: Biological Overview | References
Gene name - 14-3-3ε
Synonyms - 14-3-3epsilon
Cytological map position - 90F10-90F10
Function - signaling, protein transport
Keywords - embryonic hatching, partial redundency, oocyte determination, polarization of the A-P axis, mitosis in post-blastoderm cell cycles, mitotic timing following irradiation, wing
Symbol - 14-3-3ε
FlyBase ID: FBgn0020238
Genetic map position - 3R: 14,068,253..14,074,388 [+]
Classification - 14-3-3 protein
Cellular location - cytoplasmic and nuclear
The functional specialization or redundancy of the ubiquitous 14-3-3 proteins constitutes a fundamental question in their biology and stems from their highly conserved structure and multiplicity of coexpressed isotypes. This question was addressed in vivo using mutations in the two Drosophila 14-3-3 genes, leonardo (14-3-3ζ) and D14-3-3ε. 14-3-3ε is essential for embryonic hatching. Nevertheless, D14-3-3ε null homozygotes survive because they upregulate transcripts encoding the LeonardoII isoform at the time of hatching, compensating D14-3-3ε loss. This novel homeostatic response explains the reported functional redundancy of the Drosophila 14-3-3 isotypes and survival of D14-3-3ε mutants. The response appears unidirectional, since D14-3-3ε elevation upon Leo loss was not observed and elevation of leo transcripts was stage and tissue specific. In contrast, Leo levels are not changed in the wing discs, resulting in the aberrant wing veins characterizing D14-3-3ε mutants. Nevertheless, conditional overexpression of LeoI, but not of LeoII, in the wing disk can partially rescue the venation deficits. Thus, excess of a particular Leo isoform can functionally compensate for D14-3-3ε loss in a cellular-context-specific manner. These results demonstrate functional differences both among Drosophila 14-3-3 proteins and between the two Leo isoforms in vivo, which likely underlie differential dimer affinities toward 14-3-3 targets (Acevedo, 2007).
A fundamental issue concerning members of highly conserved protein families is the extent to which they are functionally redundant or exhibit specialized biological functions. The 14-3-3 proteins compose a highly conserved family of acidic molecules present in all eukaryotes (Aitken, 1995; Wang, 1996; Rosenquist, 2000). 14-3-3's share a common structure composed of nine antiparallel α-helices forming a horseshoe shape with a negatively charged interior surface. Interactions among particular amino acids in the first helix, with ones in helix 2 and helix 3 of another monomer, promote dimerization. Dimerization generates a tandem binding surface, which can simultaneously bind to one or two sites on one target protein or to sites on two different client molecules. The dimers bind clients containing phosphoserine- or phosphothreonine-containing motifs via highly conserved amino acids within the groove. 14-3-3 proteins can also bind targets with surfaces outside the conserved phosphopeptide-binding cleft. 14-3-3 binding may allosterically stabilize conformational changes, leading to activation or deactivation of the target or to interaction between two proteins. Furthermore, 14-3-3 binding may mask or expose interaction sites, often leading to changes in the subcellular localization of client proteins (Acevedo, 2007 and references therein).
An extraordinary feature of this protein family is the high sequence conservation among isotypes, characterized by long stretches of invariant amino acids (Wang, 1996; Gardino, 2006), suggesting functional redundancy. However, despite this extensive sequence identity, multiple 14-3-3 proteins exist in metazoans, indicating at least some functional specificity. Vertebrates contain seven distinct protein isotypes, β, ε, ζ, γ, η, θ, and σ (Aitken, 1995). In vertebrate brains where these proteins are highly abundant, there is some specificity in isotype distribution, but generally 14-3-3's are expressed in complex overlapping patterns. In addition, multiple heterodimers are possible in tissues that contain more than one isotype. It is unclear whether the presence of multiple highly similar proteins with overlapping distribution reflects functional differences among them or represents a mechanism to ensure that ample functionally redundant 14-3-3's are available to mediate the multiple essential cellular functions that require them. Thus, the question of 14-3-3 functional specificity in vivo is fundamental in understanding their biology. The highly overlapping isotype distribution in vertebrate models hinders systematic investigation of this question (Acevedo, 2007).
To address the issue of functional specificity in vivo, Drosophila, which offers a simple, but representative, genetically tractable metazoan system, was used. It is simple because it contains only two 14-3-3 genes, an ortholog of the mammalian 14-3-3ζ (88% identity) leonardo and an ortholog of the ε isotype, D14-3-3ε (Skoulakis, 1998). It is representative because the two fly genes belong to the two different 14-3-3 conservation groups (Wang, 1996; Skoulakis, 1998). leonardo encodes two nearly identical protein isoforms (Leo I and Leo II) via alternative splicing of the primary transcript, with modest tissue specificity. In contrast, D14-3-3ε encodes a single protein (Chang, 1997), apparently present in all developmental stages and tissues examined with only slight enrichment in the adult brain (Tien, 1999; Philip, 2001; Acevedo, 2007 and references therein).
Maternal Leo is required for normal chromosome separation during syncytial mitoses, whereas D14-3-3ε appears required to time them, suggesting distinct functions for the two 14-3-3's in the single-celled syncytial embryo (Su, 2001). Maternal Leo is also essential for early Raf-dependent decisions that pattern the embryo. Zygotic leo loss-of-function mutants exhibit functional impairments of their embryonic and adult nervous system. D14-3-3ε functions in photoreceptor formation and appears involved in development of the wing (Chang, 1997), but whether it is important for the function of the nervous system is unknown. Leo and D14-3-3ε appear at least partially redundant for photoreceptor formation (Karim, 1996; Chang, 1997). Furthermore, Leo and D14-3-3ε have been reported to function redundantly in anterior–posterior axis formation of the developing oocyte (Benton, 2002) and follicle cell polarity (Benton, 2003; Acevedo, 2007 and references therein).
Nevertheless, three reasons motivated a systematic investigation of potential functional specificity of the two Drosophila 14-3-3 isotypes by searching for isotype-specific phenotypes. First, studies to date used a transposon allele of D14-3-3ε (j2B10), which may not be a null allele. In fact, although D14-3-3ε has been reported dispensable for viability (Chang, 1997), a lethal deficiency uncovering this gene was used to show its involvement in Raf-mediated developmental processes in the embryo (Li, 2000). Second, leo mutations are homozygous lethal, suggesting that D14-3-3ε cannot functionally compensate for its loss, although Leo was suggested to at least partially compensate for the lack of D14-3-3ε in embryonic development (Chang, 1997). Third, the dynamic expression pattern of 14-3-3's during embryonic development and larval and adult nervous systems (Skoulakis, 1996; Tien, 1999; Philip, 2001) suggested involvement in additional processes other than photoreceptor and oocyte development, which may specifically require one but not the other. The results demonstrate 14-3-3-isotype-specific functions and a tissue- and temporal-specific transcriptional mechanism to compensate for loss of D14-3-3ε and suggest dynamic temporal and spatial interactions of the two 14-3-3 isotypes (Acevedo, 2007).
The results utilizing null alleles indicate that D14-3-3ε is not dispensable for viability, but its loss is partially compensated by elevation of endogenous leo levels. Consequently, homozygotes survive to adulthood, whose number is higher when the hypomorphic allele D14-3-3εl(3)j2B10 is used. This is the likely reason for the suggestion of previous reports that the gene is not essential (Chang, 1997; Benton, 2002). This interaction is uncovered genetically by the inability to obtain D14-3-3εex4 and D14-3-3εl(3)j2B10 homozygotes when one copy of leo is mutated (i.e., leoP1188/+; D14-3-3εl(3)j2B10/D14-3-3εl(3)j2B10 animals). Embryos homozygous for mutant alleles do not exhibit obvious morphological defects because maternally provided D14-3-3ε is likely sufficient to fulfill its requirement in syncytial cellular blastoderm and gastrulating animals (Tien, 1999; Philip, 2001; Su, 2001). D14-3-3ε mutant homozygotes die ostensibly because lack of zygotic protein from the nervous system renders them unable to hatch. Similarly, Leo accumulates in embryonic motor neurons innervating the body-wall musculature and its loss in leo mutants is the likely reason for their failure to hatch despite their apparently normal progression through development (Acevedo, 2007).
The results demonstrate that LeoII overaccumulates in late D14-3-3ε null embryos and that this elevation allows a fraction of them to hatch and survive. The conclusion is supported by the striking increase in the number of D14-3-3ε mutant homozygotes that survive upon expression of leoII transgenes in the nervous system. Because endogenous LeoII accumulates preferentially in the CNS, the data suggest that its elevation in this tissue leads to successful hatching and survival of D14-3-3ε mutant homozygotes. This 'homeostatic' response in D14-3-3ε mutants is specific to late embryogenesis after the maternally supplied D14-3-3ε, which perdures almost until stage 8, has decayed. Therefore, the response appears specific to a period when the overall level of either 14-3-3's or D14-3-3ε, specifically, is critically important for survival (Acevedo, 2007).
It appears that a mechanism sensing the absence of D14-3-3ε operates in embryos and responds by increasing the level of LeoII. Congruent with this, Leo elevation was not observed in embryos homozygous for the dominant-negative allele D14-3-3εE183K (Chang, 1997), which compromises D14-3-3ε functionally, but does not change its overall levels in the embryo. It is possible that this is the reason that D14-3-3εE183K homozygotes are never recovered. Furthermore, this response appears specific to the loss of D14-3-3ε, because levels of this protein remained normal in homozygous leo mutant embryos and adults, consistent with their strong lethal phenotype. Clearly, this sensing mechanism responds by increased accumulation of leoII transcripts by preferential utilization of one of two possible promoters and splicing of the primary transcript to include the leoII-specific exon 6′. How is lack of D14-3-3ε sensed and how could leoII transcription be increased? 14-3-3's have been reported to participate in nuclear/cytoplasmic trafficking of transcription factors. Therefore, it is possible that loss of D14-3-3ε enhances transcription from the proximal promoter of the leo gene by not mediating nuclear export of a factor that binds that site. Alternatively, D14-3-3ε may be part of a repressing complex and, upon its loss, transcription from this site is enhanced. In contrast, excessive transgenic elevation in the amount of D14-3-3ε results in recovery of few adults (<10% of expected) homozygous for strong hypomorphic leo mutations. This suggests that although D14-3-3ε can at least partially compensate for the loss of Leo in high concentrations, an endogenous molecular mechanism to elevate it in leo homozygotes does not appear to exist (Acevedo, 2007).
Although leoI transcripts accumulate in the wing disc, Leo does not appear to play a role in wing-vein formation because animals that develop with as low as 10% of normal Leo do not exhibit wing aberrations (Philip, 2001). Therefore, the venation deficits are a phenotype specific to D14-3-3ε mutant homozygotes. Interestingly, in congruence with the mechanism proposed above, the leoI transcripts normally expressed in that tissue were not upregulated and leoII transcripts were not ectopically transcribed in D14-3-3ε mutant homozygote wing disks. This is because the proposed D14-3-3ε-interacting factor(s) required for exon 1′-containing leoII transcription are likely absent from the wing disk where these transcripts do not normally accumulate. Exon 1-containing leoII transcripts do not appear to require such D14-3-3ε-interacting factor(s), since these transcripts were not upregulated in embryos. Therefore, loss of D14-3-3ε does not alter the tissue specificity of leo transcriptional regulation and specific isoform accumulation (Acevedo, 2007).
It is presently unclear whether this compensatory mechanism is operant in other systems where mutant analyses of 14-3-3's have been initiated. Interestingly, single nulls of either 14-3-3-encoding gene in Saccharomyces cerevisiae are viable, while the double mutant is lethal and similar results were obtained for the two Schizosaccharomyces pombe genes. These observations may reflect similar 14-3-3 'homeostatic' mechanisms in these species. Directed reduction of specific 14-3-3 protein levels during Xenopus laevis development yielded gastrulation and patterning defects for all proteins tested except for 14-3-3ζ. Unlike Drosophila, Xenopus 14-3-3ζ may not be essential for development, but it is also possible that loss of this isotype is specifically compensated for by elevation of the remaining 14-3-3's. Such mechanisms, if extant in mammals, are likely to hinder genetic analysis of 14-3-3 function, especially in the brain where all family members are expressed. Interestingly, mice mutant for 14-3-3ε exhibit severe brain abnormalities and die perinatally, yet a small fraction survive to adulthood appearing smaller, but otherwise normal, much like the Drosophila mutants. It is unknown whether 14-3-3ζ or other isotypes are elevated in these animals as predicted by the current results (Acevedo, 2007 and references therein).
Functional specificity of 14-3-3 family members may be the result of tissue or temporal-specific gene expression and regulation or of isotype-specific ligand selectivity. Isotypes may have redundant functions within a cell if they are able to interact with the same targets. Even then, affinity differences toward common ligands predicted by their amino-acid sequence and tertiary structure may functionally differentiate coexpressed 14-3-3's (Acevedo, 2007).
Although both leoI and leoII transgenes rescued the lethality of D14-3-3ε mutants, they clearly exhibited different efficiency. Rescue was invariably higher upon accumulation of LeoII either ubiquitously or specifically in the nervous system. However, rescue required excessive accumulation of LeoII to overcome loss of D14-3-3ε. In fact, the two- to threefold Leo elevation could be as much as a 50%-60% underestimate of the level of this protein in D14-3-3ε mutant embryos that hatch. Therefore, a large excess of Leo appears to be necessary to functionally substitute D14-3-3ε in the embryonic nervous system, which may be attained only in a small number of mutant homozygotes. This probably reflects the affinity differences that Leo dimers exhibit toward client proteins normally bound either by D14-3-3ε homodimers or by D14-3-3ε/Leo heterodimers. If so, then even a small amount of D14-3-3ε would increase the number of mutant homozygotes obtained. In agreement with this, more homozygotes were recovered from the transposon allele D14-3-3εl(3)j2B10, which likely contains residual D14-3-3 ε. Differences in ligand binding between LeoI and LeoII are likely reflected in the large difference with which the two isoforms rescue the lethality of D14-3-3ε mutants. This is the first unequivocal demonstration of functional differences between LeoI and LeoII. These differences must reside in the five unique amino acids of helix 6 that distinguish the two isoforms (Philip, 2001). It is unknown whether LeoI, LeoII, or both contribute to the reported redundancy with D14-3-3ε in photoreceptor development and oocyte polarity (Acevedo, 2007).
Interestingly, the functional redundancy of Leo isoforms with D14-3-3ε is tissue specific. In contrast to the embryonic nervous system, enhanced accumulation of LeoI, and not of LeoII, was able to compensate for anterior cross-vein deficits and partially for the posterior cross vein. Again, this suggests that D14-3-3ε ligands in the wing disk necessary for cross-vein formation can be targeted by excess LeoI (and not LeoII). Hence, LeoII can be redundant with D14-3-3ε specifically in the embryonic nervous system where it is presumed to accumulate preferentially, while in the wing disk LeoI, which is normally found in this tissue, is the potential compensating isoform. Similarly, although the two S. cerevisiae 14-3-3 genes are functionally redundant for viability, only one, Bmh1p, is required for efficient forward transport to the endoplasmic reticulum. Thus, redundancy of 14-3-3's largely depends on the specific function and interacting proteins that they engage within a particular tissue or developmental context (Acevedo, 2007).
Collectively, these data show a tissue- and temporal-specific upregulation of leo transcription that can account for the apparent functional redundancy between Leo and D14-3-3ε with respect to the lethality of D14-3-3ε mutants and possibly other processes requiring these proteins. In addition, this analysis for the first time demonstrates tissue and temporal functional differences between the two Leo isoforms. Whether these functional differences and the functional redundancy among the Drosophila 14-3-3's will also occur in the adult nervous system where they are all most abundant is currently unknown. A previous study failed to uncover differences between LeoI and LeoII with respect to learning and memory (Philip, 2001). Nevertheless, the data strongly support the notion that the existence of multiple 14-3-3 isotypes in metazoans reflects a combination of tissue- and temporal-specific isotype expression, localization, and functional specialization. Importantly, this analysis indicates that understanding the biological roles of 14-3-3's will require identification of proteins engaged by homo- and heterodimers of particular composition in a tissue- and temporal-specific manner (Acevedo, 2007).
The biochemical means through which multiple signaling pathways are integrated in navigating axons is poorly understood. Semaphorins are among the largest families of axon guidance cues and utilize Plexin (Plex) receptors to exert repulsive effects on axon extension. However, Semaphorin repulsion can be silenced by other distinct cues and signaling cascades, raising questions of the logic underlying these events. This study uncovers a simple biochemical switch that controls Semaphorin/Plexin repulsive guidance. Plexins are Ras/Rap family GTPase activating proteins (GAPs) and this study finds that the PlexA GAP domain is phosphorylated by the cAMP-dependent protein kinase (PKA). This PlexA phosphorylation generates a specific binding site for 14-3-3ε, a phospho-binding protein that is necessary for axon guidance. These PKA-mediated Plexin-14-3-3ε interactions prevent PlexA from interacting with its Ras family GTPase substrate and antagonize Semaphorin repulsion. These results indicate that these interactions switch repulsion to adhesion and identify a point of convergence for multiple guidance molecules (Yang, 2012).
Axons rely on the activation of guidance receptors for correct navigation but receptor inactivation is also thought to be a means through which growth cones integrate both attractive and repulsive guidance signals. The current results indicate that such a mechanism plays a critical role in Sema/Plex-mediated repulsive axon guidance. PlexA was found to use its GAP activity to specify axon guidance but this activity is antagonized by a PKA-mediated signaling pathway. PKA directly phosphorylates the GAP domain of PlexA and this phosphorylation provides a binding site for 14-3-3ε. 14-3-3ε is critical for axon guidance and disrupts the ability of PlexA to interact with its Ras GTPase substrate. These interactions effectively switch PlexA-mediated axonal repulsion to Integrin-mediated adhesion and provide a simple biochemical mechanism to integrate antagonistic axon guidance signals (Yang, 2012).
Genetic experiments identify a critical role for 14-3-3ε proteins in directing axon guidance events during development. The 14-3-3 proteins are a phylogentically well-conserved family of cytosolic signaling proteins including seven mammalian members that play key roles in a number of cellular processes. Interestingly, 14-3-3 family proteins were first identified because of their high level of expression in the brain, but despite considerable interest in their functions, their roles in the nervous system are still incompletely understood. For instance, 14-3-3 proteins are highly expressed in growing axons and have been found to modulate neurite extension and growth cone turning in vitro in a number of contexts. However, their necessity for directing axonal growth and guidance events in vivo are unknown as is the functional role of each family member in these neurodevelopmental processes. This study found that one of the two Drosophila 14-3-3 family members, 14-3-3ε, is required in vivo for axon guidance and plays specific roles in the pathfinding of motor and CNS axons. Moreover, previous mutant analysis has revealed that the other 14-3-3 family member in Drosophila, 14-3-3ζ (Leonardo), does not exhibit significant motor axon guidance or innervation defects but plays a critical role in synaptic transmission and learning and memory. These results indicate that individual 14-3-3 family members play specific roles in the development of the nervous system and in light of the requirement of 14-3-3ε in mammalian brain development and neuronal migration, and potential roles for 14-3-3ε (YWHAE) in human neurological disease, future work will determine if 14-3-3ε's role in axon guidance is phylogenetically conserved (Yang, 2012).
Genetic and biochemical experiments also identify a specific role for 14-3-3ε in regulating Sema/Plex-mediated repulsive axon guidance. Sema/Plex-mediated repulsive axon guidance is antagonized by increasing cAMP levels, but the mechanisms underlying these cAMP-mediated effects are poorly understood. Interestingly, Plexins associate with the cAMP-dependent protein kinase (PKA) via MTG/Nervy family PKA (A kinase) anchoring proteins (AKAPs). AKAPs position PKA at defined locations to allow for the spatially and temporally specific phosphorylation of target proteins in response to local increases in cAMP and this study now finds that PKA phosphorylates the cytoplasmic portion of PlexA. Genetic and biochemical results suggest that this phosphorylation provides a binding site for a specific 14-3-3 family member, 14-3-3ε. 14-3-3 proteins are well known as phosphoserine/threonine-binding proteins and have been found to utilize this ability to regulate the activity of specific enzymes. This study found that mutating the 14-3-3ε binding site on PlexA generates a hyperactive PlexA receptor, providing a better understanding of the molecular and biochemical events through which cAMP signaling regulates Sema/Plex repulsive axon guidance. Future work will focus on identifying the upstream extracellular signal that increases cAMP levels, although it is interesting that the axonal attractant Netrin is known to increase cAMP levels and antagonize Sema-mediated axonal repulsion (Yang, 2012).
The results also indicate that the GAP activity of PlexA is critical in vivo for repulsive axon guidance and that cAMP/PKA/14-3-3ε signaling regulates this Plexin RasGAP-mediated repulsion. Plexins are GAPs for Ras family proteins and in vitro work has revealed that the GAP activity of Plexin is important for its signaling role. This study now finds that RasGAP activity is required in vivo in neurons for Plex-mediated repulsive axon guidance. Moreover, the results indicate that 14-3-3ε binds to a single phosphoserine residue within the PlexA GAP domain and antagonizes PlexA RasGAP-mediated axon guidance. Interestingly, positive regulation of GTPase signaling may be a conserved function for 14-3-3ε since it also increases the efficiency of Ras signaling during Drosophila eye development and 14-3-3 turns off the activity of other known GAPs and enhances Ras signaling. Therefore, the results suggest a model in which Sema/Plex interactions activate PlexA GAP activity, which inactivates Ras/Rap and disables Integrin-mediated adhesion. However, these Sema/Plex-mediated effects are subject to regulation, such that increasing cAMP levels activates PlexA-bound PKA to phosphorylate PlexA and provide a binding site for 14-3-3ε. These PlexA-14-3-3ε interactions occlude PlexA GAP-mediated inactivation of Ras family GTPases and restore Integrin-dependent adhesion (Yang, 2012).
In conclusion, this study has identified a simple mechanism that allows multiple axon guidance signals to be incorporated during axon guidance. Neuronal growth cones encounter both attractive and repulsive guidance cues but the molecular pathways and biochemical mechanisms that integrate these antagonistic cues and enable a discrete steering event are incompletely understood. One way in which to integrate these disparate signals is to allow different axon guidance receptors to directly modulate each other's function. Another means is to tightly regulate the cell surface expression of specific receptors and thereby actively prevent axons from seeing certain guidance cues. Still further results are not simply explained by relatively slow modulatory mechanisms like receptor trafficking, endocytosis, and local protein synthesis but indicate that interpreting a particular guidance cue is susceptible to rapid intracellular modulation by other, distinct, signaling pathways. The results now indicate a means to allow for such intracellular signaling crosstalk events and present a logic by which axon guidance signaling pathways override one another. Given this molecular link between such key regulators of axon pathfinding as cyclic nucleotides, phosphorylation, and GTPases, the observations on silencing Sema/Plex-mediated repulsive axon guidance also suggest approaches to neutralize axonal growth inhibition and encourage axon regeneration (Yang, 2012).
Transcription regulation is mediated by enhancers that bind sequence-specific transcription factors, which in turn interact with the promoters of the genes they control. This study shows that the JIL-1 kinase is present at both enhancers and promoters of ecdysone-induced Drosophila genes, where it phosphorylates the Ser10 and Ser28 residues of histone H3. JIL-1 is also required for CREB binding protein (CBP)-mediated acetylation of Lys27, a well-characterized mark of active enhancers. The presence of these proteins at enhancers and promoters of ecdysone-induced genes results in the establishment of the H3K9acS10ph and H3K27acS28ph marks at both regulatory sequences. These modifications are necessary for the recruitment of 14-3-3, a scaffolding protein capable of facilitating interactions between two simultaneously bound proteins. Chromatin conformation capture assays indicate that interaction between the enhancer and the promoter is dependent on the presence of JIL-1, 14-3-3, and CBP. Genome-wide analyses extend these conclusions to most Drosophila genes, showing that the presence of JIL-1, H3K9acS10ph, and H3K27acS28ph is a general feature of enhancers and promoters in this organism (Kellner, 2012).
Activation of transcription in higher eukaryotes requires the interaction between transcription factors bound to distal enhancers and proteins present at the promoter. Recent findings indicate that enhancers contain a variety of histone modifications that change during the establishment of specific cell lineages suggesting that these sequences may play a more complex role in transcription than previously thought. Given the presence of common as well as specific histone marks at enhancers and promoters, it is tempting to speculate that epigenetic modifications at these sequences serve to integrate various cellular signals required to converge in order to activate gene expression. Results described in this study support this hypothesis, demonstrating that the proteins that carry out these histone modifications are necessary to establish enhancer-promoter contacts and activate transcription of ecdysone-inducible genes (Kellner, 2012).
The execution of this process in Drosophila requires the recruitment of JIL-1 by mechanisms that are not well understood. Although the direct involvement of JIL-1 in the transcription process has been brought into question due to the failure to observe recruitment of JIL-1 to heat shock genes in polytene chromosomes, results presented in this study clearly indicate that JIL-1 affects transcription at different steps in the transcription cycle. At the promoter region, phosphorylation of H3S10 by JIL-1 results in the recruitment of 14-3-3 and, subsequently, histone acetyltransferases Elp3 and MOF (Karam, 2010). This study found that JIL-1 is also able to phosphorylate H3S28 at both promoters the enhancers. The establishment of the H3K9acS10ph and H3K27acS28ph modifications correlates with the recruitment of 14-3-3 to enhancers and promoters of ecdysone-induced genes. 14-3-3 has been implicated in numerous cellular processes, where it functions as a scaffold protein). 14-3-3 is found as dimers and multimers; each monomer is capable of binding two targets and can mediate and stabilize interactions between two phosphoproteins. Additionally, acetylation facilitates the dimerization of 14-3-3 molecules and their ability to bind certain substrates. Binding assays have demonstrated that 14-3-3 interacts weakly with H3 tail peptides phosphorylated at S10 and S28, but strong binding is detected if the peptide is both phosphorylated and acetylated on the neighboring lysine residues. Given the ability of 14-3-3 to serve as a scaffold for large protein complexes, its demonstrated interactions with H3K9acS10ph and H3K27acS28ph and the presence of these two modifications at enhancers and promoters, it is possible that contacts between these two sequences are stabilized by 14-3-3. This hypothesis is supported by 3C experiments indicating that induction of transcription of the Eip75B gene is accompanied by strong enhancer-promoter interactions. These interactions are lost in JIL-1, CBP, and 14-3-3 knockdown cells. Since these proteins act several steps downstream from transcription factor binding in the pathway leading to enhancer-promoter contacts, and loss of these proteins results in the abolishment of these contacts, it appears that these proteins, rather than specific transcription factors, may be responsible for enhancer promoter interactions at the ecdysone-inducible genes (Kellner, 2012).
Genome-wide studies using ChIP-seq clearly show the presence of JIL-1, H3K9acS10ph, and H3K27acS28ph at enhancers and promoters of most Drosophila genes. There is a clear correlation between the amount of JIL-1, H3K9acS10ph, and H3K27acS28ph at promoters and the level of transcripts associated with the gene. These three marks are also present at enhancers defined by the occurrence of H3K4me1 and H3K27ac, suggesting that the JIL-1 kinase is a regulator of histone dynamics at enhancers and promoters genome-wide. JIL-1, H3K9acS10ph, and H3K27acS28ph are found at low levels at enhancers before activation, which then increase in intensity and drop in baseline when found in combination with H3K27ac, a mark of active enhancers. These conclusions are different from those previously published examining the role of JIL-1 in transcription and dosage compensation (Regnard, 2011). This study concluded that JIL-1 binds active genes along their entire length and that the levels of JIL-1 are not associated with levels of transcription. The differences in the conclusions may be due to the different cell lines used -- male S2 cells versus female Kc cells -- and the emphasis of the analysis by Regnard on the expression of dosage-compensated genes in the male X-chromosome, which may contain JIL-1 throughout the genes as a consequence of their regulation at the elongation step. In addition, the study by Regnard used ChIP-chip on custom tiling arrays of the X chromosome plus cDNA arrays containing the whole genome. This strategy may bias the conclusions and suggest the presence of JIL-1 in the coding region of genes rather than at enhancers and promoters (Kellner, 2012).
Results presented in this study extend the previous list of histone modifications characteristic of active enhancers to include H3K9acS10ph and H3K27acS28ph. Enhancers tend to be cell type-specific and are determined during differentiation with the characteristic H3K4me1 modification. It is unclear how these regions are designated before activation and what keeps them in a poised state ready for activation upon receiving the proper signal from the cell. It is tempting to speculate that the presence of JIL-1 at enhancers prior to activation might play a role in maintaining the enhancer in this poised state. An important question for future studies is the mechanistic significance of the looping between enhancers and promoters in order to achieve transcription activation. One interesting possibility is that various signaling pathways in the cell contribute to building epigenetic signatures at enhancers and promoters in the form of histone acetylation and/or phosphorylation of various Lys/Ser/Thr residues. Acetylation marks at enhancers and promoters may then cooperate to recruit BRD4 (FS(1)H in Drosophila), which contains two bromodomains each able to recognize two different acetylated Lys residues. The requirement for acetylation of histones at enhancers and promoters in order to recruit Brd4 would ensure that several different signaling events have taken place before recruitment of P-TEFb by BRD4 can release RNAPII into productive elongation (Kellner, 2012).
PAR-1 kinases are required to determine the anterior-posterior (A-P) axis in C. elegans and Drosophila, but little is known about their molecular function. Drosophila 14-3-3 proteins, 14-3-3epsilon and 14-3-3zeta/Leonardo (Leo) represent the Drosophila homologs of C. elegans PAR-5. 14-3-3 proteins have been identified as Drosophila PAR-1 interactors; PAR-1 binds a domain of 14-3-3 distinct from the phosphoserine binding pocket. PAR-1 kinases phosphorylate proteins to generate 14-3-3 binding sites and may therefore directly deliver 14-3-3 to these targets. 14-3-3 mutants display phenotypes identical to par-1 mutants in oocyte determination and the polarization of the A-P axis. Together, these results indicate that PAR-1's function is mediated by the binding of 14-3-3 to its substrates. The C. elegans 14-3-3 protein, PAR-5, is also required for A-P polarization, suggesting that this is a conserved mechanism by which PAR-1 establishes cellular asymmetries (Benton, 2002).
PAR-1 contains three conserved domains: centrally-located kinase and ubiquitin-associated (UBA) domains, and a C-terminal domain of unknown function. Since the C-terminal domain is dispensable for PAR-1 function in the germline, a yeast two-hybrid screen was performed using a bait containing the kinase and UBA domains. The largest class of preys, representing over 25% of the recovered clones, corresponded to the two Drosophila 14-3-3 proteins, 14-3-3epsilon and 14-3-3zeta/Leonardo (Leo). These interactors represent the Drosophila homologs of C. elegans PAR-5, and this interaction appears to be conserved, since PAR-5 can bind to a fragment of C. elegans PAR-1 (Benton, 2002).
To confirm this interaction by an independent assay, in vitro-synthesized, labeled full-length Drosophila PAR-1 was incubated with bacterially expressed maltose binding protein (MBP)-tagged 14-3-3 proteins bound to amylose beads. Beads containing MBP:14-3-3 fusion proteins, but not MBP alone, efficiently precipitate PAR-1, indicating that this interaction is direct (Benton, 2002).
14-3-3 proteins regulate the activity or subcellular localization of a diverse set of proteins, including several protein kinases, by binding in a phosphorylation-dependent manner to conserved motifs (RSXpSXP or RX1-2pSX2-3pS). Using the yeast two-hybrid system, it was found that 14-3-3 appears to associate with the kinase domain of PAR-1. This contrasts with the interaction of 14-3-3 with other kinases, such as Raf and Wee1, in which 14-3-3 recognizes a phosphoserine-containing motif lying outside the catalytic domain. This interaction with PAR-1 is kinase specific, since 14-3-3 does not bind to the catalytic domains of PKA or aPKC (Benton, 2002).
The region of 14-3-3epsilon that interacts with PAR-1 was determined using the molecular information of three missense alleles of 14-3-3epsilon (Chang, 1997). These alleles were isolated as suppressors of activated Ras or Raf and impair the function of 14-3-3 in Ras/Raf/MAPK signaling. One mutation, E183K, lies within the phosphoserine binding pocket and affects a residue that directly contacts phosphoserine-peptide ligands. The others, F199Y and Y214F, are both located outside this pocket in a hydrophobic region of unknown function. Each of these three mutations were introduced into a 14-3-3epsilon prey clone and their effects on the intermolecular interactions of 14-3-3epsilon were tested (Benton, 2002).
Since 14-3-3 proteins function as dimers, whether these mutations influenced the dimerization property of 14-3-3epsilon was tested. 14-3-3epsilon can form both homodimers and heterodimers with Leo, and none of the three mutations significantly affects these interactions. This is consistent with the location of these mutations in regions distinct from the dimerization interface and indicates that global protein structure and stability are not affected. Interactions were tested of these mutant proteins with a domain of Drosophila Raf that contains a conserved 14-3-3-recognition motif (R740SApSEP745). Raf binds to both Drosophila 14-3-3 isoforms, and the interaction with 14-3-3epsilon is completely abolished by the E183K mutation, but not by the F199Y and Y214F mutations, as expected for an association via the phosphoserine binding pocket (Benton, 2002).
These mutations have opposite effects upon the interaction with PAR-1: E183K does not impair binding, whereas the other mutations either result in a severe (F199Y) or a more modest (Y214F) reduction in the strength of this interaction. These results indicate that 14-3-3epsilon does not bind PAR-1 via its phosphoserine binding pocket, consistent with the lack of canonical binding motifs in PAR-1. The interaction instead appears to be mediated by a novel interface on the external surface of the 14-3-3 molecule. Since the F199 and Y214 residues are conserved in 99% of 14-3-3 sequences, this interface is likely to exist in all isoforms (Benton, 2002).
Since binding of PAR-1 to 14-3-3 should leave the phosphoserine binding pocket vacant, and PAR-1 is a serine/threonine kinase, it was reasoned that PAR-1 might be involved in regulating the phosphorylation-dependent interactions of 14-3-3 with other proteins. Whether PAR-1 can phosphorylate proteins to generate the phosphoserine epitope recognized by 14-3-3 was tested. Using either immunoprecipitated or bacterially expressed PAR-1, efficient phosphorylation was observed of the 14-3-3-interacting portion of Raf. Phosphorylation of Ser743, or the equivalent residue in Raf homologs, is essential for 14-3-3 binding and for Raf function in vivo. This residue was mutated to alanine and this was found to completely abolish the phosphorylation by PAR-1, indicating that PAR-1 specifically phosphorylates Raf to generate a 14-3-3 binding site. This activity of PAR-1 does not require the presence of 14-3-3, and addition of 14-3-3 to this assay does not detectably affect Raf phosphorylation. A mammalian PAR-1 homolog, C-TAK1, is able to phosphorylate proteins such as KSR and Cdc25C within 14-3-3 binding sites, suggesting that this specificity is a conserved property of this kinase family (Benton, 2002).
To test whether 14-3-3 proteins are involved in par-1-dependent processes in vivo, loss-of-function mutations in 14-3-3epsilon and leo were analyzed. Surprisingly, flies homozygous for a protein null allele of 14-3-3epsilon (14-3-3epsilonj2B10) are viable. However, females lay very few eggs, which fail to hatch. Most egg chambers from these females lack differentiated oocytes, as revealed by DNA staining, which distinguishes the oocyte karyosome from the 15 polyploid nurse cells. An identical phenotype is observed in ovaries from flies containing this allele over a deficiency, indicating that the phenotype is specific for this locus. To determine where 14-3-3epsilon is required, clones of this allele were generated in either the germline or somatic follicle cells. Defects in oocyte differentiation were observed only in germline clones; thus, like PAR-1, 14-3-3epsilon is required in the germline for oocyte differentiation (Benton, 2002).
Oocyte determination depends on the MT-dependent transport of specific factors, such as Orb and the germ cell centrosomes, to one cell in the cyst. These factors initially concentrate at the anterior of this cell but subsequently translocate around the nucleus and concentrate along the posterior cortex. This second step appears to require the establishment of a diffuse MTOC along the posterior of the cell and is essential for its stable determination as the oocyte. The formation of this MTOC can be visualized using an antibody to Minispindles (MSPS), a MAP that localizes to sites of MT nucleation. In wild-type egg chambers, MSPS accumulates along the posterior cortex. This accumulation is undetectable in 14-3-3epsilon mutants, indicating that the MTOC has failed to form. Orb and the centrosomes therefore do not undergo the anterior-to-posterior movement and eventually diffuse away as this cell exits meiosis and adopts a nurse cell fate. These phenotypes are indistinguishable from those of par-1 null mutant cysts, indicating that 14-3-3epsilon and PAR-1 function together in this specific step of oocyte determination (Benton, 2002).
In contrast to 14-3-3epsilon mutants, germline clones of a strong lethal allele of leo (leoP1188) display no defects in this process. Since the 14-3-3epsilon phenotype is incompletely penetrant, whether 14-3-3 proteins have partially redundant functions in the germline was tested. Removal of one copy of leo in 14-3-3epsilon mutant clones results in a fully penetrant defect in oocyte determination. Furthermore, removal of one copy of 14-3-3epsilon in leo mutant cysts uncovers an important contribution of leo in this process, since 84% of these cysts display defects in Orb localization. Thus, although 14-3-3epsilon has the predominant function in oocyte determination, Leo can partially compensate in its absence (Benton, 2002).
Polyclonal antibodies against 14-3-3epsilon and Leo were used to examine their localization in the germline. These antibodies are specific, since they do not stain tissue mutant for the corresponding isoform. 14-3-3epsilon is highly expressed in the dividing germline cells in the germarium, and colocalizes with PAR-1 on the fusome, a membranous structure that branches into each germ cell during the early germ cell divisions. The asymmetric partitioning of the fusome during these divisions results in one cell always inheriting more fusome material, which may provide an initial cue to specify this cell as the oocyte. The colocalization of PAR-1 and 14-3-3epsilon on the fusome may therefore represent a mechanism to concentrate these proteins in the future oocyte (Benton, 2002).
At later stages, 14-3-3epsilon colocalizes with PAR-1 at the ring canals, which interconnect the germline cells in each cyst. 14-3-3epsilon can be detected in the cytoplasm and around the cortex of the oocyte but, unlike PAR-1, does not accumulate at the posterior pole. Leo is also expressed in the germline and displays a similar localization to ring canals, but is expressed at very low levels in the germarium (Benton, 2002).
While the fusome and ring canals may represent sites of physical and functional association of PAR-1 with 14-3-3, its localization to these sites is not affected in 14-3-3 mutants, indicating that 14-3-3 binding does not simply act to target PAR-1 to these subcellular destinations. Mutations in the Drosophila PAR-3 homolog Baz cause similar phenotypes in oocyte determination as par-1 and 14-3-3 mutants. However, Baz concentrates at distinct sites in the germarium, in circles around each ring canal that also contain components of adherens junctions, and this localization is not detectably affected in 14-3-3 mutants (Benton, 2002).
The high early cytoplasmic concentration of 14-3-3epsilon has prevented an conclusive determination of whether the fusome localization of 14-3-3epsilon is PAR-1 dependent. However, 14-3-3epsilon is detectable at ring canals in cysts homozygous for a par-1 null allele (Benton, 2002).
To determine if 14-3-3 proteins function with PAR-1 in the repolarization of the oocyte to define the A-P axis, the distribution of osk mRNA and Stau was examined in late-stage egg chambers recovered from homozygous and hemizygous 14-3-3epsilonj2B10 females, and in germline clones of this allele. These mutants display a partially penetrant phenotype, in which osk mRNA and Stau accumulate in dots in the middle of the oocyte. Twenty seven percent of egg chambers display both ectopic and posterior accumulation of osk mRNA and Stau protein, and four percent contain only mislocalized dots. These defects are very similar to those of hypomorphic par-1 mutants and can be strongly enhanced by removal of one copy of par-1 (Benton, 2002).
Although most bcd mRNA localizes normally to the anterior cortex in 14-3-3epsilon mutants, a small proportion is mislocalized along the lateral cortex, and occasionally at the posterior. In contrast to previous observations, such defects in bcd mRNA distribution are also observed in par-1 mutants. These are more pronounced at stages 8-9 than at stage 10, which might reflect a partial recovery in bcd mRNA localization to the anterior between these stages or the diffusion of the mRNA away from the lateral and posterior cortices due to a failure in anchoring. Other mutants that affect the localization of bcd and osk mRNAs, such as gurken, also disrupt the migration of the oocyte nucleus to the dorsal-anterior corner. As in par-1 mutants, however, oocyte nucleus migration appears to be unaffected in 14-3-3epsilonj2B10 mutants (Benton, 2002).
Oocytes that are homozygous for leoP1188 do not display polarity defects. However, strong dominant genetic interactions are observed between 14-3-3 mutants. Thus, these isoforms also function partially redundantly in this process (Benton, 2002).
To gain insights into the basis for the defects in mRNA localization, the organization of the MT cytoskeleton was examined, using a MT plus end marker, Kin:ß-gal. This marker accumulates at the posterior pole in wild-type oocytes, suggesting that the majority of MT plus ends are focused on this site. In contrast, Kin:ß-gal concentrates in the center of 14-3-3epsilon mutant oocytes, indicating that MT plus ends are focused incorrectly, and providing an explanation for the defects in osk mRNA/Stau distribution. The organization of oocyte MTs was directly analyzed using both a FITC-conjugated anti-alpha-tubulin antibody and a Tau:GFP reporter of MT distribution in living egg chambers . In contrast to the wild-type anterior-to-posterior gradient of MTs, 14-3-3epsilon mutants show a uniform distribution of MTs around the oocyte cortex, with the lowest density of MTs in the center. These defects in MT organization are indistinguishable from those of par-1 mutants (Benton, 2002).
The combination of phenotypes in osk and bcd mRNA localization and MT organization is, thus far, unique to par-1 and 14-3-3 mutants, and strongly suggests that they function together in the polarization of the A-P axis (Benton, 2002).
To determine the importance of the 14-3-3 protein interaction domains in vivo, the phenotypes of the 14-3-3epsilon missense alleles, 14-3-3epsilonF199Y and 14-3-3epsilonE183K, were characterized. Neither mutation significantly affects the level or localization of the protein, as assessed by immunostainings. 14-3-3epsilonE183K displays penetrant defects in both oocyte determination and polarization. The penetrance of the latter is almost three times that observed with the protein null allele, indicating that the E183K mutant protein functions as a dominant negative, presumably through the formation of nonfunctional heterodimers with Leo. Thus, the interaction of 14-3-3 dimers with phosphorylated targets is critical for its function in the germline (Benton, 2002).
14-3-3epsilonF199Y mutant egg chambers do not exhibit significant defects in oocyte determination or polarization, consistent with previous reports that this allele only displays phenotypes under genetically sensitized conditions (Chang, 1997). In the absence of leo, however, this allele has a dominant phenotype, with 11% of leoP1188;14-3-3epsilonF199Y/+ egg chambers displaying defects in oocyte determination. Thus, the PAR-1 interaction interface is also important for 14-3-3 function (Benton, 2002).
Thus loss-of-function mutations in 14-3-3 cause phenotypes identical to par-1 mutants in both the initial polarization of the oocyte and the repolarization that defines the A-P axis. These results indicate that 14-3-3 functions as an essential cofactor for PAR-1 in the generation of polarity (Benton, 2002).
Given the diverse roles of 14-3-3 proteins, it is very surprising that the only essential requirement for 14-3-3epsilon is in PAR-1-dependent polarization events in the Drosophila germline. A similar dedication of 14-3-3 function may exist in C. elegans, where animals homozygous for hypomorphic mutations in the 14-3-3 isoform encoded by par-5 are viable but give rise to progeny with highly penetrant defects in the polarization of the A-P axis. Indeed, the discovery that 14-3-3 is required for the initial polarization of the oocyte in the germarium reveals a remarkable homology between the generation of the first A-P asymmetries in flies and worms. Mutations in 14-3-3epsilon give a very specific defect in oocyte determination, in which the oocyte is initially specified correctly but fails to establish a posterior MTOC and to translocate oocyte-specific factors from the anterior to the posterior cortex. This phenotype is identical to that of par-1 null mutants, and the colocalization of PAR-1 and 14-3-3 on the fusome supports the idea that they function together in this process (Benton, 2002).
The Baz/PAR-6/aPKC complex is also required for this step of oocyte determination but localizes to a distinct site in the germarium. Furthermore, it has recently been shown that mutants in the Drosophila homolog of PAR-4 (see Drosophila Lkb1) display this phenotype (S. Martin and D.S.J., unpublished data reported in Benton, 2002). Thus, this early polarization of the oocyte requires the Drosophila homologs of five of the six par genes that mediate the A-P polarization of the C. elegans zygote. The final gene, par-2, has no obvious homologs in other organisms and may perform some function that is unique to C. elegans (Benton, 2002).
Although the full complement of PAR proteins is necessary for the initial polarization of the Drosophila oocyte in the germarium, the Baz/PAR-6/aPKC complex does not appear to be required for the repolarization of the oocyte at stage 7. In baz and par-6 null germline clones, a few egg chambers escape the block in oocyte determination, and these complete oogenesis normally, displaying no defects in the localization of Stau to the posterior. Thus, the PAR-1/14-3-3 complex can function to polarize the oocyte independently of these other PAR proteins. PAR-1 also is required for the apical-basal polarity of the follicular epithelium, and localizes to the basolateral domain in these cells. It is interesting to note that 14-3-3epsilon concentrates basolaterally in follicle cells, raising the possibility that it functions with PAR-1 in this process as well. The PAR-1/14-3-3 complex may therefore represent a conserved polarity 'cassette' that plays an analogous role to the Baz/PAR-6/aPKC complex. This requirement is not universal, however, because PAR-1 does not appear to be necessary for the apical-basal polarization of the neuroblasts (J. Kaltschmidt and R. B., unpublished data reported in Benton, 2002), which depends upon Baz, PAR-6, and aPKC. Thus, the two PAR protein complexes may comprise distinct modules that can function either together or separately to generate polarity in different contexts (Benton, 2002 and references therein).
While the common requirement for the PAR proteins strongly suggests that the mechanisms that generate the first A-P asymmetries are conserved between flies and worms, the regulatory relationships between these proteins are not conserved. The hierarchy of PAR protein function in C. elegans has been inferred from the effects of mutants in each par gene on the localization of the other PAR proteins. This analysis places PAR-5 at the top of the hierarchy because it is required for the anterior localization of the PAR-3/PAR-6/PKC-3 complex and the posterior localization of PAR-2 and PAR-1, whereas PAR-1 lies at the bottom because par-1 mutants have little effect on the asymmetric localization of other PAR proteins. In contrast, in Drosophila, Baz and PAR-1 are localized normally in 14-3-3 mutants. Furthermore, although the localization of members of the PAR-3/PAR-6/PKC-3 complex are codependent in the C. elegans zygote, this is not the case in the Drosophila oocyte, nor are they required for the localization of PAR-1 to the fusome. The different positions of PAR-5 and PAR-1 in the C. elegans hierarchy indicate that PAR-5 functions independently of PAR-1 in the localization of the other PAR proteins, but this early requirement makes it difficult to assess whether it is also necessary at other stages in the pathway. The results in Drosophila and the observation that C. elegans PAR-5 and PAR-1 interact in yeast raise the possibility that PAR-5 also functions downstream of PAR-1 (Benton, 2002).
Although the results indicate that PAR-1 and 14-3-3 function together to polarize the oocyte at two stages of oogenesis, the mechanisms by which they generate these polarities are unknown. The repolarization of the oocyte at stage 7 principally affects the organization of the MTs. The original posterior MTOC is disassembled, and the MTs are reorganized to form an A-P gradient, in which most MTs appear to be nucleated from the anterior cortex, with their plus ends extending toward the posterior pole. In 14-3-3 and par-1 mutants the MTs are evenly distributed around the cortex, and a MT plus end marker and osk mRNA/Stau localize to the center of the oocyte. These observations led to the proposal that PAR-1 functions to recruit the plus ends to the posterior. This study shows that par-1 and 14-3-3 mutants also display mislocalization of bcd mRNA around the cortex. Since this mRNA is believed to be transported to the minus ends of MTs, this suggests that MTs are abnormally nucleated from all regions of the oocyte. Thus, PAR-1 and 14-3-3 may also contribute to the generation of the MT gradient by specifically inhibiting MT nucleation along the posterior and lateral cortices. The role of PAR-1 and 14-3-3 in the initial polarization of the oocyte in the germarium is also likely to involve MTs since their loss results in a failure in the formation of an MTOC at the posterior of the cell. The mechanisms that control the formation of this MTOC are not known, however, and it is unclear whether PAR-1 and 14-3-3 function in the same way to polarize the oocyte at both stages (Benton, 2002).
A model for 14-3-3 function with PAR-1 is presented. 14-3-3 proteins regulate the activity of numerous cellular proteins in a phosphorylation-dependent manner by binding as dimers to phosphoserine/threonine-containing motifs. In many cases, this regulation involves sequestration of the target protein in the cytoplasm. For example, 14-3-3 binding to the proapoptotic factor Bad blocks its translocation to mitochondria. 14-3-3 can also directly regulate the activity of its targets: the association of 14-3-3 with serotonin N-acetyltransferase, for example, enhances its ability to bind substrates. The interaction of 14-3-3 with PAR-1 differs from these canonical 14-3-3/target interactions in several respects: (1) the binding of 14-3-3 does not appear to regulate PAR-1 activity, since 14-3-3 mutants have no effect on PAR-1 localization or stability in vivo, or on kinase activity in vitro; (2) the PAR-1 kinase domain lacks both of the well-defined 14-3-3 binding motifs, and interacts with a novel hydrophobic region that is distinct from the phosphoserine binding pocket, which should therefore still be available to bind to other proteins. Thus, 14-3-3 may act as a cofactor for PAR-1 by binding to proteins that are phosphorylated by the kinase. In support of this, it has been demonstrated that PAR-1 can specifically phosphorylate a 14-3-3 binding site in Raf (Benton, 2002).
These observations suggest a model in which PAR-1 has a dual role in regulating 14-3-3/target interactions, first by generating the 14-3-3 binding phosphoepitope, and second by directly delivering 14-3-3 to these sites. Once 14-3-3 is bound to target proteins, its continued association with PAR-1 would maintain the kinase in close proximity to its substrate, which might ensure the stable maintenance of the phosphorylated state (Benton, 2002).
In addition to their role in establishing cell polarity, PAR-1 kinases have been implicated in a diverse range of other cellular processes. The closest mammalian homolog of PAR-1, C-TAK1, was initially purified as an activity that phosphorylates Cdc25C on Ser216. The in vivo significance of this regulation is unknown, but phosphorylation of this site by a distinct kinase, Chk1, induces 14-3-3 binding, and this inhibits Cdc25C as part of the DNA damage checkpoint. C-TAK1 also phosphorylates KSR to promote 14-3-3 binding, which sequesters KSR in the cytoplasm and inhibits EGF signaling. These biochemical activities of C-TAK1 are consistent with the data in Drosophila showing that PAR-1 phosphorylates a 14-3-3 binding site in Raf, and that 14-3-3 mutants give identical phenotypes to par-1 mutants in the germline. The ability to phosphorylate 14-3-3 binding sites may be a general property of PAR-1 kinases, which accounts for the diversity of their functions (Benton, 2002).
Consistent with this, other PAR-1 substrates have been shown to associate with 14-3-3 or contain conserved potential 14-3-3 recognition motifs. The vertebrate PAR-1 homologs, MARK1 and MARK2, were identified as kinases that phosphorylate Tau to inhibit its MT binding ability. 14-3-3 interacts with the MT binding domain of Tau and appears to compete with tubulin for Tau binding. MARK kinase regulation of Tau may therefore be mediated through 14-3-3, which physically blocks the association of Tau with MTs. PAR-1 also phosphorylates the Wingless pathway component Dishevelled. This phosphorylation has been mapped to a 30 amino acid region of the protein, which contains a putative 14-3-3 recognition motif (amino acids 234-242: RTSSYSS) that is essential for its function in planar polarity (Benton, 2002).
The intimate functional relationship between PAR-1 and 14-3-3 raises the possibility that this kinase might be involved in regulating other processes involving 14-3-3 proteins. For example, the observation that PAR-1 phosphorylates Raf to generate a 14-3-3 binding site makes it a candidate for the unidentified kinase that regulates Raf in vivo. In support of this, this study shows that the F199Y and Y214F mutations in 14-3-3epsilon that affect signaling through Raf, impair the interaction of 14-3-3epsilon with PAR-1 (Benton, 2002).
Although many of the activities of PAR-1 kinases may be mediated by inducing 14-3-3 binding, this is probably not the only mechanism by which they act. Drosophila PAR-1 has recently been proposed to have a third function in the germline, in which it phosphorylates, and so stabilizes, OSK protein at the posterior pole of the oocyte to ensure its levels are high enough to specify the germ cells. Unlike PAR-1, 14-3-3 is not detectably enriched at the posterior, suggesting that this function of the kinase might operate via a 14-3-3-independent mechanism. C. elegans PAR-1 may have a similar function in germline specification, through the regulation of P-granule stability, which does not require PAR-5 (Benton, 2002).
A major question is the nature of the target(s) of PAR-1/14-3-3 that mediate their effects on cell polarity. These are unlikely to be any of the known PAR-1 substrates, such as Dishevelled or Tau, since these are not required for axis formation in Drosophila (R. B. and D. S. J., unpublished data reported in Benton, 2002), but the results lead to the clear prediction that they will bind to 14-3-3 in a PAR-1-dependent manner (Benton, 2002).
Drosophila 14-3-3γ and 14-3-3ζ proteins have been shown to function in RAS/MAP kinase pathways that influence the differentiation of the adult eye and the embryo. Because 14-3-3 proteins have a conserved involvement in cell cycle checkpoints in other systems, it was asked (1) whether Drosophila 14-3-3 proteins also function in cell cycle regulation, and (2) whether cell proliferation during Drosophila development has different requirements for the two 14-3-3 proteins. Antibody staining for 14-3-3 family members is cytoplasmic in interphase and perichromosomal in mitosis. Using mutants of cyclins, Cdk1 and Cdc25string to manipulate Cdk1 activity, it was found that the localization of 14-3-3 proteins is coupled to Cdk1 activity and cell cycle stage. Relocalization of 14-3-3 proteins with cell cycle progression suggested cell-cycle-specific roles. This notion is confirmed by the phenotypes of 14-3-3γ and 14-3-3ζ mutants: 14-3-3γ is required to time mitosis in undisturbed post-blastoderm cell cycles and to delay mitosis following irradiation; 14-3-3ζ is required for normal chromosome separation during syncytial mitoses. A model is suggested in which 14-3-3 proteins act in the undisturbed cell cycle to set a threshold for entry into mitosis by suppressing Cdk1 activity, to block mitosis following radiation damage and to facilitate proper exit from mitosis (Su, 2001).
In a previous study of 14-3-3γ localization in the embryo, this protein was reported to become nuclear-localized in infolding cells (Tien, 1999). However, a close examination of the published data revealed that the localization was in pre-mitotic cells (the publication featured mitotic domain 14 that borders the ventral furrow). In fact, a close correspondence of cells that show nuclear-localized 14-3-3γ in this publication (Tien, 1999) and cells that compose the mitotic domains is what led to further examination of the role of 14-3-3 proteins in the cell cycle. Using the same antibody and the same conditions, similar staining patterns were demonstrated (Tien, 1999). A different interpretation of these data is being offered. No correlation of the localized staining with the movement of cells or folding of the epithelium was found. Instead, the findings that 14-3-3 proteins localize to the perichromosomal region during mitosis and that this localization is coupled to Cdk1 activity demonstrate that localization is coupled to cell cycle progression and suggest that 14-3-3 proteins have a cell cycle role (Su, 2001).
One striking set of data presented in this study concern the localization of 14-3-3 proteins to the neighborhood of chromosomes in mitosis. Although the perinuclear localization of Drosophila 14-3-3 proteins is unprecedented, the interphase location and activity are consistent with reports from other systems. S. pombe Rad24 remains exclusively cytoplasmic throughout the cell cycle and this localization appears to be important for blocking mitosis upon checkpoint activation. Similarly, it has been proposed that cytoplasmic human 14-3-3sigma inhibits mitosis by retaining Cdk1/cyclin B in the cytoplasm (Chan, 1999). Like their homologs in other systems, Drosophila 14-3-3 proteins are cytoplasmic in interphase, and analysis of mutations suggests that Drosophila 14-3-3γ also inhibits entry into mitosis in response to activation of DNA damage checkpoint in embryos. This is in agreement with its proposed role in other species and consistent with a recent report (Brodsky, 2000) of a role for 14-3-3γ in preventing mitosis after DNA damage in Drosophila larvae (Su, 2001).
In addition, observations indicate a role for 14-3-3γ in the normal timing of embryonic mitoses. The precise schedule of mitotic times of cells in various positions in the Drosophila embryo made possible detection of deviations from normal timing that are as small as a few minutes. Defects can occur in the normally rigid stereotypical order with which different regions of the embryo progress into mitosis. For example, recent reports described the premature mitosis of mesodermal cells, normally domain 10, in a mutant tribbles. When embryos deficient in 14-3-3γ were examined, a different type of timing defect was found. The normal order of the mitotic domains was retained, but the entire schedule of mitosis was advanced relative to germ-band extension, a major morphological marker of developmental progression. Because there was no detectable slowing of germ-band extension in 14-3-3γ mutant embryos, it is infered that mitosis is advanced in embryos that lack 14-3-3γ. Thus, 14-3-3γ might set physiologically relevant thresholds for entry into mitosis in Drosophila, and this activity might be amplified in response to irradiation. S. pombe mutants in a 14-3-3 homolog show smaller cell size at division; because cellular growth in this organism occurs mainly in G2, it has been proposed that G2 is shorter in these 14-3-3 mutants (Ford, 1994), although precise measurements of this period have not been reported. Thus, it remains to be seen whether 14-3-3 proteins have a similar ability to set the threshold for normal mitosis in other species where only its checkpoint function has been detected (Su, 2001).
14-3-3ζ mutants show defective mitoses in the syncytium, indicating a requirement for this protein in syncytial divisions. Embryos that lack checkpoint functions such as Grapes (Chk1 homolog) and Mei-41 (an ATR homolog) also show mitotic defects, and it has been proposed that these defects are secondary to entry into mitosis with unreplicated DNA. However, loss of 14-3-3ζ functions affects early cycles. By contrast, the dramatic phenotypes of checkpoint defects occur at later syncytial stages (around cycle 12) when checkpoints are thought to become essential to postpone mitosis as S phase takes longer to complete. Thus, the early phenotype of 14-3-3ζ mutant embryos suggests that 14-3-3ζ has roles beyond its likely function in the checkpoint. Perhaps, like 14-3-3γ, 14-3-3ζ might contribute to the normal timing of mitosis even when checkpoints are not operating. Alternatively, incomplete separation of chromosomes in 14-3-3ζ mutants could indicate a more direct involvement of 14-3-3ζ in mitotic progression, an idea that is supported by the localization of the proteins around the mitotic chromosomes and their dispersal after chromosome separation. A direct test of these models will require specific inactivation of 14-3-3ζ in mitosis (as opposed to interphase) (Su, 2001).
Drosophila 14-3-3γ and 14-3-3ζ have documented roles in RAS signaling. Recent data implicate a MAP kinase pathway in cell cycle control in Xenopus, raising the possibility that Drosophila 14-3-3 proteins function through a MAPK pathway to affect their cell cycle roles. This is thought to be unlikely because treatment of Drosophila embryos with pharmacological inhibitors of MAPK pathway did not phenocopy either 14-3-3γ or 14-3-3ζ mutations (Su, 2001).
Regardless of the mechanism of action of 14-3-3ζ, it is notable that it has essential cell cycle roles in the absence of perturbations that normally provoke checkpoint responses. This reinforces other findings in Drosophila and in mammals that suggest that functions normally considered to be checkpoint functions have essential roles in regulating the cell cycle early in development (Su, 2001).
Based on the cytoplasmic localization of 14-3-3γ and cyclin/Cdk1 during interphase, it is proposed that 14-3-3γ acts to keep Cdk1 in check during interphase. As Cdk1 becomes active (owing to the accumulation of its activator Stg or after recovery from DNA damage) and cells enter mitosis, accumulating cyclin/Cdk1 activity promotes and maintains, probably indirectly, 14-3-3 protein localization near chromosomes. Upon the transition to anaphase, the localized 14-3-3 proteins can contribute to chromosome separation. The decline in Cdk1 activity allows 14-3-3 proteins to return to their interphase distribution. Thus, during interphase, 14-3-3γ can act to keep Cdk1 inactive in the cytoplasm but, once Cdk1 is active, it can act in turn to localize 14-3-3 proteins in preparation for their action during the exit from mitosis. No physical interaction has been detected between 14-3-3 proteins and Drosophila homologs of cell cycle regulators known to interact with 14-3-3 proteins in other systems (Cdc25string and cyclin B). Thus, understanding the mechanism of 14-3-3 action might require the identification of novel target molecules (Su, 2001).
The results do not rule out the possibility that 14-3-3ζ also functions to regulate the entry into mitosis in cellular embryos. This possibility cannot be addressed because 14-3-3ζ mutants arrest before G2/M control is first seen in embryogenesis, and the fraction of embryos that do progress to cellular stages are too defective with respect to cell cycle progression and gastrulation. In addition, the fact that these embryos progressed to cellular stages might reflect an incomplete loss of maternal 14-3-3ζ, thus precluding meaningful experiments. What is certain, however, is that 14-3-3γ cannot substitute for 14-3-3ζ during the nuclear divisions of syncytial stages, and that 14-3-3ζ cannot substitute 14-3-3γ for regulating the entry into mitosis during cellular stages (Su, 2001).
In summary, three lines of data indicate that Drosophila 14-3-3 proteins function in normal cell cycle progression, in addition to checkpoint regulation. These are: (1) cell cycle stage specific localization, which is dictated by Cdk1; (2) advancement of mitotic entry in 14-3-3γ mutants; and (3) defective mitoses in 14-3-3ζ mutants. This is the first clear evidence for the requirement for 14-3-3 proteins in normal mitosis in a eukaryote. Furthermore, the fact that mutations in two 14-3-3 proteins lead to different outcomes and at different stages in embryogenesis indicates that these proteins are not functionally redundant. Instead, the results provide strong evidence that, during metazoan development, cell division and its regulation might have different requirements for two members of the 14-3-3 family (Su, 2001).
Protein complexes have largely been studied by immunoaffinity purification and (mass spectrometric) analysis. Although this approach has been widely and successfully used it is limited because it has difficulties reliably discriminating true from false protein complex components, identifying post-translational modifications, and detecting quantitative changes in complex composition or state of modification of complex components. A protocol has been developed that enables determination, in a single LC-MALDI-TOF/TOF analysis, the true protein constituents of a complex, to detect changes in the complex composition, and to localize phosphorylation sites and estimate their respective stoichiometry. The method is based on the combination of fourplex iTRAQ (isobaric tags for relative and absolute quantification) isobaric labeling and protein phosphatase treatment of substrates. It was evaluated on model peptides and proteins and on the complex Ccl1-Kin28-Tfb3 isolated by tandem affinity purification from yeast cells. The two known phosphosites in Kin28 and Tfb3 could be reproducibly shown to be fully modified. The protocol was then applied to the analysis of samples immunopurified from Drosophila melanogaster cells expressing an epitope-tagged form of the insulin receptor substrate homologue Chico. These experiments allowed identification 14-3-3ε, 14-3-3zeta, and the insulin receptor as specific Chico interactors. In a further experiment, the immunopurified materials obtained from tagged Chico-expressing cells that were either treated with insulin or left unstimulated were cmpared. This analysis showed that hormone stimulation increases the association of 14-3-3 proteins with Chico and modulates several phosphorylation sites of the bait, some of which are located within predicted recognition motives of 14-3-3 proteins (Pflieger, 2008: Full text of article).
The two 14-3-3 proteins ε and zeta were identified as interactors of Chico, and their association appeared to increase upon insulin stimulation of cells. The mammalian homologues of Chico, IRS-1 as well as IRS-2 and IRS-4, were also shown to bind to 14-3-3 proteins. IRS-1 was proven to interact with 14-3-3β in 3T3L1 adipocytes, and this binding was shown to increase with insulin treatment. In contrast, another study did not observe a significant change of interaction between 14-3-3ε and IRS-1 upon hormonal stimulation in HepG2 cells; nevertheless this observation relied on Western blotting, which provides less accurate quantitative data than MS-based approaches and may not have been able to detect changes at or below 2-fold, such as those observed here using mass spectrometry techniques. In NIH-3T3 cells, 14-3-3ε was shown to interact with IRS-1 and protein kinase C-α, thus modulating insulin signaling and degradation. This study also observed an increased association of Chico and IR after a 7-min insulin treatment, which reflects activation of the insulin pathway involving tyrosine phosphorylation of Chico by IR (Pflieger, 2008).
Kc cells were stimulated with an insulin concentration and within a time window previously established to give a robust induction of the whole pathway. As a result, several insulin-dependent phosphosites, mainly phosphoserines, were identified, in Chico. The roles of phosphoserines/phosphothreonines in the mammalian homologue IRS-1 have been studied with regard to the regulation of the insulin pathway. Some serine residues, when phosphorylated, participate in the negative control of insulin signaling, whereas others appear to have a positive regulatory function. The homology of the Chico sequence to the mammalian IRS homologues is too weak to allow precise comparison of phosphosites. Nonetheless it is worth mentioning that some serine residues were shown previously to become partially or fully phosphorylated in rat and mouse IRS-1 after 5-min stimulation with 80-100 nM insulin, which is in agreement with the current observations. Among the phosphorylated residues identified in Chico, several appear to correlate with insulin stimulation either positively or negatively. Most interestingly, five sequences overlap with predicted recognition motives of 14-3-3 proteins. All but one of them were shown to become more highly phosphorylated upon stimulation, which correlates well with an enhanced association of the two 14-3-3 proteins with Chico. The differences of phosphorylation levels measured in samples Chico3 and Chico4 may be, at least in part, due to the different cell densities reached before induction. Despite differences in absolute phosphorylation levels, similar variations of the phosphorylation states (increase or decrease) were observed in the two samples upon insulin stimulation (Pflieger, 2008).
Phosphorylations on tyrosine residues were also expected at least upon insulin treatment. The presence of phosphotyrosine-containing peptides could not be conclusively established by the MS data. Nonetheless the intact protein Chico could be shown to contain phosphorylated tyrosines: a fraction of the samples Chico3 and Chico4 was analyzed by Western blot using an anti-phosphotyrosine antibody, and signal was detected in both insulin conditions with increased signal in the +INS case as expected (Pflieger, 2008).
Mutations have been isolated in the gene encoding a Drosophila 14-3-3 epsilon protein as suppressors of the rough eye phenotype caused by the ectopic expression of RAS1(V12). Using a simple loss-of-function 14-3-3 epsilon mutation, it was shown that 14-3-3 epsilon acts to increase the efficiency of RAS1 signaling. The 14-3-3 epsilon protein appears to function in multiple RTK pathways, suggesting that it is a general component of RAS1 signaling cascade. Sequence analysis of three dominant-negative alleles defines two regions of 14-3-3 epsilon that participate in RAS1 signaling. Evidence is presentedthat 14-3-3 epsilon and 14-3-3 zeta, two 14-3-3 protein family members, are partially redundant for RAS1 signaling in photoreceptor formation and in animal viability. These genetic data suggest that 14-3-3 epsilon functions downstream of or parallel to RAF, but upstream of nuclear factors in RAS1 signaling (Chang, 1997).
Search PubMed for articles about Drosophila 14-3-3epsilon
Acevedo, S. F., Tsigkari, K. K., Grammenoudi, S. and Skoulakis, E. M. (2007). In vivo functional specificity and homeostasis of Drosophila 14-3-3 proteins. Genetics 177(1): 239-53. PubMed ID: 17660572
Aitken, A. (1995). 14-3-3 proteins on the MAP. Trends Biochem. Sci. 20: 95-97. PubMed ID: 7709434
Benton, R., Palacios, I. M. and St. Johnston, D. (2002). Drosophila 14-3-3/PAR-5 is an essential mediator of PAR1 function in axis formation. Dev. Cell 3: 659-671. PubMed ID: 12431373
Brodsky, M. H., Sekelsky, J. J., Tsang, G., Hawley, R. S. and Rubin, G. M. (2000). mus304 encodes a novel DNA damage checkpoint protein required during Drosophila development. Genes Dev. 14: 666-678. PubMed ID: 10733527
Chan, T. A., Hermeking, H., Lengauer, C., Kinzler, K. W. and Vogelstein, B. (1999). 14-3-3Sigma is required to prevent mitotic catastrophe after DNA damage. Nature 401: 616-620. PubMed ID: 10524633
Chang, H. C. and Rubin, G. M. (1997). 14-3-3 epsilon positively regulates Ras-mediated signaling in Drosophila. Genes Dev. 11(9): 1132-9. PubMed ID: 9159394
Ford, J. C., al-Khodairy, F., Fotou, E., Sheldrick, K. S., Griffiths, D. J. and Carr, A. M. (1994). 14-3-3 protein homologs required for the DNA damage checkpoint in fission yeast. Science 265: 533-535. PubMed ID: 8036497
Gardino, A. K., Smerdon, S. J. and Yaffe, M. B. (2006). Structural determinants of 14-3-3 binding specificities and regulation of subcellular localization of 14-3-3-ligand complexes: a comparison of the X-ray crystal structures of all human 14-3-3 isoforms. Semin. Cancer Biol. 16: 173-182. PubMed ID: 16678437
Karam, C. S., Kellner, W. A., Takenaka, N., Clemmons, A. W. and Corces, V. G. (2010). 14-3-3 mediates histone cross-talk during transcription elongation in Drosophila. PLoS Genet. 6: e1000975. PubMed ID: 20532201
Karim, F. D., et al. (1996). A screen for genes that function downstream of Ras1 during Drosophila eye development. Genetics 143: 315-329. PubMed ID: 8722784
Kellner, W. A., Ramos, E., Van Bortle, K., Takenaka, N. and Corces, V. G. (2012). Genome-wide phosphoacetylation of histone H3 at Drosophila enhancers and promoters. Genome Res. 22: 1081-1088. PubMed ID: 22508764
Li, W., Noll, E. and Perrimon, N. (2000). Identification of autosomal regions involved in Drosophila Raf function. Genetics 156: 763-774. PubMed ID: 11014822
Philip, N., Acevedo, S., and Skoulakis, E. M. C. (2001). Conditional rescue of olfactory learning and memory defects in mutants of the 14-3-3ζ gene leonardo. J. Neurosci. 21: 8417-8425. PubMed ID: 11606630
Pflieger, D., et al. (2008). Quantitative proteomic analysis of protein complexes: concurrent identification of interactors and their state of phosphorylation. Mol. Cell Proteomics. 7(2): 326-46. PubMed ID: 17956857
Regnard, C., (2011). Global analysis of the relationship between JIL-1 kinase and transcription. PLoS Genet 7: e1001327. PubMed ID: 21423663
Rosenquist, M., et al. (2000). Evolution of the 14-3-3 protein family: Does the large number of isoforms in multicellular organisms reflect functional specificity? J. Mol. Evol. 51: 446-458. PubMed ID: 11080367
Skoulakis, E. M. C. and Davis, R. L. (1996). Olfactory learning deficits in mutants for leonardo, a Drosophila gene encoding a 14-3-3 protein. Neuron 17: 931-944. PubMed ID: 8938125
Skoulakis, E. M. C. and Davis, R. L. (1998) 14-3-3 proteins in neuronal development and function. Mol. Neurobiol. 16: 269-284. PubMed ID: 9626666
Su, T. T., et al. (2001). Cell cycle roles for the two 14-3-3 proteins during Drosophila development. J. Cell Sci. 114: 3445-3462. PubMed ID: 11682604
Tien, A.-C., Hsei, H.-Y. and Chien, C.-T. (1999). Dynamic expression and cellular localization of the Drosophila 14-3-3e during embryonic development. Mech. Dev. 81: 209-212. PubMed ID: 10330502
Wang, W., and Shakes, D. (1996). Molecular evolution of the 14-3-3 family. Mol. Evol. 43: 384-398. PubMed ID: 8798343
Yang, T. and Terman, J. R. (2012). 14-3-3ε couples protein kinase A to semaphorin signaling and silences plexin RasGAP-mediated axonal repulsion. Neuron 74(1): 108-21. PubMed ID: 22500634
date revised: 30 December 2012
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