. Rivkin C.J. Sessions R.A. Feldmann K.A. Zambryski P.C. Cell. 1993; 75: 939-950Abstract Full Text PDF PubMed Scopus (146) Google Scholar). TOUSLED is constitutively localized in the nucleus of plant cells and is thought to play a role in cell cycle regulation (26Roe J.L. Durfee T. Zupan J.R. McLean B.G. Zambryski P.C. J. Biol. Chem. 1997; 272: 5838-5845Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar).DISCUSSION14-3-3 proteins are ubiquitously expressed intracellular dimeric proteins that regulate several aspects of cellular physiology and bind to signaling, cell cycle, cytoskeletal, and apoptotic proteins (1Aitken A. Trends Cell Biol. 1996; 6: 341-347Abstract Full Text PDF PubMed Scopus (348) Google Scholar,2Aitken A. Jones D. Soneji Y. Howell S. Biochem. Soc. Trans. 1995; 23: 605-611Crossref PubMed Scopus (114) Google Scholar). The varied biochemical functions of 14-3-3 are dependent on binding to a partner protein; this binding may alter the enzymatic activity of the partner (e.g. tyrosine hydroxylase, protein kinase C, and Raf-1) (1Aitken A. Trends Cell Biol. 1996; 6: 341-347Abstract Full Text PDF PubMed Scopus (348) Google Scholar, 2Aitken A. Jones D. Soneji Y. Howell S. Biochem. Soc. Trans. 1995; 23: 605-611Crossref PubMed Scopus (114) Google Scholar, 7Fantl W.J. Muslin A.J. Kikuchi A. Martin J.A. MacNicol A.M. Gross R.W. Williams L.T. Nature. 1994; 371: 612-614Crossref PubMed Scopus (309) Google Scholar, 8Freed E. Symons M. Macdonald S.G. McCormick F. Ruggieri R. Science. 1994; 265: 1713-1716Crossref PubMed Scopus (352) Google Scholar, 9Irie K. Gotoh Y. Yashar B.M. Errede B. Nishida E. Matsumoto K. Science. 1994; 265: 1716-1719Crossref PubMed Scopus (255) Google Scholar, 10Fu H. Xia K. Pallas D.C. Cui C. Conroy K. Narsimhan R.P. Mamon H. Collier R.J. Roberts T.M. Science. 1994; 266: 126-129Crossref PubMed Scopus (242) Google Scholar), sequester it (e.g. BAD) (19Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2241) Google Scholar), enhance its solubility (e.g. keratin K8) (35Liao J. Omary M.B. J. Cell Biol. 1996; 133: 345-357Crossref PubMed Scopus (179) Google Scholar), link it to other signaling proteins (e.g. BCR and Raf-1) (23Braselmann S. McCormick F. EMBO J. 1995; 14: 4839-4848Crossref PubMed Scopus (177) Google Scholar), or promote its nuclear export (e.g. Cdc25) (24Lopes-Girona A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (501) Google Scholar). 14-3-3 preferentially binds to proteins that contain serine-phosphorylated residues (14Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar, 15Thorson J.A., Yu, L.W.K. Hsu A.L. Shih N.Y. Graves P.R. Tanner J.W. Allen P.M. Shaw A.S. Mol. Cell. Biol. 1998; 18: 5229-5238Crossref PubMed Scopus (184) Google Scholar, 20Furukawa Y. Ikuta N. Omata S. Yamauchi T. Isobe T. Ichimura T. Biochem. Biophys. Res. Commun. 1993; 194: 144-149Crossref PubMed Scopus (87) Google Scholar, 21Michaud N.R. Fabian J.R. Mathes K.D. Morrison D.K. Mol. Cell. Biol. 1995; 15: 3390-3397Crossref PubMed Scopus (188) Google Scholar, 22Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar, 36Liu Y.-C. Liu Y. Elly C. Yoshida H. Lipowitz S. Altman A. J. Biol. Chem. 1997; 272: 9979-9985Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 37Zhang S.-H. Kobayashi R. Graves P.R. Piwnica-Worms H. Tonks N.K. J. Biol. Chem. 1997; 272: 27281-27287Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), a requirement that suggests that serine kinases play a critical role in the regulation of 14-3-3 binding. Indeed, in the case of the apoptosis-promoting protein BAD, the serine kinase Akt or other serine kinases may be required for BAD phosphorylation that leads to 14-3-3 binding (38del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1978) Google Scholar). Not only do serine kinases regulate 14-3-3 binding, but it also appears that 14-3-3 regulates the activity of a variety of serine kinases, such as Raf-1 and protein kinase C.In this work, we sought new binding partners of 14-3-3 by performing a yeast two-hybrid screen. The carboxyl-terminal portion of a murine serine/threonine kinase, named PKUα, was found to interact with 14-3-3ζ. PKUα is homologous to an A. thaliana protein, TOUSLED, that is required for normal flower and leaf development (25Roe J.L. Rivkin 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 identity of the signal transduction cascade in which TOUSLED participates is unclear; there is a homologue of TOUSLED in C. elegans, but the function of the worm protein is unknown.In this study, we documented in Northern blot experiments that the PKUα gene is highly expressed throughout murine embryonic development and is widely expressed in adult murine tissues. GST/14-3-3β and GST/14-3-3ζ fusion proteins were used to determine that PKUα binds to 14-3-3 in vitro. Coimmunoprecipitation experiments demonstrated that PKUα and 14-3-3 form a complex in vivo.PKUα is found in the cytoplasmic intermediate filament system of cells at the G1/S border, in the perinuclear area of S phase cells, and in the nucleus of late G2 cells. This localization differs from that of TOUSLED protein, which is found entirely in the nuclei of plant cells at all phases of the cell cycle (26Roe J.L. Durfee T. Zupan J.R. McLean B.G. Zambryski P.C. J. Biol. Chem. 1997; 272: 5838-5845Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). TOUSLED lacks a putative 14-3-3-binding site and this may explain the difference in subcellular localization. In transfected COS1 cells, previous work has demonstrated that overexpressed PKUβ is found in the nucleus with some cytoplasmic localization (30Yamakawa A. Kameoka Y. Hashimoto K. Yoshitake Y. Nishikawa K. Tanihara K. Date T. Gene (Amst.). 1997; 202: 193-201Crossref PubMed Scopus (10) Google Scholar), but the subcellular localization of native PKUβ and PKUα has not been previously determined.In order to test the ability of 14-3-3 to regulate the subcellular localization of PKUα, NIH/3T3 cells were transfected with a dominant negative form of 14-3-3η that is mutated at two arginine residues (R56A and R60A). Dominant negative forms of 14-3-3 were first identified by a genetic screen in Drosophila melanogaster, where Chang and Rubin (4Chang H.C. Rubin G.M. Genes Dev. 1997; 11: 1132-1139Crossref PubMed Scopus (123) Google Scholar) demonstrated that three missense mutant forms of Dm14-3-3ε inhibited wild type 14-3-3. Subsequent mutagenesis studies with human 14-3-3η and 14-3-3ζ established that additional mutant forms of 14-3-3, including the R56A and R60A double mutant form of 14-3-3η, were potent at inhibiting the activity of wild type 14-3-3 (15Thorson J.A., Yu, L.W.K. Hsu A.L. Shih N.Y. Graves P.R. Tanner J.W. Allen P.M. Shaw A.S. Mol. Cell. Biol. 1998; 18: 5229-5238Crossref PubMed Scopus (184) Google Scholar, 38del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1978) Google Scholar). Previous work has demonstrated that arginine 56 and arginine 60 are located in the phosphoserine binding pocket of 14-3-3 and that mutating these residues does not inhibit the ability of 14-3-3 monomers to dimerize nor does it result in the production of an unstable protein (39Wang H. Zhang L. Liddington R. Fu H. J. Biol. Chem. 1998; 273: 16297-16304Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The presumed mechanism of dominant negative forms of 14-3-3 is that they form inactive heterodimers with wild type 14-3-3 proteins (40Jones D.H. Ley S. Aitken A. FEBS Lett. 1995; 368: 55-58Crossref PubMed Scopus (206) Google Scholar), although this remains to be proved. Our findings indicate that transfection of cultured cells with a dominant negative form of 14-3-3η promotes the nuclear localization of PKUα, and this is consistent with the attachable nuclear export signal model of 14-3-3 action (24Lopes-Girona A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (501) Google Scholar). However, these results do not exclude the possibility that dominant negative 14-3-3η indirectly causes PKUα to accumulate in the nucleus.Recently, a leucine-rich nuclear export signal (NES)-like sequence in the fission yeast 14-3-3 protein Rad24 was described that regulates the subcellular localization of Cdc25 (24Lopes-Girona A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (501) Google Scholar). The nuclear export factor Crm1 binds to NES-like sequences, but it has not been established whether Crm1 binds to 14-3-3 (24Lopes-Girona A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (501) Google Scholar). The NES-like sequence in Rad24 is conserved in mammalian forms of 14-3-3, and crystallographic analysis suggests that several key residues, including leucine-216 and leucine-220 of 14-3-3ζ, are located on one side of the amphipathic groove that binds to phosphoserine-containing peptide motifs (39Wang H. Zhang L. Liddington R. Fu H. J. Biol. Chem. 1998; 273: 16297-16304Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Mutation of leucine 220 of 14-3-3ζ to aspartic acid abrogates binding to Raf-1 kinase, and this demonstrates that residues in the NES-like sequence are important for phosphoserine motif binding (39Wang H. Zhang L. Liddington R. Fu H. J. Biol. Chem. 1998; 273: 16297-16304Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). One hypothetical model that explains our results is that wild type 14-3-3 forms a oligomeric complex with PKUα and Crm1 in the nucleus of cultured cells that mediates PKUα export into the cytoplasm and that DN-14-3-3 forms inactive heterodimers that are unable to bind simultaneously to both PKUα and Crm1. Experiments are ongoing to test this model of 14-3-3-mediated nuclear export.The intranuclear substrates of PKUα and TOUSLED, if any, have not been identified. The intranuclear biochemical function of TOUSLED is obscure, although its role in proliferative events in plant development suggests that it may have a cell cycle-related activity (25Roe J.L. Rivkin C.J. Sessions R.A. Feldmann K.A. Zambryski P.C. Cell. 1993; 75: 939-950Abstract Full Text PDF PubMed Scopus (146) Google Scholar, 26Roe J.L. Durfee T. Zupan J.R. McLean B.G. Zambryski P.C. J. Biol. Chem. 1997; 272: 5838-5845Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Further studies are needed to investigate this possibility. 14-3-3 proteins are intracellular, acidic dimeric molecules that play a role in signal transduction pathways (1Aitken A. Trends Cell Biol. 1996; 6: 341-347Abstract Full Text PDF PubMed Scopus (348) Google Scholar, 2Aitken A. Jones D. Soneji Y. Howell S. Biochem. Soc. Trans. 1995; 23: 605-611Crossref PubMed Scopus (114) Google Scholar). They have been identified in many eukaryotic organisms, including plants and fungi, and are primarily found in the cytoplasmic compartment of eukaryotic cells. The biological function of 14-3-3 is best modeled in the budding yeast Saccharomyces cerevisiae. Certain yeast strains that lack both 14-3-3 homologues, BMH1 and BMH2, are inviable (3Gelperin D. Weigle J. Nelson K. Roseboom P. Irie K. Matsumoto K. Lemmon S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11539-11543Crossref PubMed Scopus (146) Google Scholar). Furthermore, strains that lack BMH1 and BMH2 can be partially “rescued” by overexpression of the Ras-stimulated kinase TPK1 or by overexpression of clathrin heavy chain. These results suggest that BMH proteins play a role in both the Ras pathway and the membrane sorting pathway. In Drosophila, 14-3-3 proteins positively regulate Ras signaling in R7 photoreceptor development (4Chang H.C. Rubin G.M. Genes Dev. 1997; 11: 1132-1139Crossref PubMed Scopus (123) Google Scholar, 5Kockel L. Vorbruggen G. Jackle H. Mlodzik M. Bohmann D. Genes Dev. 1997; 11: 1140-1147Crossref PubMed Scopus (80) Google Scholar). Genetic epistasis analyses in Drosophila suggest that 14-3-3 acts between Ras and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (4Chang H.C. Rubin G.M. Genes Dev. 1997; 11: 1132-1139Crossref PubMed Scopus (123) Google Scholar). In vertebrate organisms, 14-3-3 proteins regulate several facets of cell physiology, including binding to and promotion of the activation of tyrosine and tryptophan hydroxylases that are important in neurotransmitter synthetic pathways (6Ichimura T. Isobe T. Okuyama T. Takahashi N. Araki K. Kuwano R. Takahashi Y. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7084-7088Crossref PubMed Scopus (295) Google Scholar). 14-3-3 proteins bind to the protein kinases Raf-1 (7Fantl W.J. Muslin A.J. Kikuchi A. Martin J.A. MacNicol A.M. Gross R.W. Williams L.T. Nature. 1994; 371: 612-614Crossref PubMed Scopus (309) Google Scholar, 8Freed E. Symons M. Macdonald S.G. McCormick F. Ruggieri R. Science. 1994; 265: 1713-1716Crossref PubMed Scopus (352) Google Scholar, 9Irie K. Gotoh Y. Yashar B.M. Errede B. Nishida E. Matsumoto K. Science. 1994; 265: 1716-1719Crossref PubMed Scopus (255) Google Scholar, 10Fu H. Xia K. Pallas D.C. Cui C. Conroy K. Narsimhan R.P. Mamon H. Collier R.J. Roberts T.M. Science. 1994; 266: 126-129Crossref PubMed Scopus (242) Google Scholar), KSR-1 (11Xing H. Kornfeld K. Muslin A.J. Curr. Biol. 1997; 7: 294-300Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), BCR (12Reuther G.W. Fu H. Cripe L.D. Collier R.J. Pendergast A.M. Science. 1994; 266: 129-133Crossref PubMed Scopus (209) Google Scholar), and protein kinase C (13Aitken A. Howell S. Jones D. Madrazo J. Martin H. Patel Y. Robinson K. Mol. Cell. Biochem. 1995; 149/150: 41-49Crossref Scopus (60) Google Scholar) and are thought to modulate the activity of these kinases. In the case of protein kinase C, most data demonstrate that 14-3-3 binding inhibits its activity (13Aitken A. Howell S. Jones D. Madrazo J. Martin H. Patel Y. Robinson K. Mol. Cell. Biochem. 1995; 149/150: 41-49Crossref Scopus (60) Google Scholar). The interaction of 14-3-3 with Raf-1 is required for the Ras-dependent activation of Raf (14Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar, 15Thorson J.A., Yu, L.W.K. Hsu A.L. Shih N.Y. Graves P.R. Tanner J.W. Allen P.M. Shaw A.S. Mol. Cell. Biol. 1998; 18: 5229-5238Crossref PubMed Scopus (184) Google Scholar, 16Tzivion G. Luo Z. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (386) Google Scholar, 17Roy S. McPherson R.A. Apollini A. Yan J. Lane A. Clyde-Smith J. Hancock J.F. Mol. Cell. Biol. 1998; 18: 3947-3955Crossref PubMed Scopus (115) Google Scholar). 14-3-3 also interacts with the cell cycle protein phosphatase Cdc25c (18Peng C.-Y. Graves P.R. Thomas R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1178) Google Scholar) and the apoptosis-promoting protein BAD (19Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2241) Google Scholar). These interactions may play an important role in the regulation of apoptosis and the cell cycle. 14-3-3 preferentially binds to serine-phosphorylated proteins (14Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar, 15Thorson J.A., Yu, L.W.K. Hsu A.L. Shih N.Y. Graves P.R. Tanner J.W. Allen P.M. Shaw A.S. Mol. Cell. Biol. 1998; 18: 5229-5238Crossref PubMed Scopus (184) Google Scholar,20Furukawa Y. Ikuta N. Omata S. Yamauchi T. Isobe T. Ichimura T. Biochem. Biophys. Res. Commun. 1993; 194: 144-149Crossref PubMed Scopus (87) Google Scholar, 21Michaud N.R. Fabian J.R. Mathes K.D. Morrison D.K. Mol. Cell. Biol. 1995; 15: 3390-3397Crossref PubMed Scopus (188) Google Scholar, 22Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar), but the biochemical significance of this is not clear, and there are several models of 14-3-3 “behavior” that are not mutually exclusive. In one, 14-3-3 binding alters the conformation of a target protein, altering its enzymatic activity. The ability of 14-3-3 to promote the activation of tyrosine and tryptophan hydroxylases in vitro supports this hypothesis (6Ichimura T. Isobe T. Okuyama T. Takahashi N. Araki K. Kuwano R. Takahashi Y. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7084-7088Crossref PubMed Scopus (295) Google Scholar). In another model, 14-3-3 functions as a “competitive inhibitor” that prevents the binding of other proteins to the target. This model is supported by data demonstrating that 14-3-3 binding to BAD inhibits the ability of BCL-XL to bind to BAD (19Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2241) Google Scholar). Another possibility is that 14-3-3 is a scaffolding protein that promotes the assembly of oligomeric signaling complexes. Indeed, Raf-1 and BCR can form a complex that is mediated by 14-3-3 protein (23Braselmann S. McCormick F. EMBO J. 1995; 14: 4839-4848Crossref PubMed Scopus (177) Google Scholar). A fourth possibility is that 14-3-3 is an attachable nuclear export signal that promotes the ability of binding partners to translocate out of the nucleus (24Lopes-Girona A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (501) Google Scholar). In an attempt to identify additional 14-3-3-binding partners, we performed a yeast two-hybrid screen with human 14-3-3ζ as a bait. One interacting clone was found to encode a serine/threonine kinase, named protein kinase U-α (PKUα).1 This protein kinase is homologous to a plant protein, TOUSLED, that is required for normal flower and leaf development (25Roe J.L. Rivkin C.J. Sessions R.A. Feldmann K.A. Zambryski P.C. Cell. 1993; 75: 939-950Abstract Full Text PDF PubMed Scopus (146) Google Scholar). TOUSLED is constitutively localized in the nucleus of plant cells and is thought to play a role in cell cycle regulation (26Roe J.L. Durfee T. Zupan J.R. McLean B.G. Zambryski P.C. J. Biol. Chem. 1997; 272: 5838-5845Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). DISCUSSION14-3-3 proteins are ubiquitously expressed intracellular dimeric proteins that regulate several aspects of cellular physiology and bind to signaling, cell cycle, cytoskeletal, and apoptotic proteins (1Aitken A. Trends Cell Biol. 1996; 6: 341-347Abstract Full Text PDF PubMed Scopus (348) Google Scholar,2Aitken A. Jones D. Soneji Y. Howell S. Biochem. Soc. Trans. 1995; 23: 605-611Crossref PubMed Scopus (114) Google Scholar). The varied biochemical functions of 14-3-3 are dependent on binding to a partner protein; this binding may alter the enzymatic activity of the partner (e.g. tyrosine hydroxylase, protein kinase C, and Raf-1) (1Aitken A. Trends Cell Biol. 1996; 6: 341-347Abstract Full Text PDF PubMed Scopus (348) Google Scholar, 2Aitken A. Jones D. Soneji Y. Howell S. Biochem. Soc. Trans. 1995; 23: 605-611Crossref PubMed Scopus (114) Google Scholar, 7Fantl W.J. Muslin A.J. Kikuchi A. Martin J.A. MacNicol A.M. Gross R.W. Williams L.T. Nature. 1994; 371: 612-614Crossref PubMed Scopus (309) Google Scholar, 8Freed E. Symons M. Macdonald S.G. McCormick F. Ruggieri R. Science. 1994; 265: 1713-1716Crossref PubMed Scopus (352) Google Scholar, 9Irie K. Gotoh Y. Yashar B.M. Errede B. Nishida E. Matsumoto K. Science. 1994; 265: 1716-1719Crossref PubMed Scopus (255) Google Scholar, 10Fu H. Xia K. Pallas D.C. Cui C. Conroy K. Narsimhan R.P. Mamon H. Collier R.J. Roberts T.M. Science. 1994; 266: 126-129Crossref PubMed Scopus (242) Google Scholar), sequester it (e.g. BAD) (19Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2241) Google Scholar), enhance its solubility (e.g. keratin K8) (35Liao J. Omary M.B. J. Cell Biol. 1996; 133: 345-357Crossref PubMed Scopus (179) Google Scholar), link it to other signaling proteins (e.g. BCR and Raf-1) (23Braselmann S. McCormick F. EMBO J. 1995; 14: 4839-4848Crossref PubMed Scopus (177) Google Scholar), or promote its nuclear export (e.g. Cdc25) (24Lopes-Girona A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (501) Google Scholar). 14-3-3 preferentially binds to proteins that contain serine-phosphorylated residues (14Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar, 15Thorson J.A., Yu, L.W.K. Hsu A.L. Shih N.Y. Graves P.R. Tanner J.W. Allen P.M. Shaw A.S. Mol. Cell. Biol. 1998; 18: 5229-5238Crossref PubMed Scopus (184) Google Scholar, 20Furukawa Y. Ikuta N. Omata S. Yamauchi T. Isobe T. Ichimura T. Biochem. Biophys. Res. Commun. 1993; 194: 144-149Crossref PubMed Scopus (87) Google Scholar, 21Michaud N.R. Fabian J.R. Mathes K.D. Morrison D.K. Mol. Cell. Biol. 1995; 15: 3390-3397Crossref PubMed Scopus (188) Google Scholar, 22Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar, 36Liu Y.-C. Liu Y. Elly C. Yoshida H. Lipowitz S. Altman A. J. Biol. Chem. 1997; 272: 9979-9985Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 37Zhang S.-H. Kobayashi R. Graves P.R. Piwnica-Worms H. Tonks N.K. J. Biol. Chem. 1997; 272: 27281-27287Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), a requirement that suggests that serine kinases play a critical role in the regulation of 14-3-3 binding. Indeed, in the case of the apoptosis-promoting protein BAD, the serine kinase Akt or other serine kinases may be required for BAD phosphorylation that leads to 14-3-3 binding (38del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1978) Google Scholar). Not only do serine kinases regulate 14-3-3 binding, but it also appears that 14-3-3 regulates the activity of a variety of serine kinases, such as Raf-1 and protein kinase C.In this work, we sought new binding partners of 14-3-3 by performing a yeast two-hybrid screen. The carboxyl-terminal portion of a murine serine/threonine kinase, named PKUα, was found to interact with 14-3-3ζ. PKUα is homologous to an A. thaliana protein, TOUSLED, that is required for normal flower and leaf development (25Roe J.L. Rivkin 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 identity of the signal transduction cascade in which TOUSLED participates is unclear; there is a homologue of TOUSLED in C. elegans, but the function of the worm protein is unknown.In this study, we documented in Northern blot experiments that the PKUα gene is highly expressed throughout murine embryonic development and is widely expressed in adult murine tissues. GST/14-3-3β and GST/14-3-3ζ fusion proteins were used to determine that PKUα binds to 14-3-3 in vitro. Coimmunoprecipitation experiments demonstrated that PKUα and 14-3-3 form a complex in vivo.PKUα is found in the cytoplasmic intermediate filament system of cells at the G1/S border, in the perinuclear area of S phase cells, and in the nucleus of late G2 cells. This localization differs from that of TOUSLED protein, which is found entirely in the nuclei of plant cells at all phases of the cell cycle (26Roe J.L. Durfee T. Zupan J.R. McLean B.G. Zambryski P.C. J. Biol. Chem. 1997; 272: 5838-5845Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). TOUSLED lacks a putative 14-3-3-binding site and this may explain the difference in subcellular localization. In transfected COS1 cells, previous work has demonstrated that overexpressed PKUβ is found in the nucleus with some cytoplasmic localization (30Yamakawa A. Kameoka Y. Hashimoto K. Yoshitake Y. Nishikawa K. Tanihara K. Date T. Gene (Amst.). 1997; 202: 193-201Crossref PubMed Scopus (10) Google Scholar), but the subcellular localization of native PKUβ and PKUα has not been previously determined.In order to test the ability of 14-3-3 to regulate the subcellular localization of PKUα, NIH/3T3 cells were transfected with a dominant negative form of 14-3-3η that is mutated at two arginine residues (R56A and R60A). Dominant negative forms of 14-3-3 were first identified by a genetic screen in Drosophila melanogaster, where Chang and Rubin (4Chang H.C. Rubin G.M. Genes Dev. 1997; 11: 1132-1139Crossref PubMed Scopus (123) Google Scholar) demonstrated that three missense mutant forms of Dm14-3-3ε inhibited wild type 14-3-3. Subsequent mutagenesis studies with human 14-3-3η and 14-3-3ζ established that additional mutant forms of 14-3-3, including the R56A and R60A double mutant form of 14-3-3η, were potent at inhibiting the activity of wild type 14-3-3 (15Thorson J.A., Yu, L.W.K. Hsu A.L. Shih N.Y. Graves P.R. Tanner J.W. Allen P.M. Shaw A.S. Mol. Cell. Biol. 1998; 18: 5229-5238Crossref PubMed Scopus (184) Google Scholar, 38del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1978) Google Scholar). Previous work has demonstrated that arginine 56 and arginine 60 are located in the phosphoserine binding pocket of 14-3-3 and that mutating these residues does not inhibit the ability of 14-3-3 monomers to dimerize nor does it result in the production of an unstable protein (39Wang H. Zhang L. Liddington R. Fu H. J. Biol. Chem. 1998; 273: 16297-16304Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The presumed mechanism of dominant negative forms of 14-3-3 is that they form inactive heterodimers with wild type 14-3-3 proteins (40Jones D.H. Ley S. Aitken A. FEBS Lett. 1995; 368: 55-58Crossref PubMed Scopus (206) Google Scholar), although this remains to be proved. Our findings indicate that transfection of cultured cells with a dominant negative form of 14-3-3η promotes the nuclear localization of PKUα, and this is consistent with the attachable nuclear export signal model of 14-3-3 action (24Lopes-Girona A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (501) Google Scholar). However, these results do not exclude the possibility that dominant negative 14-3-3η indirectly causes PKUα to accumulate in the nucleus.Recently, a leucine-rich nuclear export signal (NES)-like sequence in the fission yeast 14-3-3 protein Rad24 was described that regulates the subcellular localization of Cdc25 (24Lopes-Girona A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (501) Google Scholar). The nuclear export factor Crm1 binds to NES-like sequences, but it has not been established whether Crm1 binds to 14-3-3 (24Lopes-Girona A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (501) Google Scholar). The NES-like sequence in Rad24 is conserved in mammalian forms of 14-3-3, and crystallographic analysis suggests that several key residues, including leucine-216 and leucine-220 of 14-3-3ζ, are located on one side of the amphipathic groove that binds to phosphoserine-containing peptide motifs (39Wang H. Zhang L. Liddington R. Fu H. J. Biol. Chem. 1998; 273: 16297-16304Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Mutation of leucine 220 of 14-3-3ζ to aspartic acid abrogates binding to Raf-1 kinase, and this demonstrates that residues in the NES-like sequence are important for phosphoserine motif binding (39Wang H. Zhang L. Liddington R. Fu H. J. Biol. Chem. 1998; 273: 16297-16304Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). One hypothetical model that explains our results is that wild type 14-3-3 forms a oligomeric complex with PKUα and Crm1 in the nucleus of cultured cells that mediates PKUα export into the cytoplasm and that DN-14-3-3 forms inactive heterodimers that are unable to bind simultaneously to both PKUα and Crm1. Experiments are ongoing to test this model of 14-3-3-mediated nuclear export.The intranuclear substrates of PKUα and TOUSLED, if any, have not been identified. The intranuclear biochemical function of TOUSLED is obscure, although its role in proliferative events in plant development suggests that it may have a cell cycle-related activity (25Roe J.L. Rivkin C.J. Sessions R.A. Feldmann K.A. Zambryski P.C. Cell. 1993; 75: 939-950Abstract Full Text PDF PubMed Scopus (146) Google Scholar, 26Roe J.L. Durfee T. Zupan J.R. McLean B.G. Zambryski P.C. J. Biol. Chem. 1997; 272: 5838-5845Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Further studies are needed to investigate this possibility. 14-3-3 proteins are ubiquitously expressed intracellular dimeric proteins that regulate several aspects of cellular physiology and bind to signaling, cell cycle, cytoskeletal, and apoptotic proteins (1Aitken A. Trends Cell Biol. 1996; 6: 341-347Abstract Full Text PDF PubMed Scopus (348) Google Scholar,2Aitken A. Jones D. Soneji Y. Howell S. Biochem. Soc. Trans. 1995; 23: 605-611Crossref PubMed Scopus (114) Google Scholar). The varied biochemical functions of 14-3-3 are dependent on binding to a partner protein; this binding may alter the enzymatic activity of the partner (e.g. tyrosine hydroxylase, protein kinase C, and Raf-1) (1Aitken A. Trends Cell Biol. 1996; 6: 341-347Abstract Full Text PDF PubMed Scopus (348) Google Scholar, 2Aitken A. Jones D. Soneji Y. Howell S. Biochem. Soc. Trans. 1995; 23: 605-611Crossref PubMed Scopus (114) Google Scholar, 7Fantl W.J. Muslin A.J. Kikuchi A. Martin J.A. MacNicol A.M. Gross R.W. Williams L.T. Nature. 1994; 371: 612-614Crossref PubMed Scopus (309) Google Scholar, 8Freed E. Symons M. Macdonald S.G. McCormick F. Ruggieri R. Science. 1994; 265: 1713-1716Crossref PubMed Scopus (352) Google Scholar, 9Irie K. Gotoh Y. Yashar B.M. Errede B. Nishida E. Matsumoto K. Science. 1994; 265: 1716-1719Crossref PubMed Scopus (255) Google Scholar, 10Fu H. Xia K. Pallas D.C. Cui C. Conroy K. Narsimhan R.P. Mamon H. Collier R.J. Roberts T.M. Science. 1994; 266: 126-129Crossref PubMed Scopus (242) Google Scholar), sequester it (e.g. BAD) (19Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2241) Google Scholar), enhance its solubility (e.g. keratin K8) (35Liao J. Omary M.B. J. Cell Biol. 1996; 133: 345-357Crossref PubMed Scopus (179) Google Scholar), link it to other signaling proteins (e.g. BCR and Raf-1) (23Braselmann S. McCormick F. EMBO J. 1995; 14: 4839-4848Crossref PubMed Scopus (177) Google Scholar), or promote its nuclear export (e.g. Cdc25) (24Lopes-Girona A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (501) Google Scholar). 14-3-3 preferentially binds to proteins that contain serine-phosphorylated residues (14Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar, 15Thorson J.A., Yu, L.W.K. Hsu A.L. Shih N.Y. Graves P.R. Tanner J.W. Allen P.M. Shaw A.S. Mol. Cell. Biol. 1998; 18: 5229-5238Crossref PubMed Scopus (184) Google Scholar, 20Furukawa Y. Ikuta N. Omata S. Yamauchi T. Isobe T. Ichimura T. Biochem. Biophys. Res. Commun. 1993; 194: 144-149Crossref PubMed Scopus (87) Google Scholar, 21Michaud N.R. Fabian J.R. Mathes K.D. Morrison D.K. Mol. Cell. Biol. 1995; 15: 3390-3397Crossref PubMed Scopus (188) Google Scholar, 22Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar, 36Liu Y.-C. Liu Y. Elly C. Yoshida H. Lipowitz S. Altman A. J. Biol. Chem. 1997; 272: 9979-9985Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 37Zhang S.-H. Kobayashi R. Graves P.R. Piwnica-Worms H. Tonks N.K. J. Biol. Chem. 1997; 272: 27281-27287Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), a requirement that suggests that serine kinases play a critical role in the regulation of 14-3-3 binding. Indeed, in the case of the apoptosis-promoting protein BAD, the serine kinase Akt or other serine kinases may be required for BAD phosphorylation that leads to 14-3-3 binding (38del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1978) Google Scholar). Not only do serine kinases regulate 14-3-3 binding, but it also appears that 14-3-3 regulates the activity of a variety of serine kinases, such as Raf-1 and protein kinase C. In this work, we sought new binding partners of 14-3-3 by performing a yeast two-hybrid screen. The carboxyl-terminal portion of a murine serine/threonine kinase, named PKUα, was found to interact with 14-3-3ζ. PKUα is homologous to an A. thaliana protein, TOUSLED, that is required for normal flower and leaf development (25Roe J.L. Rivkin 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 identity of the signal transduction cascade in which TOUSLED participates is unclear; there is a homologue of TOUSLED in C. elegans, but the function of the worm protein is unknown. In this study, we documented in Northern blot experiments that the PKUα gene is highly expressed throughout murine embryonic development and is widely expressed in adult murine tissues. GST/14-3-3β and GST/14-3-3ζ fusion proteins were used to determine that PKUα binds to 14-3-3 in vitro. Coimmunoprecipitation experiments demonstrated that PKUα and 14-3-3 form a complex in vivo. PKUα is found in the cytoplasmic intermediate filament system of cells at the G1/S border, in the perinuclear area of S phase cells, and in the nucleus of late G2 cells. This localization differs from that of TOUSLED protein, which is found entirely in the nuclei of plant cells at all phases of the cell cycle (26Roe J.L. Durfee T. Zupan J.R. McLean B.G. Zambryski P.C. J. Biol. Chem. 1997; 272: 5838-5845Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). TOUSLED lacks a putative 14-3-3-binding site and this may explain the difference in subcellular localization. In transfected COS1 cells, previous work has demonstrated that overexpressed PKUβ is found in the nucleus with some cytoplasmic localization (30Yamakawa A. Kameoka Y. Hashimoto K. Yoshitake Y. Nishikawa K. Tanihara K. Date T. Gene (Amst.). 1997; 202: 193-201Crossref PubMed Scopus (10) Google Scholar), but the subcellular localization of native PKUβ and PKUα has not been previously determined. In order to test the ability of 14-3-3 to regulate the subcellular localization of PKUα, NIH/3T3 cells were transfected with a dominant negative form of 14-3-3η that is mutated at two arginine residues (R56A and R60A). Dominant negative forms of 14-3-3 were first identified by a genetic screen in Drosophila melanogaster, where Chang and Rubin (4Chang H.C. Rubin G.M. Genes Dev. 1997; 11: 1132-1139Crossref PubMed Scopus (123) Google Scholar) demonstrated that three missense mutant forms of Dm14-3-3ε inhibited wild type 14-3-3. Subsequent mutagenesis studies with human 14-3-3η and 14-3-3ζ established that additional mutant forms of 14-3-3, including the R56A and R60A double mutant form of 14-3-3η, were potent at inhibiting the activity of wild type 14-3-3 (15Thorson J.A., Yu, L.W.K. Hsu A.L. Shih N.Y. Graves P.R. Tanner J.W. Allen P.M. Shaw A.S. Mol. Cell. Biol. 1998; 18: 5229-5238Crossref PubMed Scopus (184) Google Scholar, 38del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1978) Google Scholar). Previous work has demonstrated that arginine 56 and arginine 60 are located in the phosphoserine binding pocket of 14-3-3 and that mutating these residues does not inhibit the ability of 14-3-3 monomers to dimerize nor does it result in the production of an unstable protein (39Wang H. Zhang L. Liddington R. Fu H. J. Biol. Chem. 1998; 273: 16297-16304Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The presumed mechanism of dominant negative forms of 14-3-3 is that they form inactive heterodimers with wild type 14-3-3 proteins (40Jones D.H. Ley S. Aitken A. FEBS Lett. 1995; 368: 55-58Crossref PubMed Scopus (206) Google Scholar), although this remains to be proved. Our findings indicate that transfection of cultured cells with a dominant negative form of 14-3-3η promotes the nuclear localization of PKUα, and this is consistent with the attachable nuclear export signal model of 14-3-3 action (24Lopes-Girona A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (501) Google Scholar). However, these results do not exclude the possibility that dominant negative 14-3-3η indirectly causes PKUα to accumulate in the nucleus. Recently, a leucine-rich nuclear export signal (NES)-like sequence in the fission yeast 14-3-3 protein Rad24 was described that regulates the subcellular localization of Cdc25 (24Lopes-Girona A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (501) Google Scholar). The nuclear export factor Crm1 binds to NES-like sequences, but it has not been established whether Crm1 binds to 14-3-3 (24Lopes-Girona A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (501) Google Scholar). The NES-like sequence in Rad24 is conserved in mammalian forms of 14-3-3, and crystallographic analysis suggests that several key residues, including leucine-216 and leucine-220 of 14-3-3ζ, are located on one side of the amphipathic groove that binds to phosphoserine-containing peptide motifs (39Wang H. Zhang L. Liddington R. Fu H. J. Biol. Chem. 1998; 273: 16297-16304Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Mutation of leucine 220 of 14-3-3ζ to aspartic acid abrogates binding to Raf-1 kinase, and this demonstrates that residues in the NES-like sequence are important for phosphoserine motif binding (39Wang H. Zhang L. Liddington R. Fu H. J. Biol. Chem. 1998; 273: 16297-16304Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). One hypothetical model that explains our results is that wild type 14-3-3 forms a oligomeric complex with PKUα and Crm1 in the nucleus of cultured cells that mediates PKUα export into the cytoplasm and that DN-14-3-3 forms inactive heterodimers that are unable to bind simultaneously to both PKUα and Crm1. Experiments are ongoing to test this model of 14-3-3-mediated nuclear export. The intranuclear substrates of PKUα and TOUSLED, if any, have not been identified. The intranuclear biochemical function of TOUSLED is obscure, although its role in proliferative events in plant development suggests that it may have a cell cycle-related activity (25Roe J.L. Rivkin C.J. Sessions R.A. Feldmann K.A. Zambryski P.C. Cell. 1993; 75: 939-950Abstract Full Text PDF PubMed Scopus (146) Google Scholar, 26Roe J.L. Durfee T. Zupan J.R. McLean B.G. Zambryski P.C. J. Biol. Chem. 1997; 272: 5838-5845Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Further studies are needed to investigate this possibility. We are very grateful to John Cooper, Mike Olszowy, Helen Piwnica-Worms, Andrey Shaw, and Steve Weintraub for technical advice and helpful discussions.