Tatsis N, Lannigan D A, Macara I G
Center for Cell Signaling, University of Virginia, Charlottesville, Virginia 22908, USA.
J Biol Chem. 1998 Dec 18;273(51):34631-8. doi: 10.1074/jbc.273.51.34631.
p190 is a GTPase-activating protein (GAP) for the Rho family of GTPases. The GAP domain of p190 is at the C terminus of the protein. At its N terminus, p190 contains a GTP binding domain of unknown significance. We have introduced a mutation (Ser36 --> Asn) into this domain of p190 that decreased its ability to bind guanine nucleotide when expressed as a hemagglutinin (HA)-tagged protein in COS cells. In vitro, both the wild type and S36N mutant HA-p190 proteins showed similar GAP activities toward RhoA, but when expressed in NIH 3T3 fibroblasts only wild type p190 appeared able to function as a RhoGAP. Wild type HA-p190 induced a phenotype of rounded cells with long, beaded extensions similar to that seen when Rho function is disrupted by ADP-ribosylation. HA-p190(S36N), although expressed at a similar level to the wild type protein, had no discernible effect on the cells. The beaded extension phenotype induced by wild type HA-p190 required GAP function. A GAP-defective mutant, p190(R1283A), had no effect on cell morphology. Moreover, the beaded extension phenotype could be suppressed by co-expression of a gain-of-function Rho mutant, RhoA(G14V), or Rac mutant, Rac1(G12V). Activation of the Jun kinase (JNK) via muscarinic receptors was inhibited by wild type HA-p190, but JNK activity was enhanced by the S36N mutant. Co-expression of HA-p190 with a fragment containing only the mutated GTP binding domain partially inhibited the beaded extension phenotype, suggesting that it may sequester a factor required for p190 function. Taken together these data demonstrate that within the cell, the Rho/Rac GAP activity of p190 can be regulated by the N-terminal GTP binding domain.
p190是一种针对Rho家族GTP酶的GTP酶激活蛋白（GAP）。p190的GAP结构域位于该蛋白的C末端。在其N末端，p190含有一个意义不明的GTP结合结构域。我们在p190的这个结构域中引入了一个突变（Ser36→Asn），当它作为血凝素（HA）标记的蛋白在COS细胞中表达时，该突变降低了其结合鸟嘌呤核苷酸的能力。在体外，野生型和S36N突变型HA-p190蛋白对RhoA都表现出相似的GAP活性，但当在NIH 3T3成纤维细胞中表达时，只有野生型p190似乎能够发挥RhoGAP的功能。野生型HA-p190诱导出一种细胞变圆并带有长的、串珠状延伸的表型，类似于Rho功能被ADP-核糖基化破坏时所观察到的表型。HA-p190（S36N）虽然与野生型蛋白表达水平相似，但对细胞没有明显影响。野生型HA-p190诱导的串珠状延伸表型需要GAP功能。一个GAP缺陷型突变体p190（R1283A）对细胞形态没有影响。此外，功能获得型Rho突变体RhoA（G14V）或Rac突变体Rac1（G12V）的共表达可以抑制串珠状延伸表型。通过毒蕈碱受体激活Jun激酶（JNK）受到野生型HA-p190的抑制，但JNK活性被S36N突变体增强。HA-p190与仅包含突变GTP结合结构域的片段共表达部分抑制了串珠状延伸表型，表明它可能隔离了p190功能所需的一个因子。综上所述，这些数据表明在细胞内，p190的Rho/Rac GAP活性可由N末端GTP结合结构域调节。
