Inositol 1,4,5-Triphosphate Receptor-binding Protein Released with Inositol 1,4,5-Triphosphate (IRBIT) Associates with Components of the mRNA 3′ Processing Machinery in a Phosphorylation-dependent Manner and Inhibits Polyadenylation
Inositol 1,4,5-Triphosphate Receptor-binding Protein Released with Inositol 1,4,5-Triphosphate (IRBIT) Associates with Components of the mRNA 3′ Processing Machinery in a Phosphorylation-dependent Manner and Inhibits Polyadenylation
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DOI：
10.1074/jbc.m807136200
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发表时间：
2009-04-17
影响因子：
4.8
通讯作者：
Mikoshiba, Katsuhiko
中科院分区：
文献类型：
--
作者：
Kiefer, Helene;Mizutani, Akihiro;Mikoshiba, Katsuhiko
IRBIT是新近发现的一种蛋白质，可调节肌醇1，4，5-三磷酸受体和胰脏Na+/HCO3-协同转运蛋白1的活性，而IRBIT的多位点磷酸化是实现这一调节作用所必需的。在这里，我们报告了切割和多聚腺苷酸化特异性因子(CPSF)的鉴定，CPSF是一种参与mRNA前体3‘加工的多蛋白复合体，作为IRBIT的额外结合伙伴。我们发现Irbit与CPSF相互作用，并被募集到含有外源多聚腺苷信号的RNA中。IRBIT在CPSF中的主要靶点是Fip1亚基，为了在体外与Fip1直接结合以及将Fip1重新分布到完整细胞的细胞质中，IRBIT富含丝氨酸区的磷酸化是必需的。此外，氧化应激诱导剂叔丁基对苯二酚(TBHQ)可增加体内IRBIT的磷酸化水平，同时增强IRBIT与CPSF的相互作用，促进内源性Fip1的胞质分布。除CPSF外，Irbit还在体外与聚(A)聚合酶(PAP)相互作用，PAP是CPSF招募的延长聚(A)尾巴的酶，并以磷酸化依赖的方式抑制PAP的活性。这些发现提出了这样一种可能性，即IRBIT通过控制Fip1的细胞质/核分配和抑制PAP活性来调节特定mRNAs的多聚腺苷酸化状态，以响应改变其磷酸化状态的刺激。
IRBIT is a recently identified protein that modulates the activities of both inositol 1,4,5-triphosphate receptor and pancreas-type Na+/HCO3- cotransporter 1, and the multi-site phosphorylation of IRBIT is required for achieving this modulatory action. Here, we report the identification of the cleavage and polyadenylation specificity factor (CPSF), which is a multi-protein complex involved in 3' processing of mRNA precursors, as an additional binding partner for IRBIT. We found that IRBIT interacted with CPSF and was recruited to an exogenous polyadenylation signal-containing RNA. The main target for IRBIT in CPSF was Fip1 subunit, and the phosphorylation of the serine-rich region of IRBIT was required both for direct association with Fip1 in vitro and for redistribution of Fip1 into the cytoplasm of intact cells. Furthermore, tert-butylhydroquinone (tBHQ), an agent that induces oxidative stress, increased the phosphorylation level of IRBIT in vivo and in parallel enhanced the interaction between IRBIT and CPSF and promoted the cytoplasmic distribution of endogenous Fip1. In addition to CPSF, IRBIT interacted in vitro with poly(A) polymerase (PAP), which is the enzyme recruited by CPSF to elongate the poly(A) tail, and inhibited PAP activity in a phosphorylation-dependent manner. These findings raise the possibility that IRBIT modulates the polyadenylation state of specific mRNAs, both by controlling the cytoplasmic/nuclear partitioning of Fip1 and by inhibiting PAP activity, in response to a stimulus that alters its phosphorylation state.