14-3-3基因家族在斑馬魚胚胎發育之表現及其過度表現所引發之神經纖維生長

Abstract

14-3-3是一種酸性蛋白，大小約25-30kDa，主要存在於腦部和神經性統中。它的功能廣泛，包含代謝，細胞週期的控制，訊息傳導，細胞凋亡，蛋白運送，轉錄，壓力反應及惡性轉型等。目前已知它和超過兩百種的受質結合，涵蓋細胞內幾乎所有的反應。本實驗根據其它物種比對所得之14-3-3序列來選殖斑馬魚14-3-3基因，並且收集資料庫中之其它已知的斑馬魚14-3-3序列，最後得到九種斑馬魚14-3-3蛋白家族成員，包括ζ，θ，β1，β2，β3，η，γ，ε1，以及ε2。經過胺基酸排序分析，親緣演化樹分析，基因結構分析，以及成對比較之結果，推論ζ，θ，β1，β2，及β3親緣關係較接近，屬於第一群；η和γ為第二群；ε1及ε2則為第三群。利用全胚胎原位雜交法分析斑馬魚14-3-3家族之mRNA在受精後六小時到一百二十小時的胚胎表現分佈位置，結果發現它們在腦部之表現量最多，其次則是視網膜，鰓，以及腸道。將斑馬魚14-3-3的九個基因以顯微注射方式表現在斑馬魚早期胚胎中，結果在受精後48小時，只有14-3-3 β3可觀察到明顯的神經纖維生長現象。將14-3-3 β3作R56A及R60A雙突變以破壞14-3-3和受質的結合，或是將14-3-3 β3與蛋白質去磷酸酶PP2A一同表現以使受質去磷酸化，均可有效抑制神經纖維生長的情形。而隨著MEK (MAPKK) 抑制劑PD98059的增加，神經纖維生長的抑制越明顯。將斑馬魚的Rho GTPase家族中的Rac-1及Cdc42均作T17N突變，和14-3-3 β3一同注射到斑馬魚胚胎中，也是隨著注射劑量增加，抑制神經纖維生長的效果越明顯。故由以上結果，推測斑馬魚14-3-3 β3和受質結合後，是經由MEK及MAPK路徑而使神經纖維生長，然而Rac-1及Cdc42在路徑中作用的位置則尚未明瞭。

14-3-3 family proteins are adaptor proteins that specifically bind to a discrete phosphoserine or phosphothreonine motif in the substrate proteins. They play important roles in metabolism, cell-cycle control, signal transduction, apoptosis, protein trafficking, transcription, stress responses, and malignant transformation. They are found only in eukaryotes. In this study, I cloned and characterized 9 genes encoding nine members of zebrafish 14-3-3 family, designated theta (θ), zeta (ζ), beta1 (β1), beta2 (β2), beta3 (β3), eta (η), gamma (γ), epsilon1 (ε1), epsilon2 (ε2). Amino acid sequence alignment and phylogenetic tree analysis indicated that all zebrafish 14-3-3 members can be divided into three group. The ζ, θ, β1, β2 and β3 belong to Group I, while η and γ Group II. In addition, ε1 and ε2 are the third. Using whole-mount in situ hybridization to analyze the mRNA expression profile of zebrafish 14-3-3 family from 6 hpf to 120 hpf, the signal was found primarily in the brain while moderate in retina, gill and gut. Expression of GFP-fusion protein of each isoform of zebrafish 14-3-3 family by a neuron-specific HuC promoter in zebrafish embryos indicated that only 14-3-3-beta3 isoform could promote neurite outgrowth. A double mutant of 14-3-3-beta3 (R56A and R60A) abolished this effect. Coinjection of pHuC-14-3-3-beta3-HuC-GFP with protein phosphatase 2A (PP2A), Cdc42 dominant negative form (Cdc42-T17N), Rac1 dominant negative form (Rac1-T17N) and MAPKK inhibitor PD98059, respectively, reduced the neurite outgrowth induced by 14-3-3-beta3.