TARGETING OF A DISTINCTIVE PROTEIN-SERINE PHOSPHATASE TO THE PROTEIN KINASE-LIKE DOMAIN OF THE ATRIAL-NATRIURETIC-PEPTIDE RECEPTOR
TARGETING OF A DISTINCTIVE PROTEIN-SERINE PHOSPHATASE TO THE PROTEIN KINASE-LIKE DOMAIN OF THE ATRIAL-NATRIURETIC-PEPTIDE RECEPTOR
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DOI：
10.1073/pnas.91.23.11075
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发表时间：
1994-11-08
影响因子：
11.1
通讯作者：
CHINKERS, M
中科院分区：
文献类型：
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作者：
CHINKERS, M
未知功能的蛋白激酶相关结构域存在于蛋白酪氨酸激酶的JAK家族和受体/鸟苷酸环化酶中。我使用酵母双杂交系统筛选与心房利钠肽（ANP）受体/鸟苷酸环化酶的激酶样结构域相互作用的蛋白质。构建酵母菌株，其表达该激酶样结构域与莱克萨DNA结合结构域的融合体，并含有在莱克萨上游激活序列控制下的HIS 3基因。这些酵母细胞用与VP 16转录激活结构域融合的小鼠胚胎cDNA片段的质粒文库转化。通过在不存在组氨酸的情况下生长来选择含有与lexA激酶样结构域融合蛋白相互作用的VP 16融合蛋白的细胞。通过使用这种方法分离的部分长度的cDNA克隆编码的蛋白质，该蛋白质与ANP受体蛋白激酶样结构域在酵母细胞和体外特异性相互作用。与该cDNA杂交的2.2 kb mRNA的组织特异性表达证实了已知的ANP受体mRNA表达模式。预测从大鼠肺文库中分离的全长cDNA克隆编码55 kDa的蛋白质，其氨基末端含有与ANP受体激酶样结构域结合的靶向结构域，其羧基末端含有推定的蛋白丝氨酸磷酸酶结构域。该蛋白是参与ANP受体脱敏的磷酸酶的可能候选者。靶向调节蛋白可能是蛋白激酶样结构域的重要功能。
Protein kinase-related domains of unknown function are present in the JAK family of protein tyrosine kinases and in receptor/guanylyl cyclases. I used the yeast two-hybrid system to screen for proteins interacting with the kinase-like domain of the atrial natriuretic peptide (ANP) receptor/guanylyl cyclase. A yeast strain was constructed expressing a fusion of this kinase-like domain to the lexA DNA-binding domain and containing a HIS3 gene under the control of lexA upstream activating sequences. These yeast cells were transformed with a plasmid library of mouse embryo cDNA fragments fused to the VP16 transcriptional activation domain. Cells containing VP16-fusion proteins interacting with the lexA-kinase-like domain fusion protein were selected by growth in the absence of histidine. A partial-length cDNA clone isolated by using this approach encoded a protein that interacted specifically with the ANP-receptor protein kinase-like domain both in yeast cells and in vitro. Tissue-specific expression of a 2.2-kb mRNA hybridizing to this cDNA paralleled the known pattern of ANP-receptor mRNA expression. A full-length cDNA clone isolated from a rat lung library was predicted to encode a 55-kDa protein containing at its amino terminus a targeting domain that binds to the ANP-receptor kinase-like domain and containing at its carboxyl terminus a putative protein-serine phosphatase domain. This protein is a possible candidate for the phosphatase involved in desensitizing the ANP receptor. Targeting of regulatory proteins may be an important function of protein kinase-like domains.