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A novel 14-3-3 gene is osmoregulated in gill epithelium of the euryhaline teleost Fundulus heteroclitus

Dietmar Kültz^1,2,*, Devulapalli Chakravarty¹ and Tadepalli Adilakshmi¹

¹ The Whitney Laboratory, University of Florida, 9505 Ocean Shore Boulevard, St Augustine, FL 32080, USA and
² Mount Desert Island Biological Laboratory, Old Bar Harbor Road, PO Box 35, Salisbury Cove, ME 04672, USA

View larger version (65K): [in a new window]   Fig. 1. Nucleotide (black) and deduced amino acid (red) sequences of the 14-3-3.a gene from the euryhaline teleost Fundulus heteroclitus. The nucleotide sequence is numbered on the left side. The partial 5' UTR (34 nucleotides), the complete CDS (741 nucleotides plus 3 nucleotides for the stop codon corresponding to 247 amino acid residues), and the complete 3' UTR (881 nucleotides) are shown.

View larger version (77K): [in a new window]   Fig. 2. Alignment of the 3' UTR nucleotide sequences of Fundulus heteroclitus 14-3-3.a (Fish), Rana rugosa 14-3-3 (Frog), and Homo sapiens 14-3-3 (Human) mRNAs. Nucleotides in red are identical in all three sequences. Nucleotides in blue are identical only in two of the sequences. The polyadenylation signal is underlined in all three sequences. Nucleotide sequences are numbered on the left.

View larger version (84K): [in a new window]   Fig. 3. Radial phylogenetic tree depicting the evolutionary relationship of Fundulus heteroclitus 14-3-3.a to the seven known mammalian and some Xenopus laevis 14-3-3 isoforms. 14-3-3 proteins from different vertebrates (labeled with species name) that are of the same isoform-type (either , ß, , , , , or ) cluster together (gray background). Such clusters are labeled with the isoform-type of the corresponding 14-3-3 proteins in each cluster. F. heteroclitus 14-3-3.a (boxed in red) may be evolutionarily distinct from the 14-3-3 isoforms that are known from higher vertebrates or be a homolog of 14-3-3 or 14-3-3ß.

View larger version (95K): [in a new window]   Fig. 4. Multiple sequence alignment of the amino acid sequences of Fundulus heteroclitus 14-3-3.a (in bold) and all seven isoforms of 14-3-3 from mammals (Mus musculus). Amino acid residues that are identical in all the 14-3-3 proteins are shown on gray background. The locations of helices 3, 5, 7 and 9 are indicated by black bars above the corresponding residues. These regions, which form the amphipathic substrate binding groove, are highly conserved in their amino acid sequence. Important domains are indicated by colored background: blue, protein kinase C phosphorylation site; purple, casein kinase II phosphorylation site; green, protein kinase A phosphorylation site; orange, tyrosine kinase phosphorylation site; yellow, Asn glycosylation site. Please note that the corresponding residues are colored in the 14-3-3ß sequence when two sites utilize the same residues in the 14-3-3.a sequence.

View larger version (52K): [in a new window]   Fig. 5. Three-dimensional structure model of Fundulus heteroclitus 14-3-3.a. The protein structure is shown from Ser 4 to Asn 234 as a ribbon model with gray loop regions and colored helices. The first 3 amino acids of the amino terminus and the last 13 amino acids of the carboxy terminus are not shown because they are too different from the 14-3-3 proteins that have been used as templates during the modeling process. Helices are labeled (H1–H9). H9 corresponds to the 14-3-3 signature motif 2 (S2). Red helices form the outer coat of the protein while the four green helices (H3, H5, H7, H9) form the amphipathic substrate binding groove. The structural arrangement of the substrate binding groove is highly conserved in 14-3-3.a, suggesting that it binds the same set of phosphorylated proteins as other 14-3-3 proteins despite its overall distinct primary structure.

View larger version (39K): [in a new window]   Fig. 6. Tissue-specific and osmotic regulation of 14-3-3.a gene transcription. 14-3-3.a mRNA abundance in various tissues of Fundulus heteroclitus was measured using northern blot analysis. (A) Northern blot of 14-3-3.a from gill epithelium RNA of F. heteroclitus acclimated to FW, SW and 2xSW for 24h. Below the 14-3-3.a mRNA blot are the corresponding bands for the two rRNAs that were used for normalization. (B) Northern blot of 14-3-3.a with samples from different tissues of F. heteroclitus acclimated to SW. Below the 14-3-3.a mRNA blot are the corresponding bands for the two rRNAs that were used for normalization. (C) Quantification of 14-3-3.a mRNA expression in gill epithelium of F. heteroclitus acclimated to various salinities for 24h. Values are normalized for 14-3-3.a mRNA abundance in SW fish (=1) that represent the pre-acclimation conditions. Values are means ± S.E.M., N=4; an asterisk indicates a significant difference from SW data. (D) Quantification of the effect of salinity acclimation on 14-3-3.a mRNA abundance in different tissues of F. heteroclitus. Data are normalized for 14-3-3.a mRNA abundance in SW fish (=1). Values are means ± S.E.M., N=4, except for testes and ovary where N=2. An asterisk indicates a significant difference from SW data.

View larger version (26K): [in a new window]   Fig. 7. Abundance of F. heteroclitus 14-3-3 protein in gill epithelium of fish acclimated to either SW or for 24h or 48h to FW. (A) Example of a western blot developed with an antibody against a synthetic peptide that is 100% conserved in the N terminus of 14-3-3.a. This antibody recognizes two bands with apparent molecular mass of 28kDa and 29kDa. The abundance of both 14-3-3 bands increases after transfer of fish from SW to FW. (B) Quantification of 14-3-3 in gill epithelium of F. heteroclitus acclimated to SW and after transfer to FW. DVU, densitometric volume units; values are means ± S.E.M.; N=4. An asterisk indicates a significant difference to the respective 14-3-3 band in the SW sample (P<0.05).