Yeast 14‐3‐3 proteins

14-3-3 proteins, first isolated from bovine brain andnamed after their behaviour in 2-D chromatography, form a group of highly conserved, abundant, acidic, dimeric proteins with a subunit molecular mass of around 30 kDa. In most higher eukaryotes multiple isoforms are found, e.g. nine in man and at least 10 in Arabidopsis. Despite a vast list of binding partners, currently more than 60, and processes in which 14-3-3 proteins are involved, such as apoptosis, signal transduction and cell cycle regulation (reviewed in Fu et al.15, van Hemert), their exact function remains unclear. Some general themes start to emerge (van Hemert, in preparation): (i) some 14-3-3 proteins function as adapter molecules or scaffolds in anumber of processes. (ii) 14-3-3 proteins may also act as localization anchors, changing the sub-cellular localization of binding partners. (iii) 14-3-3s play a role in transcription, either by their intrinsic transcriptional activation activity or by associating with larger multiprotein transcription factor complexes. (iv) 14-3-3 proteins are directly involved in the regulation of enzyme activity. In many cases (de)phosphorylation of the 14-3-3 binding partners modulates their localization, complex formation, enzyme activation or conformational changes through 14-3-3 association or dissociation. The binding of some target proteins is regulated by the cell cycle or is controlled by signal transduction. Phosphorylation of 14-3-3 itself also modulates the association with binding partners. Although the majority of these data arise from human, mammalian and plant studies, the structure and functions appear to be strongly conserved in yeast 14-3-3 proteins. In this review we will discuss the various functions found so far for the yeast 14-3-3 proteins. Best studied are the 14-3-3s in baker's yeast, but genes encoding these proteins have also been reported for Schizosaccharomyces pombe, 14 Candida albicans, Trichoderma harzianum and Lentinus edodes.25 Saccharomyces cerevisiae and Sz. pombe both contain two isoforms.14, 43, 45 The two S. cerevisiae 14-3-3 proteins, encoded by BMH1 and BMH2, 17, 43, 45 are more than 60% identical to the mammalian ϵ isoform. Bmh1p and Bmh2p are 91% identical and 97% identical, respectively, over the first 250 residues, therefore any isoform-specific function probably resides in the divergent C-termini. Single disruptions of BMH1 or BMH2 have little effect, except for a slight reduction in growth rate.45 Cells overexpressing BMH1 grow poorly on media with acetate or glycerol as the sole carbon source.45 However, transcriptional regulation of BMH1 and BMH2 has not been observed in either of the transcriptome analyses reported so far, with the exception of a 13-fold increase of BMH1 transcription after salt stress.33 Although the S.cerevisiae 14-3-3 proteins can be phosphorylated in vitro by mammalian kinases, in vivo phosphorylation has not been reported.12 A bmh1 bmh2 disruption is lethal in most laboratory strains, 43 but not in Σ1288-derived strains.34 A bmh1 bmh2 disruption in the latter strain results in increased sensitivity to environmental stresses such as heat, high osmolarity and caffeine. In addition, it does not grow on galactose, accumulates high amounts of glycogen, is unable to sporulate and exhibits an abnormal budding pattern.34 This phenotype suggests that many processes are affected in these strains. It might be interesting to identify the gene(s) that allow this strain to (barely) survive, in contrast to other bmh1 bmh2 laboratory strains. In other strains, a conditional lethal mutant has a highly increased percentage of large budded cells under restrictive conditions.43 After prolonged growth under restrictive conditions, chains of cells and cells with abnormal bud morphology are formed. Many of these cells lack DAPI-stainable nuclei but contain small spots instead, suggesting a disturbed nuclear organization or continued chromosome segregation.43 This implies a role for the yeast 14-3-3 proteins in regulating mitosis and/or cell–cell separation. The lethal bmh1 bmh2 disruption can be complemented by at least four Arabidopsis isoforms, by a human and by a Dictyostelium isoform.44 This demonstrates that 14-3-3 isoforms, even from different organisms, all share at least some basic functions. Similarly, a mutation in the Sz. pombe pat1 gene, coding for a kinase that controls meiosis, could be rescued both by overexpression of rad24, an endogenous 14-3-3 encoding gene, and five Arabidopsis isoforms26. In addition to their putative role in cell division, 14-3-3 proteins in cooperation with other proteins play a role in many other processes (Tables 1 and 2). BMH2 and to a lesser extent BMH1 could rescue inviable strains with mutations in the clathrin heavy-chain gene, CHC1.17 Clathrin is a vesicular coat protein involved in receptor-mediated endocytosis andprotein sorting at the late Golgi compartment. A null mutation of CHC1 is lethal in certain strains and leads to slow growth, morphological and membrane trafficking defects in other strains.17 Only a genetic interaction between CHC1 and BMH1 and BMH2 was demonstrated and there is no evidence for physical interactions. Deletion of the C-terminal region abolishes the ability of 14-3-3 to suppress a lethal chc1 mutation.17 It is likely that the C-termini of the yeast isoforms, which are longer compared to mammalian isoforms and contain a glutamine stretch, are involved in regulation of vesicle transport. The 14-3-3 proteins are involved in exocytosis in mammalian cells through rearrangement of the actin cytoskeleton. In yeast a similar involvement was found. Cells overexpressing the C-terminal region of Bmh2p exhibited a failure in polarizing vesicular transport and showed a disrupted actin cytoskeleton.35 Only polarization seems to be affected since the total level of exocytosis did not change.35 This suggests that, like in mammalian cells, the 14-3-3 proteins are involved in organization of the actin cytoskeleton to regulate vesicle targeting and do not affect vesicle docking or fusion to membranes. BMH1 and BMH2 are required for Ras/MAPK signalling during pseudohyphal development, but are not essential for the mating MAPK pathway.34 Bmh2p directly associates with Ste20p, an upstream activator of the Ste11p–Ste7p–Ste12p, Tec1p signalling pathway that activates genes with a FRE, filamentous response element.34 Others have also found evidence for involvement of 14-3-3s in yeast Ras signalling. Overexpression of the BMH genes suppresses mutations in CDC25 and TPK1.17 This suggests that the yeast 14-3-3 proteins participate in the Ras/PKA signalling pathway. In addition, BMH1 is required for Ras-dependent activation of human Raf-1 in a reconstituted yeast system, through direct physical interaction with Raf-1.21 The yeast 14-3-3 proteins positively regulate rapamycin-sensitive signalling.5 The TOR (target of rapamycin) proteins, Tor1p and Tor2p, are phosphatidylinositol kinase-related kinases. TOR-mediated phosphorylation prevents nuclear accumulation of a number of nutrient-regulated transcription factors. The TOR proteins are essential for progression through G1 and treating yeast with rapamycin or deleting (TOR1 and) TOR2 causes growth arrest and several physiological changes that are characteristic of starvation (reviewed in Hall, Schmelzle and Hall.38) BMH1 and BMH2 are multicopy suppressors of the growth inhibitory phenotype caused by rapamycin.5 Conversely, deletion of BMH1 and BMH2 causes hypersensitivity to rapamycin, although there is no direct physical interaction between 14-3-3 and the TOR proteins.5 The 14-3-3s act further downstream in the TOR pathway. Bmh2p binds to the transcription factors Msn2p and Msn4p and retains the phosphorylated forms in the cytoplasm.4 Rapamycin treatment causes rapid translocation of these transcription factors to the nucleus.4 In addition to the transcription factors mentioned above, interaction of 14-3-3 proteins with two other transcription factors was found. Mutations in RTG3 and SIN4 were identified as suppressors of a lethal bmh2ts mutation (van Heusden and Steensma, in preparation). Sin4p is a global transcription regulator. RTG3 codes for a basic helix–loop–helix transcription factor, which is involved in retrograde regulation and probably other processes.23 It was recently shown to shuttle between the cytoplasm and the nucleus in response to the functional state of the mitochondria as well as environmental stress.39 A physical, phosphorylation-dependent interaction between Rtg3p and the 14-3-3s was found in vitro, suggesting that 14-3-3 proteins inhibit Rtg3p-dependent activation by binding the phosphorylated form (van Heusden and Steensma, in preparation). The suppressing effect of the SIN4 and RTG3 mutations was reduced by mild overexpression of RTS1, encoding a regulatory subunit of protein phosphatases 2A. This strongly suggests that for Sin4p phosphorylation also plays a role, although a physical interaction with 14-3-3 proteins could not be shown (van Heusden and Steensma, in preparation). A human 14-3-3 isoform was able to suppress the growth defect of a S. cerevisiae mutant with impaired myo-inositol metabolism.30 In the same screen, a protein phosphatase type 2A subunit and the TGF-β type II receptor were isolated.30 This suggests that the human TGF-β receptor, 14-3-3 proteins and protein phosphatase 2A affect a signalling process that also involves myo-inositol. It remains unclear why the endogenous yeast 14-3-3 proteins were not able to suppress the growth defect. It might be explained by a gene dosage effect or by a specific feature of mammalian 14-3-3s. In a large scale two-hybrid screen, three genes were identified that encode proteins that physically interact with Bmh2p, viz. ECM13, KCS1 and BOP3.42 ECM13 encodes a 28.5 kDa acidic protein that is possibly involved in cell wall structure or biogenesis.42 KCS1 encodes 120 kDa inositol (1,2,3,4,5,6) hexaphosphate (Ins6p) kinase, which was previously identified as a suppressor of a temperature-sensitive pkc1-4 mutation.20, 37 A mutation in BOP3 can bypass PAM1, which is a multicopy suppressor of the loss of protein phosphatase 2A activity. Pam1p, contains two putative coiled coil regions and a highly basic C-terminus. Another protein with a putative coiled coil region in the C-terminus, Fin1p, was not detected in this study but was identified in a different two-hybrid screen with Bmh2p as bait (van Hemert et al., in preparation). Dephosphorylation of Fin1p reduced the in vitro binding to 14-3-3 proteins. Like Fin1p, overexpression of Pam1p inhibits growth and induces morphological changes.19 Although it appears that Bmh2p, Bop3p, Pam1p and protein phosphatase 2A are all involved in the same process, there is no evidence for a physical interaction between Bmh2p and Pam1p. In the same two-hybrid screen mentioned above, Gcr2p, a regulator of glycolysis, 40, 41 was identified. Binding to Bmh2p was confirmed in vitro but was not affected by dephosphorylation of Gcr2p (van Hemert et al., in preparation). The Sz. pombe 14-3-3 genes rad24 and rad25 arehighly homologous to each other except for their C-termini, as in S. cerevisiae. Disruption of rad24, and to a lesser extent rad25, leads to premature entry into mitosis. Simultaneous rad24 rad25 disruption is lethal.14 The genes play a role in timing of mitosis and are essential for cell cycle arrest in response to radiation-induced DNA damage.14 This effect is probably mediated through Cdc25p. A cdc25 mutant unable to bind 14-3-3 shows loss of DNA damage and replication checkpoints and exhibits an increased nuclear localization of Cdc25p. Rad24p overexpression leads to increased nuclear export of Cdc25p. This suggests that one function of Rad24p is to keep Cdc25p out of the nucleus.49 Rad24p contains an attachable nuclear export signal (NES) that is essential for moving Cdc25p out of the nucleus in a Crm1-dependent manner in response to DNA damage. Crm1 is a highly conserved nuclear-export factor.28 Cdc25p activity is also regulated through phosphorylation by kinases such as Chk1p and Cds1p6, 7, 13 Cds1p, for instance, can phosphorylate Cdc25p on 12 residues creating putative 14-3-3 binding sites, although these do not exactly match the RSXSXP consensus.49 Chk1p can inactivate Cdc25p by phosphorylation of Ser-216.31 In cells with undamaged DNA, most Cdc25p is bound to 14-3-3 and kept in the cytoplasm by a Chk1-independent mechanism.28 However, a mutation in the putative NES of 14-3-3 also leads to impaired Cdc25p binding. Therefore, the phenotype of increased nuclear Cdc25p concentrations could also be explained by reduced cytoplasmic retainment instead of reduced nuclear export.49 Cdc25p is a phosphatase that activates Cdc2p, a B-type mitotic cyclin-dependent kinase. During interphase and after DNA damage, the activity of Cdc2p is suppressed through phosphorylation by the Wee1 and Myt1/Mik1 kinases. Dephosphorylation of Cdc2p by Cdc25p and subsequent activation of Cdc2p leads to entry into mitosis. The 14-3-3s Rad24p and Rad25p also associate with Chk1p, preferentially with the phosphorylated form, which is generated in response to DNA damage.8 Association with 14-3-3 could target Chk1p to a certain subcellular location or to proteins like Cdc25p (see above). Overall, it appears that 14-3-3s in Sz. pombe are involved in checkpoint control by keeping Cdc25p out of the nucleus, either by cytoplasmic sequestering and/or nuclear export. 14-3-3 has no effect on the catalytic activity of Cdc25p.10 In Sz. pombe, the 14-3-3 together with phospholipase C (PLC) play a role in developing resistance to UV light. Both rad24 and plc1 mutants are hypersensitive to UV radiation.1 Phosphoinositide-specific phospholipase C catalyses the hydrolysis of phosphatidylinositol 4,5-bisphosphate and produces the second messengers inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG), which cause a rise in intracellular Ca2+ and activation of protein kinase C, respectively. PLC isoforms are present in many organisms and are regulated by a wide variety of extracellular signals and signalling pathways (fora review, see Katan.24) The Sz. pombe Plc1p interacts physically with the 14-3-3 proteins Rad24 and Rad25, 1 although the 14-3-3s do not appear to regulate the activity of Plc1p. Contrary to other reports, Andoh et al.1 could not find an effect of Rad24p on the DNA damage checkpoint. This might be due to the difference in the source of radiation used or by the pleiotropic phenotype of a rad24 mutant. Plc1p and 14-3-3 may participate in an UV-induced signalling pathway that also involves activation of a MAPK cascade. In higher eukaryotes a number of different motifs have been identified in proteins that bind to 14-3-3 proteins. Many of these motifs contain a phosphoserine (pS) residue flanked by an arginine and proline, such as RSXpSXP, 29, 48 RX1-2 SX2-3pS, 27 RX(Y/F)XpSXP48 or RXSX(S/T)XP.48 In addition, several apparently unrelated motifs for binding 14-3-3 proteins have been described.2, 11, 16, 22, 32, 46, 47 For the yeast proteins that bind to 14-3-3 proteins, consensus sequences as mentioned above have not yet been reported and it is unclear which sequences are responsible for binding in yeast. Interestingly, theheterologous Raf-1p, which binds S. cerevisiae 14-3-3 proteins, contains a functional RSXpSXP motif.21 A two-hybrid study using deletions of Fin1p showed that for binding to Bmh2p both the N- and C-terminus were required (van Hemert et al., in preparation). Despite the large number of studies dealing with 14-3-3 proteins in higher eukaryotes (over 500 publications at the end of the year 2000), relatively little has been reported on the mechanisms by which these proteins achieve their effects. Given the strong conservation of these proteins, not only with respect to sequence but also to function, as exemplified by several complementation studies with heterologous 14-3-3s, data obtained in yeast will have great relevance for the higher eukaryotic systems. The data presented above may have illustrated that use of the powerful genetic and biochemical techniques available for both S. cerevisiae and Sz. pombe mayadvance our knowledge on the highly intriguing 14-3-3 proteins much further.