【作者】 张永夏；

【导师】 邢苗；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2011， 博士

【摘要】 SR蛋白（Serine/Arginine-rich protein）是一组结构相关、进化保守的non-snRNP剪接因子,在调节剪接复合体组装和调节pre-mRNA组成型剪接和选择型剪接方面发挥重要作用。多头绒泡菌（Physarum polycephalum）是原核生物与真核生物之间的过渡态真核古生物,但尚未见到多头绒泡菌SR蛋白的研究报道。本文从多头绒泡菌分离到一个SR类似蛋白cNDA,其编码蛋白的C-端含有一个富含精氨酸/丝氨酸二肽序列（Arginine/Serine motif）的结构域（RS domian）,RS domain的N-端毗邻一个SR蛋白激酶锚定结构域（Docking motif）,与其它物种SR蛋白的C-端结构类似,故将该蛋白命名为PSR（Physarum SR）。本文以[γ-³²P] ATP为底物,通过放射自显影检测了大肠杆菌（E. Coli）表达的多头绒泡菌SR蛋白激酶PSRPK（Physarum Serine/Arginine Protein-specific Kinase）对大肠杆菌表达的PSR及其残缺肽和突变肽的磷酸化作用,发现PSRPK能在体外磷酸化PSR以及拥有完整RS domain和Docking motif的PSR残缺肽。研究还发现,PSRPK磷酸化作用位点位于PSR C-端连续的RS二肽重复序列（5×RS）上,Docking motif在调节PSRPK磷酸化PSR上扮演重要角色,PSRPK的ATP结合区（ATP-binding region, ABR）序列⁶²LGWGHFSTVWLAIDEKNGGREVALK86和丝氨酸/苏氨酸激酶活性位点特征序列（serine/threonine protein kinases active- site signature, AS）⁽184(IIHTDLKPENVLL¹⁹⁶是影响PSRPK磷酸化PSR的关键序列,Lys⁸⁶和Asp¹⁸⁸分别是ABR和AS序列的关键氨基酸。进化保守的14-3-3蛋白广泛参与细胞的各种生命活动。已有的研究显示,植物14-3-3蛋白在调节SR蛋白SRp38应答热胁迫时扮演着重要角色。P14-3-3是多头绒泡菌14-3-3类似蛋白。PSR是以P14-3-3为饵蛋白分离的。本文通过FRET检测了PSR与P14-3-3在哺乳动物细胞内的相互作用,发现PSR在细胞内的分布受P14-3-3的调节,与P14-3-3共表达的PSR由聚集在细胞核转变为主要分布在细胞质。酵母双杂交检测结果显示,PSR需要保持其C-端的完整Docking motif和连续的RS重复序列才能与P14-3-3作用;而P14-3-3 C-端的217-237aa肽段和N-端的76-109aa肽段是其与PSR作用的关键序列,C-端的235TS236磷酸化位点是其与PSR作用的关键位点。更多还原

【Abstract】 SR proteins are a group of structurally related, evolutionary conserved non-snRNP splicing factors, which participate in the maturation of the splieosome. These proteins contain one or two RNA recognition motifs and a C-terminal domain rich in Arg-Ser repeats （RS domain）. SR proteins are phosphorylated at numerous serines in the RS domain by the SR-specific protein kinase （SRPK） family of protein kinases. RS domain phosphorylation is necessary for entry of SR proteins into the nucleus, and may also play important roles in alternative splicing, mRNA export, and other processing events. At present, studies regarding SR protein and SRPKs are mainly on higher eukaryotes. In our previous work, we isolated an SR protein kinase PSRPK from Physarum polycephalum, which can phosphorylate the RS domain of ASF/SF2 both in vivo and in vitro. There is no report of SR protein in P. polycephalum.The aim of this study was to clarify whether SR protein exists in lower eukaryotes, such as P. polycephalum, and to explore its phosphorylation mechanism and interacting proteins. Here, a novel cDNA encoding a serine/arginine （SR）-rich protein, designated PSR, was isolated from the true slime mold P. polycephalum by yeast two hybridization and 5’-RLM-RACE. The deduced amino acid （aa） sequence reveals that PSR contains RS repeats at its C-terminus, similar to the conventional PSRPK substrate ASF/SF2. To study the novel protein, we generated a variety of mutant constructs by PCR and site-directed mutagenesis. Autoradiography indicated that the purified recombinant PSR was phosphorylated by PSRPK in vitro, and the SR-rich domain （aa 460–469） in the PSR protein was required for phosphorylation. In addition, removal of the docking motif （aa 424–450） from PSR significantly reduced the overall catalytic efficiency of the phosphorylation reaction. We also found that the conserved ATP-binding region （ABR） ⁶²⁰LGWGHFSTVWLAIDEKNGGREVALK86 and the serine/threonine protein kinases active-site signature （AS） 184IIHTDLKPENVLL¹⁹⁶ of PSRPK played a crucial role in substrate phosphorylation, and Lys⁸⁶ and Asp¹⁸⁸ were crucial for PSRPK phosphorylation of PSR.14-3-3 proteins associate with phosphorylated SRp38 resulting in protection from dephosphorylation under non-stress conditions, but dissociate in response to heat shock （Shi and Manley, 2007）. However, little is known about how the distribution of SR proteins is affected by 14-3-3. To study the interaction between PSR and P14-3-3, we generated a variety of truncated and mutant peptides of PSR and P14-3-3 and co-expression of them in the mammalian cell strain L929 and yeast strain AH109. The distribution of PSR was affected by co-expression with P14-3-3 in the mammalian cell strain L929 using confocal microscopy. Our results demonstrated that P14-3-3 can modulate the distribution of PSR and that the integrated dock motif combined with the RS domain is necessary for PSR interactions with P14-3-3. Furthermore, the peptides 76KGNENHVKRIKEYRNKVEKELSDICQDILNVLD108 （includingα-helix D） and 217YKDSTLIMQLLRDNLTLWTSD237 （includingα-helix I and the phosphorylation site 235ThrSer236） are involved in P14-3-3 interactions with PSR. The phosphorylation site 235ThrSer236 plays a key role in regulating P14-3-3 interactions with phosphorylated PSR, and thereby modulates the distribution of PSR. This study provides further insight into the mechanism of transcriptional regulation of PSR in the true slime mold.更多还原