Analysis of the role of the leucine zipper motif in regulating the ability of AFAP-110 to alter actin filament integrity
Analysis of the role of the leucine zipper motif in regulating the ability of AFAP-110 to alter actin filament integrity
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DOI：
10.1002/jcb.10725
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发表时间：
2004-02-15
影响因子：
4
通讯作者：
Flynn, DC
中科院分区：
文献类型：
--
作者：
Qian, Y;Gatesman, AS;Flynn, DC
AFAP-110作为肌动蛋白丝交联蛋白具有改变肌动蛋白丝完整性的内在能力。这种能力是由一个羧基末端亮氨酸拉链（Lzip）基序。Lzip基序促进自缔合稳定AFAP-110多聚体。Lzip基序（AFAP-110（Deltalzip））的缺失降低了AFAP-110多聚体的稳定性，并伴随地增加了其在体外交联肌动蛋白丝的能力，以及在体内激活cSrc并改变肌动蛋白丝完整性的能力。我们试图确定Lzip基序如何调节AFAP-110功能。预测c-Fos Lzip基序取代AFAP-110 Lzip基序（AFAP-110（fos））在改变序列的同时保留α-螺旋结构。为了改变α-螺旋的结构，在AFAP-110 α-螺旋Lzip基序（AFAP-110（581 P））中产生亮氨酸至脯氨酸的突变，这在很大程度上保留了序列。螺旋突变体，AFAP-110（Deltalzip），AFAP-110（fos），和AFAP-110（581 P），表现出降低的多聚体稳定性与增加的能力，交联肌动蛋白丝，在体外，相对于AFAP-110。相反的结合位点的分析表明，羧基末端/Lzip基序可以接触的氨基末端普列克底物蛋白同源性（PH 1）域内的序列，表明调节多聚体稳定性和肌动蛋白丝交联的自抑制机制。在体内，只有AFAP-110（Deltalzip）和AFAP-110（581 P）激活cSrc并改变细胞肌动蛋白丝的完整性。这些数据表明，AFAP-110交联肌动蛋白丝的固有能力取决于Lzip基序的序列和结构，而Lzip基序调节AFAP-110指导的cSrc活化和体内肌动蛋白丝完整性变化的能力取决于Lzip基序的结构或存在。我们假设AFAP-110交联肌动蛋白丝或激活cSrc的内在能力是不同的功能。(C)2003 Wiley-Liss，Inc.
AFAP-110 has an intrinsic ability to alter actin filament integrity as an actin filament cross I inking protein. This capability is regulated by a carboxy terminal leucine zipper (Lzip) motif. The Lzip motif facilitates self-association stabilizing the AFAP-110 multimers. Deletion of the Lzip motif (AFAP-110(Deltalzip)) reduces the stability of the AFAP-110 multimer and concomitantly increases its ability to crosslink actin filaments, in vitro, and to activate cSrc and alter actin filament integrity, in vivo. We sought to determine how the Lzip motif regulates AFAP-110 function. Substitution of the c-Fos Lzip motif in place of the AFAP-110 Lzip motif (AFAP-110(fos))was predicted to preserve the alpha-helical structure while changing the sequence. To alter the structure of the alpha-helix, a leucine to proline mutation was generated in the AFAP-110 alpha-helical Lzip motif (AFAP-110(581P)), which largely preserved the sequence. The helix mutants, AFAP-110(Deltalzip), AFAP-110(fos), and AFAP-110(581P), demonstrated reduced multimer stability with an increased capacity to crosslink actin filaments, in vitro, relative to AFAP-110. An analysis of opposing binding sites indicated that the carboxy terminus/Lzip motif can contact sequences within the amino terminal pleckstrin homology (PH1) domain indicating an auto-inhibitory mechanism for regulating multimer stability and actin filament crosslinking. In vivo, only AFAP-110(Deltalzip) and AFAP-110(581P) were to activate cSrc and to alter cellular actin filament integrity. These data indicate that the intrinsic ability of AFAP-l 10 to crosslink actin filaments is dependent upon both the sequence and structure of the Lzip motif, while the ability of the Lzip motif to regulate AFAP-110-directed activation of cSrc and changes in actin filament integrity in vivo is dependent upon the structure or presence of the Lzip motif. We hypothesize that the intrinsic ability of AFAP-110 to crosslink actin filaments or activate cSrc are distinct functions. (C) 2003 Wiley-Liss, Inc.