Gene name - 14-3-3zeta
Synonyms - leonardo, Par-5
Cytological map position - 46E1--46E9
Function - phosphoserine/threonine interacting protein
Symbol - 14-3-3zeta
Genetic map position - 2-
Classification - 14-3-3-protein
Cellular location - cytoplasmic
14-3-3 proteins carry out numerous physiological functions in a variety of species. Leonardo is one of two known Drosophila isoforms. In vitro the 14-3-3 proteins activate tyrosine and tryptophan hydroxylases, the rate-limiting enzymes in the biosynthesis of catecholamines and serotonin respectively. They function in Ca2+ regulated exocytosis, cell cycle control and in protein kinase C (PKC) regulation. They associate physically with PI-3 kinase, CDC25 phosphatase (see Drosophila String), and RAF-1 kinase. The association with this apparently diverse group of proteins may be due to the intriguing ability of 14-3-3 to bind to residues sites phosphorylated on serine or threonine residues. Their interaction with diverse signaling proteins suggests that these proteins function as modulators of activity or specificity of various kinases, or as coordinators for the assembly of signaling complexes for different cascades (Sokoulakis, 1996).
The two Drosophila 14-3-3 isoforms, Leonardo (14-3-3zeta) and 14-3-3eta, have been shown to positively regulate Ras-mediated signaling in the development of the compound eye. 14-3-3eta was identified as a suppressor of activated Ras1 in eye development. Suppressor of Ras1 3-9 (SR3-9) alleles act as dominant suppressors of sev-Ras1, that is, of Ras1 expressed ectopically in the eye disc using a sevenless promoter, a treatment that produces a rough eye phenotype. The eyes of flies carrying SR3-9 are less rough than those of flies expressing sev-Ras1. It is known that SR3-9 functions either downstream of or in parallel to Raf kinase, since SR3-9 dominantly suppresses the rough eye defect caused by an activated raf expression construct (Chang, 1997).
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. When Leonardo antisense RNA is expressed in eye imaginal discs in postmitotic photoreceptors, a weakly penetrant but reproducible loss-of-photoreceptor phenotype results. Since the artificial activation of Raf rescues the nonviability caused by leonardo mutation and permits photoreceptor development, it has been 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).
Of particular interest is the involvement of leonardo in facilitating olfactory learning in the fly. leonardo was identified in an enhancer detector screen for genes preferentially expressed in mushroom bodies of the brain: a lacZ reporter construct was found to have been inserted in a gene expressed in mushroom bodies, the seat of olfactory learning in insects. Isolation of multiple insertion strains was carried out to obtain viable leonardo mutant alleles. All the viable alleles were found to be confined to genomic excisions of insertions in the first intron. Since introns do not code for the primary sequence of proteins but instead are involved in splicing or carry regulatory sequences, this suggests that these excisions disrupt regulatory elements necessary for mushroom body expression or RNA splicing (Skoulakis, 1996).
To examine associative learning in these flies, a Pavlovian olfactory conditioning assay was used (see Dunce for a more complete description of this procedure). Briefly, flies are trained by exposure to electroshock paired with one odor (octanol or methylcyclohexanol) and subsequently exposed to a second odor without electroshock. Immediately after training, learning is measured by forcing flies to choose between the two odors used during training. No preference between odors results in a performance index of zero (no learning), as is the case for naive flies. Avoidance of the odor previously paired with electroshock, however, yields a performance index greater than O. Alleles of leonardo classed as modest by immunohistochemical criteria (meaning that there is a decrease in Leonardo protein relative to the controls, but that some remains) exhibit a 20-25% decrease in 3 minute memory (as compared to non-mutant controls), whereas alleles nearly devoid of Leo in mushroom bodies exhibit a increased decrement of 30-35% relative to the controls. It is concluded that Leo is involved in olfactory learning in flies (Skoulakis, 1996).
Longer-term memory of the conditioned association was assessed for a selected set of mutants at 45, 90 and 240 min after training. One excision mutant exhibited a 30% reduction in 3 minute memory and retained this highly significant difference over time through four hours. Other leo insertion mutants exhibit significant differences for initial memory and at 45 min, although their performance at 90 and 240 min was not significantly different from that of controls. As reduction in leonardo expression does not precipitate developmental abnormalites in the brain anatomy, it is concluded that the behavioral deficits observed do not have an anatomical basis, but instead involve deficits in signal processing (Skoulakis, 1996).
Since 14-3-3 proteins are not known to participate in the cAMP cascade, the results suggest that Leo protein is a member of an additional signaling cascade that mediates learning and memory. This function of Leonardo is consistent with its role in protein kinase C-mediated processes or alternatively in an interaction with Raf-1 in the mitogen-activated protein kinase (MAPK) signal transdution cascade (Skoulakis, 1996). How does it happen that the reduction of the leonardo gene product does not cause proliferation or differentiation defects in the mushroom bodies? leonardo codes for an adult-specific 2.9-kb splicing variant that is strongly enriched or even exclusively expressed in the head. Thus, it might be possible that the different splice variants operate in different signal transducton pathways not necessarily linked to Raf function. Alternatively, the strong reduction in leonardo expression may affect only acquisition of memory, whereas low residual levels of expression are sufficient to mediate the cellular aspects of differentiaton and proliferation, processes that are severely affected in the lack-of-function mutations (Kockel,1997).
Five different types of Leonardo cDNAs have been isolated. The transcripts differ by the alternative use of exons I or I' and VI or VI' as well as by use of three different alternative sites of addition of poly(A) onto the 3' terminus of the mRNA. Switching of exons VI and VII' affects the open reading frame and causes sequence differences in the respective translation products (Kockel, 1997).
Bases in 3' UTR - 309 (1.3 kb transcript) (Swanson, 1992)
A cloned 1.3-kb cDNA hybridizes to genomic clone 549, containing genes predominantly expressed in the head of Drosophila melanogaster. DNA sequencing shows that the cDNA-encoded protein is similar to a family of mammalian proteins, called 14-3-3, which activate tyrosine hydroxylase (TyrOHase) and tryptophan hydroxylase (TrpOHase), the two key enzymes regulating biosynthesis of biogenic monoamine neurotransmitters, such as dopamine and serotonin, in the brain. The putative D. melanogaster 14-3-3 protein (D14-3-3) shares 72.4, 74.3 and 78.3% amino acid (aa) sequence identity and 83.5, 87.7 and 85.9% aa sequence similarity with the beta, gamma and eta forms of bovine 14-3-3 protein, respectively. A lower (71%), but significant level of aa sequence identity was also found between D14-3-3 and sheep brain protein kinase C inhibitor protein (KCIP) (Swanson, 1992).
The variant amino acids caused by the switching of exons VI and VI' are predicted to lie in alpha-helix 6 on the outside of the groove-shaped 14-3-3 dimer. Interestingly, helix 6 is composed of the sequences that are least conserved throughout the 14-3-3 protein family. This suggests that helix 6 might confer specificity to 14-3-3 interactions with target proteins. Both exons VI and VI' encode a potential phosphorylation site characterized previously in mammalian 14-3-3 beta and zeta (Kockel, 1997).
Multiple alignments were constructed from forty-six 14-3-3 sequences retrieved from the GenBank and SwissProt databases. The alignments constructed reveal five highly conserved sequence blocks. Blocks 2-5 correlate well with the alpha helices 3, 5, 7, and 9, which form the proposed internal binding domain in the three-dimensional structure model of the functioning dimer. Amino acid differences within the functional and structural domains of plant and animal 14-3-3 proteins were identified, which may account for the functional diversity among isoforms. Epsilon isoforms from the animal lineage form a distinct grouping, which suggests an early divergence from the other animal isoforms. Epsilons were found to be more similar to yeast and plant isoforms than other animal isoforms at numerous amino acid positions, and thus epsilon may have retained functional characteristics of the ancestral protein. The known invertebrate proteins are most properly grouped with the nonepsilon mammalian isoforms. Most of the current 14-3-3 isoform diversity probably arose through independent duplication events after the divergence of the major eukaryotic kingdoms. Divergence of the seven mammalian isoforms beta, zeta, gamma, eta, epsilon, tau, and sigma occurred before the divergence of mammalian and perhaps before the divergence of vertebrate species. A possible ancestral 14-3-3 sequence has been proposed (W. Wang, 1996).
date revised: 4 June 97
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