MECHANISM OF MULTIHORMONAL REGULATION OF RAT GENE 33

大鼠基因33的多激素调节机制

• 批准号: 6107345
• 负责人: CARMEN LYDIA CADILLA
• 金额: $ 12.07万
• 依托单位: UNIVERSITY OF PUERTO RICO MED SCIENCES
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6107345
• 关键词: SDS polyacrylamide gel electrophoresis; affinity chromatography; biological signal transduction; cell growth regulation; chimeric proteins; clone cells; cyclic AMP; gene expression; growth factor; hormone regulation /control mechanism; immunofluorescence technique; laboratory rabbit; nucleic acid sequence; polymerase chain reaction; protein structure function; protein tyrosine kinase; regulatory gene; transfection; western blottings

The long term objective of this research project is to contribute to a better understanding of the mechanisms by which growth factor act to control growth in mammalian cells. The specific gene to be studied, g33, has been cloned and characterized at the sequence level. This gene has many interesting properties, including widespread tissue distribution (6, and L.A. Balogh, K._L. Lee, and F.T. Kenney, unpublished observations) and inducibility, and the potential for producing two discrete protein products. The function of the g33 proteins has remained elusive. Sequence analysis of the predicted protein sequence revealed that g33 protein contains several regions that are similar to functional or regulatory domains of other known proteins. Using amino acid sequence derived from the PROSITE database we compared g33 protein (PIR entry number S03116) to numerous entries in PROSITE using the PROSIS protein analysis software. We found several potential PKC phosphorylation sites, casein kinase II phosphorylation sites. cAMP/cGMP dependent kinase phosphorylation site, N- myristolation site, tyrosine sulfatation site, a region similar to a DEAD box, and a Class I SH3 domain- binding site. No SH3 or SH2 domains or SH2 domain binding sites we found. The strongest homology to protein sequences in the NCB1 database found using the BLAST algorithm is tot he human non- receptor tyrosine kinase called ACK that inhibits the GTPase activity of p21 cdc42 (23), and the homology is restricted to portions of the C- terminal half of the protein including a Class I SH3 domain binding site, and do not include the kinase catalytic domain, the SH3 domain or the Cdc42Hs binding site. The region in g33 protein where the Class I SH3- domain binding site is located has some homology to the ACK protein sequence, which also possesses a Class I SH3 domain binding site. Other homologies that come out in the protein sequence analysis are to proline-rich protein, but the alignments are poor and scattered and restricted mainly to the proline rich regions. The biological function of the gene 33 protein products cannot be inferred from the amino acid sequence and needs to be elucidated experimentally. G33 cDNAs were re- isolated by two different research groups that study genes whose expression increases independent of de novo protein synthesis during the transition from quiescence to proliferation. Such genes, known as immediate-early genes, are postulated to play regulatory roles in the growth response. G33 sequences were cloned from regenerating rat liver cDNA library and from serum-stimulated NIH 3T3 cells, and was shown to function as an immediate early gene in regenerating liver and in mitogen treated H-35 and Balb/C 3T3 cells. These results suggest that g33 participates in the transition from quiescence to proliferation in many mitogen-treated cells in addition to its previously reported involvement in hormone responses. The widespread and perhaps ubiquitous tissue distribution of g33 suggests that this gene may participate in a number of different responses perhaps as a member of a common signal transduction pathway. The presence of possible post- transcriptional modification sites (such as PKC and cAMP dependent protein kinase sites) and protein-protein interaction domains such as the SH3 domain binding site give strength to these hypotheses and suggest experimental targets for future studies on g33 function. In order to analyze in depth the function and expression of this multihormonally- regulated gene, we will express g33 protein in three different expression vectors in order to prepare polyclonal antibodies for analysis of g33 expression at the protein level; express g33 protein as a green fluorescent protein fusion protein to study the localization of g33 protein within the cell in vivo and if it varies in response to known mRNAg33 regulators; and to determine whether g33 protein interacts with other cellular proteins through its SH3 domain binding site or other regions, and whether protein phosphorylation plays a role in those interactions. The proposed project will enable students to be trained in basic research techniques and to give them an experience in studying the effects of structure on function, which will serve as a catalyst for a future research career in biomedical sciences. The students will involve themselves in all aspects of scientific research.

该研究项目的长期目标是为 更好地了解生长因子的作用机制 控制哺乳动物细胞的生长。 要研究的具体基因，g33， 已在序列水平上进行克隆和表征。 这个基因有 许多有趣的特性，包括广泛的组织分布（6， 和 L.A. Balogh，K._L。李和 F.T.肯尼，未发表的观察）和 诱导性，以及产生两种离散蛋白质的潜力 产品。 g33 蛋白的功能仍然难以捉摸。 顺序 对预测的蛋白质序列的分析表明，g33 蛋白质 包含几个与功能或监管相似的区域 其他已知蛋白质的结构域。 使用衍生自的氨基酸序列 我们将 PROSITE 数据库中的 g33 蛋白（PIR 条目号 S03116）与 PROSITE 中使用 PROSIS 蛋白质分析软件的大量条目。 我们 发现了几个潜在的 PKC 磷酸化位点，酪蛋白激酶 II 磷酸化位点。 cAMP/cGMP 依赖性激酶磷酸化位点，N- 肉豆蔻酰化位点，酪氨酸硫酸化位点，类似于DEAD的区域 框和 I 类 SH3 域结合位点。 无 SH3 或 SH2 域或 SH2 我们发现的域结合位点。 与蛋白质序列最强的同源性 在NCB1数据库中发现使用BLAST算法是人类非 受体酪氨酸激酶称为 ACK，可抑制 GTP 酶活性 p21 cdc42 (23)，同源性仅限于 C- 的部分 蛋白质的末端一半包括 I 类 SH3 结构域结合位点， 并且不包括激酶催化结构域、SH3 结构域或 Cdc42Hs 结合位点。 g33 蛋白中 I 类 SH3- 所在的区域 结构域结合位点所在位置与 ACK 蛋白有一定的同源性 序列，还具有 I 类 SH3 结构域结合位点。 蛋白质序列分析中出现的其他同源性是 富含脯氨酸的蛋白质，但排列较差且分散， 主要限于脯氨酸丰富的区域。 的生物学功能 基因33的蛋白质产物不能从氨基酸推断出来 的顺序，需要通过实验来阐明。 G33 cDNA 被重新 由两个不同的研究小组分离出来，研究其表达的基因 在转变过程中增加独立于从头蛋白质合成 从静止到增殖。 这种基因被称为即早基因 基因被认为在生长反应中发挥调节作用。 G33 从再生大鼠肝脏 cDNA 文库中克隆序列 血清刺激的 NIH 3T3 细胞，并被证明可以作为立即 再生肝脏和经丝裂原处理的 H-35 和 Balb/C 3T3 中的早期基因 细胞。 这些结果表明 g33 参与了从 除了许多经有丝分裂原处理的细胞中的增殖静止 先前报道其参与激素反应。 广泛流传的 也许 g33 无处不在的组织分布表明该基因 可能作为成员参与许多不同的反应 共同的信号转导途径。 可能存在的后 转录修饰位点（例如 PKC 和 cAMP 依赖性蛋白 激酶位点）和蛋白质-蛋白质相互作用结构域，例如 SH3 域结合位点增强了这些假设并表明 g33功能未来研究的实验目标。 为了 深入分析这种多激素的功能和表达 调控基因，我们将以三种不同的表达方式表达g33蛋白 载体以制备用于 g33 分析的多克隆抗体 蛋白质水平的表达；将 g33 蛋白表达为绿色荧光 蛋白质融合蛋白，用于研究 g33 蛋白在 体内细胞以及它是否响应已知的 mRNAg33 调节剂而变化；和 确定 g33 蛋白是否与其他细胞蛋白相互作用 通过其 SH3 结构域结合位点或其他区域，以及蛋白质是否 磷酸化在这些相互作用中发挥作用。 拟议的项目将使学生能够接受基础研究方面的培训 技术并给予他们研究影响的经验 功能结构，这将成为未来研究的催化剂 生物医学科学的职业生涯。 学生将全身心投入 科学研究的各个方面。