14-3-3zeta/leonardo
The D14-3-3 gene (14-3-3zeta or Leonardo) expresses 1.0-, 1.9- and 2.9-kb mRNAs, all of which show differential expression patterns. While the 2.9-kb mRNA is expressed only in the head, the other two mRNAs are found both in the head and body. Compared to the 1.9- and 2.9-kb mRNAs, the 1.0-kb mRNA is more abundant in the ovary and is probably maternally inherited. The 1.9-kb mRNA is the most predominant species in the embryo; its level peaks between 6-15 h of embryogenesis. The D14-3-3 gene is predominantly expressed in the ventral nerve cord of the embryo, and in the neural tissues of the head (Swanson, 1992).
In addition to a strong 14-3-3zeta expression in the central nervous system, an enrichment of the transcript is seen in the region posterior to the progressing morphogenic furrow of the developing eye imaginal disc. 14-3-3zeta (Leonardo) is expressed in most, if not all cells of the eye disc. Strong antibody staining levels are found in the region posterior to the morphogenetic furrow where cells undergo neuronal induction and differentiation as photoreceptors. Within these cells, the distribution of protein appears to be concentrated apically, showing a subcellular pattern similar to the distribution of proteins that act upstream of Raf, such as Boss, Sevenless, EGF receptor, Drk, Sos and Dos (Kockel, 1997)
Antisense probes produce remarkably intense signal in mushroom body cells. Lower levels of hybridization are observed in cells of the subesophageal ganglion, optic lobes, antennal lobes, and the central brain. The gene is also expressed in cells of the thoracic ganglia, nurse cells, and maturing oocytes of female flies (Sokoulakis, 1996).
Antibody preferentially decorates the neuropil of the mushroom bodies. The antigen is detected in the dendritic projections (calyces), the cytoplasm of the parikarya, and the axonal projections that form the peduncle and lobes of the mushroom body neurons. In addition, the antibody decorates the ellipsoid body, a neuropil structure of the central complex and a group of cell bodies residing just anterior to the "heel" of the mushroom bodies. These cells appear to be the ring neurons that project to the ellipsoid body. The antibody also decorates neuropil and cell bodies in the antennal lobes, albeit with lower intensity. In sagittal sections, modest staining is observed in thoracic ganglia and throughout the cytoplasm of nurse cells and oocytes (Sokoulakis, 1996).
Immunological localization of the Leonardo protein shows that it is expressed at synaptic
connections and enriched in presynaptic boutons of the neuromuscular junction (NMJ). Null leonardo
mutants die as mature embryos. Electrophysiological assays of the mutant NMJ demonstrate that basal
synaptic transmission is reduced by 30% and that transmission amplitude, fidelity, and fatigue
resistance properties are reduced at elevated stimulation frequencies and in low external [Ca2+].
Moreover, transmission augmentation and post-tetanic potentiation (PTP) are disrupted in the mutant.
These results suggest that Leonardo plays a role in the regulation of synaptic vesicle dynamics, a
function which may underlie synaptic modulation properties enabling learning (Broadie, 1997).
14-3-3 proteins have been shown to interact with Raf-1 and cause its activation when
overexpressed. However, their precise role in Raf-1 activation is still enigmatic, as
they are ubiquitously present in cells and found to associate with Raf-1 in vivo
regardless of Raf's activation state. The function of the Drosophila
14-3-3 gene leonardo (leo) has been analyzed in the Torso (Tor) receptor tyrosine kinase (RTK)
pathway. In the syncytial blastoderm embryo, activation of Tor triggers the
Ras/Raf/MEK pathway that controls the transcription of tailless (tll). In
the absence of Tor, overexpression of leo is sufficient to activate tll expression. The
effect of leo requires D-Raf and Ras1 activities but not KSR or DOS, two recently
identified essential components of Drosophila RTK signaling pathways. Tor signaling
is impaired in embryos derived from females lacking maternal expression of leo. It is
proposed that binding to 14-3-3zeta/Leonardo by Raf is necessary but not sufficient for the activation
of Raf and that overexpressed Drosophila 14-3-3zeta requires Ras1 to activate D-Raf (Li, 1997).
Studies of Drosophila and other insects have indicated an essential role for the mushroom bodies in learning and memory. The leonardo gene encodes a Drosophila protein highly homologous to the vertebrate 14-3-3 zeta isoform. The gene is expressed abundantly and preferentially in mushroom body neurons. Mutant alleles that reduce Leonardo protein levels in the mushroom bodies significantly decrease the capacity for olfactory learning, but do not affect sensory modalities or brain neuroanatomy that are requisite for conditioning. These results establish a biological role for 14-3-3 proteins in mushroom bodymediated learning and memory processes, and suggest that proteins known to interact with them, such as RAF-1 or other protein kinases, may also have this biological function (Skoulakis, 1996).
Leonardo exhibits similar involvement in the Raf/Ras pathway. Clones of mutant leonardo show a loss of photoreceptors. Ommatidia lack outer as well as inner photoreceptors. This phenotype is reminiscent of clones homozygous for Drosophila ras or raf hypomorphic alleles. Artificial activation of Raf rescues the nonviability causes by leonardo mutation and permits photoreceptor development. It is concluded that leonardo acts downstream of Ras and upstream of Raf in the signaling pathway that controls cell proliferation in the Drosophila eye imaginal disc (Kockel, 1997).
The reduction of leonardo gene dose appears to worsen the phenotype of 14-3-3eta mutants, suggesting that the different 14-3-3 proteins are partially redundant. However, reduction of leonardo gene does not detectably modify the rough eye phenotype caused by activated Ras1 expression (Chang, 1997).
Members of the ubiquitous 14-3-3 family of proteins are abundantly expressed in metazoan neurons. The Drosophila 14-3-3zeta gene leonardo is preferentially expressed in adult mushroom bodies, centers of insect learning and memory. Mutants
exhibit defects in olfactory learning and memory and physiological neuroplasticity at the neuromuscular junction. Because strong mutations in this gene are lethal, the nature of the defects that precipitate the learning and memory deficit and the role of the two protein isoforms encoded by leonardo in these processes were investigated. The behavioral deficit in the mutants is not caused by aberrant development, Leonardo protein is acutely required for learning and memory, and both protein isoforms can function equivalently in embryonic development and behavioral neuroplasticity (Philip, 2001).
The leonardo gene encodes three size classes of
transcripts attributable to use of alternative promoters and three
polyadenylation sites. Alternative splicing of exon 6 or 6' into the mRNA results in two protein isoforms (LeoI and LeoII) that differ by five amino acids. Because exons 6 and 6' are similar in size, alternative inclusion into the mRNA does not contribute to size heterogeneity. RT-PCR was used to determine the spatial and temporal expression of mRNAs that contain exon 6 (leoI) and 6' (leoII) (Philip, 2001).
Both leoI and leoII transcripts are present in embryos before activation of the zygotic genome, suggesting that they are deposited in the oocytes maternally. Exclusive presence of leoII transcripts in stage 10-12
embryos indicates preferential splicing of exon 6' into the mRNA, which
may underlie a specific contribution of LeoII to early development. In
contrast, both leoI and leoII transcripts are found in late embryos and all larval stages. In adult animals, although both isoforms are present in heads and abdomens, leoI is absent from the thorax (Philip, 2001).
To determine whether head tissues that require leo function
exhibit differential isoform expression, flies carrying the eyes-absent
mutation and wild-type animals were subjected to mushroom body ablation with
hydroxyurea. Lack of eye tissues does not
eliminate one of the isoforms differentially, but leoII is
specifically absent from the brains of mushroom body-ablated animals.
The results indicate that leoII transcripts are specific to
the mushroom bodies, whereas although leoI may be present in these neurons, it is more broadly expressed in the brain. Outside the
mushroom bodies, Leo protein is preferentially distributed in the
ellipsoid body neurons of the central complex. Because ellipsoid body neurons are not ablated by hydroxyurea and retain Leo immunoreactivity, they must contain only leoI transcripts. Presence of D14-3-3epsilon in all tissues and
stages tested suggests a broad role in basic cellular functions, and
possible colocalization with Leo isoforms may result in heterodimer
formation. Together, the differential expression of the two
leonardo mRNAs in embryos and adult tissues suggests
functional differences between the two Leo protein isoforms. Therefore,
a functional investigation of potential differences between LeoI and
LeoII isoforms was necessary before experiments aimed at rescuing the
learning-memory deficit of leo mutations (Philip, 2001).
To investigate potential functional differences of the putative
Leo isoforms, conditional rescue of lethality associated
with strong leo alleles was attempted. The level of Leo
protein induced in heads of rescued leoP1375 homozygotes was estimated. These homozygotes contained ~75%-80%
the amount of Leo present in similarly treated wild-type animals.
Because both LeoI and LeoII can support development to adulthood, under the conditions used the isoforms do not exhibit functional specificity. Furthermore, both LI and LII transgenes are highly inducible and allow manipulation of Leo
levels in adult heads over a wide range, and animals thus obtained do
not exhibit morphological defects. In addition, these experiments
identified highly inducible leo transgenes and methods to
obtain animals for behavioral testing (Philip, 2001).
The differential distribution of leo transcripts in adult heads suggested potentially differential roles for LeoI and LeoII in olfactory learning and memory. To investigate whether the behavioral deficit of leonardo viable alleles could be reversed by conditional induction of the leo transgenes, both transgenes were introduced into
Df(1)yw67c23;leo23,
(leo23) and Df(1)yw67c23;leoX1,
(leoX1) flies. To ascertain that the transgenes remained inactive during development, all animals including the Df(1)yw67c23 (yw) control strains were raised at 18°C. Because leonardo expression in tissues other than the mushroom bodies and ellipsoid body appears normal in these alleles, quantitative Western blots were not used to monitor Leo levels in the heads of these animals. Transgene induction in animals raised at 18°C was achieved by two 30 min heat shocks delivered 6 hr apart, followed by a 5-6 hr rest period. Accumulation of LeoI and LeoII in the mushroom bodies of leo23;LI and leo23;LII animals after the rest period was monitored by immunohistochemistry using the anti-Leo antibody. Very low levels of Leo protein are present in the mushroom bodies of leo23;LI and leo23;LII animals. A significant increase of both protein isoforms in the mushroom bodies and ellipsoid body neurons was observed during induction of the respective transgenes, although final accumulation did not equal the amount of Leo in controls. Similar results were obtained with leoX1;LI and leoX1;LII animals. Moreover, lack of Leo during development did not precipitate neuroanatomical aberrations in the brains of mutant animals raised at 18°C (Philip, 2001).
Animals raised at 18°C and ones subjected to the induction and rest
period were transferred to 23-24°C 2 hr before behavioral experiments. The growth conditions and temperature shift did not affect
the ability of the mutants to perceive the stimuli used for olfactory
conditioning compared with similarly treated controls. To further
investigate their olfactory acuity, the performance of mutants and
controls toward an attractive odor, geraniol, was measured using a
modified olfactory trap assay. Although an attractive odor is not used in conditioning, this test is a good measure of olfactory acuity. Flies seek and navigate toward the source of an attractive odor, a more complex olfactory task than simple avoidance of an aversive odor. The performance of experimental animals was not significantly different from controls. Performance of the animals after olfactory conditioning was assessed immediately after training or 90 min later to investigate memory. The performance of leo23;LI,
leo23;LII, leoX1;LI, and
leoX1;LII animals exhibited a significant
30% decrement compared with controls both immediately and 90 min after
training, similar to the decrement observed with
leo23 and leoX1 animals raised under similar
conditions. In contrast, learning and 90 min retention were not
significantly different from controls during transgene induction before
conditioning. The results suggest that LeoI and LeoII accumulation in
the mushroom bodies after transgene induction fully restores the
learning and memory deficit of leo23and
leoX1 mutants. Interestingly, under the
conditions used, both LeoI and LeoII isoforms appear equivalent in
rescuing the behavioral deficit of the mutants. Collectively, the
results indicate strongly that leonardo gene products are acutely required for mushroom body-dependent olfactory learning and memory (Philip, 2001).
Given the behavioral rescue of leo mutants, it was of interest to determine whether the learning and memory deficit exhibited by leo
viable alleles represents the maximal contribution of Leo-mediated
processes in mushroom body-dependent olfactory learning. The
ability to obtain animals that harbor very low levels of Leo throughout their heads was used to address this question. The nearly complete lack of Leo throughout the adult brain did not
result in neuroanatomical anomalies judged by histological and
immunohistochemical analyses using multiple markers to examine the morphology of the mushroom bodies and central brain (Philip, 2001).
To determine whether lack of Leo throughout these rescued animals precipitated general sensory deficits, they were subjected
to behavioral control tests. These leo mutants exhibited
normal attraction to geraniol, avoidance of electric shock (US), and
avoidance of both aversive odors (benzaldehyde and 3-octanol) used as
CS. However, all rescued animals exhibit a
25%-30% decrement in olfactory learning. Significantly, the decrease in learning
exhibited by the rescued lethal homozygotes and heteroallelics was
similar in magnitude with that of leo23;LI
animals. Therefore, near lack of Leo throughout the head does not
reduce learning further than exhibited by
leo23 animals, which lack Leo only in the
mushroom bodies. This suggests that the
leo23 and
leoX1 mutations represent strong mutant
alleles with respect to the behavioral phenotype. As with
leo23;LI animals, the learning deficit of
lethal homozygotes and heteroallelics was fully rescued to control
levels by multiple inductions of either LI or LI/LII transgenes. To
determine whether restoration of learning ability results from
permanent changes attributable to the elevation of Leo, animals were
kept at 18°C after induction and trained and tested along similarly
treated and aged controls. Restoration of learning during transgene
induction decayed back to mutant levels 60-70 hr later compared with
age-matched control animals (Philip, 2001).
These results indicate that induction of Leo to levels sufficient to
restore learning does not precipitate permanent changes but rather that
the available amount of protein is acutely essential for this process.
Furthermore, elevated Leo expression outside the mushroom bodies and
ellipsoid body observed in controls and abrogated in the mutants does
not appear essential for learning, the sensory inputs used in these
experiments, or for the neuroanatomical integrity of the brain (Philip, 2001).
Thus, acute induction of either LI or LII transgenes completely restores
learning and memory in leo23 and
leoX1 mutant flies. The behavioral
deficit in these animals is unlikely the result of sensory or
developmental defects below the threshold of detection but rather are
attributable to an acute requirement for Leo in learning-memory. This
conclusion is further supported by the reversible rescue of the
learning deficit exhibited by lethal homozygotes and heteroallelics. In
contrast to leo23 and
leoX1 mutants, which lack Leo in mushroom
body and ellipsoid body neurons, minimally rescued animals contain a negligible amount of Leo throughout their
heads. This lack of Leo in lethal homozygotes and heteroallelics should
exaggerate putative developmental or sensory deficits present in
leo23 and
leoX1. However, neither sensory deficits
nor anatomical aberrations were detectable in the later, despite the
lack of transgene induction in larval or pupal stages. Therefore,
either the 10%-15% residual Leo suffices for normal larval development
and the reorganization of the brain at pupariation or Leo isoforms are
not required for these processes. Because transgene induction and Leo
accumulation restores the learning deficit of the lethal alleles to
control levels, but this recovery is eliminated during the decay of the
protein, Leo is acutely necessary for learning (Philip, 2001).
Involvement of 14-3-3 proteins in multiple cellular processes may be at
least in part the result of multiple isotypes or isoforms present
within one cell. This may
be of particular importance in vertebrates in which nine isotypes
exhibit primarily overlapping expression patterns, especially in
neuronal tissues. Similarly, because Leo isoforms and D14-3-3epsilon
appear to be at least partially overlapping, heterodimerization among
the three 14-3-3 proteins is possible. In fact, genetic analysis
suggested interactions between leonardo and D14-3-3epsilon gene products critical to embryonic and eye development (Philip, 2001).
The distinct expression of leo transcripts in adult
ellipsoid body and thorax indicates that LeoI and LeoII may have
isoform-specific functions in these tissues and suggest that structural
differences between the two isoforms may be reflected in functional
specificity. The two Leo isoforms differ by five amino acids in the
variable sixth alpha helix. The first two unique amino acids in LeoII (K, N in place of Q, T) are never found at that position among metazoan 14-3-3 isotypes. The
third substitution (E in place of D) is present in the vertebrate zeta,
beta, tau, eta and sigma isotypes and the two Caenorhabditis
elegans isotypes. Finally, the last two amino acids (A, T in place
of S, G) are present in both yeast isotypes but not among animal 14-3-3s. Thus, the LeoII isoform appears to be a unique zeta isotype. Helix 6 does not appear to be involved in phosphopeptide binding or dimerization. It is unclear then whether the differences between LeoI and LeoII result in differential dimerization or substrate engagement. The mushroom bodies apparently contain both
LeoI and LeoII isoforms and the ellipsoid body contains only LeoI.
However, both isoforms rescue equally the olfactory learning and memory
deficits of leo mutants; thus, they do not appear to have
isoform-specific functions pertinent to these processes. Alternatively,
subtle functional differences may have been concealed by elevated
transgene expression and the accumulation of a single isoform in the
mushroom bodies (Philip, 2001).
Collectively, the data indicate that monomers and homodimers of either
Leo isoform and/or heterodimers with D14-3-3epsilon are capable of similar
physiological roles essential for learning and memory. The results
demonstrate that Leo proteins do not contribute to postembryonic developmental processes in the brain. This is expected to enable investigation and identification of signaling molecules engaged by each isoform in the adult mushroom body and ellipsoid body, which in turn may reveal functional differences among them. The role of Raf1 and the Ras/Raf cascade, which requires leonardo function for signaling in developmental processes, is of particular interest. Furthermore, these results establish an acute role for 14-3-3 proteins in behavioral neuroplasticity, and, by virtue of the high degree of conservation and similarly elevated neuronal expression, are directly applicable to 14-3-3 function in vertebrates (Philip, 2001).
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).
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
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. 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, 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. 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. It is representative because the two fly genes belong to the two different 14-3-3 conservation groups. 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, apparently present in all developmental stages and tissues examined with only slight enrichment in the adult brain (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. 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, 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. 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 (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, a lethal deficiency uncovering this gene was used to show its involvement in Raf-mediated developmental processes in the embryo. 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. Third, the dynamic expression pattern of 14-3-3's during embryonic development and larval and adult nervous systems 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. 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). 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, 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. 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. 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). 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).
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