Protein phosphatase 6 regulatory subunits composed of ankyrin repeat domains
Protein phosphatase 6 regulatory subunits composed of ankyrin repeat domains
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DOI：
10.1021/bi7022877
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发表时间：
2008-02-05
期刊：
影响因子：
2.9
通讯作者：
Brautigan, David L.
中科院分区：
文献类型：
--
作者：
Stefansson, Bjarki;Ohama, Takashi;Brautigan, David L.
蛋白磷酸酶6(PP6)是真核生物中保守的一种重要的丝氨酸/苏氨酸磷酸酶。酿酒酵母PP6的同源物Sit4依赖于与SAPS结构域亚单位的结合。本研究使用人SAPS结构域亚单位标志-PP6R1来鉴定内源相互作用蛋白。质谱仪鉴定共沉淀蛋白为PP6催化亚基和3个锚定蛋白重复蛋白(Ankrd28、Ankrd44和Ankrd52)。这些蛋白质彼此具有广泛的序列一致性，但在脊椎动物物种的系统发育树上分成不同的分支，表明了各自的生物学功能。标记的Ankrd28与PP6共沉淀，而不与PP2A或PP4以及SAPS结构域亚单位PP6R1和PP6R3共沉淀。标记的PP6共沉淀内源SAPS结构域亚单位和Ankrd28。PP6R1的C-末端区域足以共沉淀Ankrd28，但不能共沉淀PP6，表明PP6R1作为支架，有单独的区域与PP6和Ankrd28结合。含有PP6R1和PP6R3亚基的内源PP6全酶被DEAE层析分离，并与Ankrd28一起在M-r&Gt；440 kDa处从Superose 12中洗脱。敲除PP6R1或Ankrd28，而不是PP6R3，产生了同样的促进I kappa B epsilon降解的反应。结果表明，PP6具有杂三聚体的功能，由PP6催化亚基结合到与Ankrd28相关的SAPS结构域支架亚基组成。我们认为，SAPS和锚蛋白重复调控亚基决定了PP6的功能和特异性。
Protein phosphatase 6 (PP6) is an essential Ser/Thr phosphatase conserved among eukaryotes. The Saccharomyces cerevisiae homologue of PP6 called Sit4 depends on association with SAPS domain subunits. This study used a human SAPS domain subunit FLAG-PP6R1 to identify endogenous interacting proteins. Mass spectrometry identified coprecipitating proteins as PP6 catalytic subunit and three ankyrin repeat proteins (Ankrd28, Ankrd44, and Ankrd52). These proteins have extensive sequence identity to one another but segregate into separate branches on a phylogenetic tree for vertebrate species, suggesting individual biological functions. Tagged Ankrd28 coprecipitated with PP6, not with PP2A or PP4, and with SAPS domain subunits PP6R1 and PP6R3. Tagged PP6 coprecipitated endogenous SAPS domain subunits and Ankrd28. The C-terminal region of PP6R1 was sufficient to coprecipitate Ankrd28, but not PP6, demonstrating that PP6R1 acts as a scaffold with separate regions for binding to PP6 and to Ankrd28. Endogenous PP6 holoenzymes with PP6R1 and PP6R3 subunits were resolved by DEAE chromatography and eluted together with Ankrd28 at M-r > 440 kDa from Superose 12. Knockdown of PP6R1 or Ankrd28, but not PP6R3, produced equivalent enhancement of I kappa B epsilon degradation in response to TNF alpha. The results suggest that PP6 functions as a heterotrimer, composed of the PP6 catalytic subunit bound to a SAPS domain scaffold subunit that associates with Ankrd28. We propose that the SAPS and ankyrin repeat regulatory subunits determine the function and specificity of PP6.