A kinase anchoring protein (AKAP) interaction and dimerization of the RIα and RIβ regulatory subunits of protein kinase A in vivo by the yeast two hybrid system
A kinase anchoring protein (AKAP) interaction and dimerization of the RIα and RIβ regulatory subunits of protein kinase A in vivo by the yeast two hybrid system
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DOI：
10.1016/s0022-2836(03)00093-7
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发表时间：
2003-03-28
影响因子：
5.6
通讯作者：
Taskén, K
中科院分区：
文献类型：
--
作者：
Carlson, CR;Ruppelt, A;Taskén, K
蛋白激酶A （PKA）调控亚基通过n端基序二聚化。这种二聚化对于结合PKA锚定蛋白（AKAPs）和将PKA靶向到其作用位点是必要的。在本研究中，我们使用酵母双杂交系统作为体内生物报告者实验，分析了riα和riβ的同源和异源二聚体复合物的形成以及RI二聚体的AKAP结合。酵母提取物的天然聚丙烯酰胺凝胶电泳（PAGE）证实了双杂交的数据。观察到riα -和riβ同型二聚体以及riα: riβ异源二聚体。在riα和riβ亚基中引入单、双和一个三重突变，分析突变体的二聚化特性。与先前的报道一致，RIalpha（C37H）二聚化，尽管二硫桥被破坏，而F47或F52的额外突变消除了二聚化。RIbeta中相应的突变（C38H, F48A, F53A）不足以消除RIbeta二聚化，表明额外或其他氨基酸很重要。riα：突变体的riβ异源二聚体在中等强度下形成。通过酵母双杂交系统对三元配合物进行分析，发现RIalpha和RIbeta同型二聚体以及RIalpha:RIbeta异源二聚体和一些突变体能够结合到AKAP149/D-AKAP1的r结合结构域。此外，将RIbeta引入HEK293细胞后，鉴定出RIbeta: AKAP149复合物。重要的是，RIbeta显示出与RIalpha相似的AKAP结合特性，表明RIbeta全酶可能是锚定的。2003爱思唯尔科学有限公司版权所有。
Protein kinase A (PKA) regulatory (R) subunits dimerize through an N-terminal motif. Such dimerization is necessary for binding to PKA anchoring proteins (AKAPs) and targeting of PKA to its site of action. In the present study, we used the yeast two-hybrid system as an in vivo bioreporter assay and analyzed the formation of homo- and heterodimeric complexes of RIalpha and RIbeta as well as AKAP binding of RI dimers. Native polyacrylamide gel electrophoresis (PAGE) of yeast extracts confirmed the two-hybrid data. Both RIalpha- and RIbeta homodimers as well as an RIalpha:RIbeta heterodimer were observed. Single, double and one triple mutation were introduced into the RIalpha and RIbeta subunits and dimerization properties of the mutants were analyzed. Consistent with previous reports, RIalpha(C37H) dimerized, although the disulfide bridges were disrupted, whereas the additional mutation of F47 or F52 abolished the dimerization. Corresponding mutations (C38H, F48A, F53A) in RIbeta were not sufficient to abolish the RIbeta dimerization, indicating that additional or other amino acids are important. RIalpha:RIbeta heterodimers of the mutants were formed at intermediate stringency. Analysis of ternary complexes by the yeast two-hybrid system revealed that RIalpha and RIbeta homodimers as well as an RIalpha:RIbeta heterodimer and several of the mutants were able to bind to the R-binding domain of AKAP149/D-AKAP1. Furthermore, an RIbeta: AKAP149 complex was identified following introduction of RIbeta into HEK293 cells. Importantly, RIbeta revealed AKAP binding properties similar to those of RIalpha, indicating that RIbeta holoenzymes may be anchored. (C) 2003 Elsevier Science Ltd. All rights reserved.