Müller R T, Honnert U, Reinhard J, Bähler M
Friedrich-Miescher-Laboratorium in der Max-Planck Gesellschaft, Tübingen, Germany.
Mol Biol Cell. 1997 Oct;8(10):2039-53. doi: 10.1091/mbc.8.10.2039.
myr 5 is an unconventional myosin (class IX) from rat that contains a Rho-family GTPase-activating protein (GAP) domain. Herein we addressed the specificity of the myr 5 GAP activity, the molecular mechanism by which GAPs activate GTP hydrolysis, the consequences of myr 5 overexpression in living cells, and its subcellular localization. The myr 5 GAP activity exhibits a high specificity for Rho. To achieve similar rates of GTPase activation for RhoA, Cdc42Hs, and Rac1, a 100-fold or 1000-fold higher concentration of recombinant myr 5 GAP domain was needed for Cdc42Hs or Rac1, respectively, as compared with RhoA. Cell lysates from Sf9 insect cells infected with recombinant baculovirus encoding myr 5 exhibited increased GAP activity for RhoA but not for Cdc42Hs or Rac1. Analysis of Rho-family GAP domain sequences for conserved arginine residues that might contribute to accelerate GTP hydrolysis revealed a single conserved arginine residue. Mutation of the corresponding arginine residue in the myr 5 GAP domain to a methionine (M1695) virtually abolished Rho-GAP activity. Expression of myr 5 in Sf9 insect cells induced the formation of numerous long thin processes containing occasional varicosities. Such morphological changes were dependent on the myr 5 Rho-GAP activity, because they were induced by expression the myr 5 tail or just the myr 5 Rho-GAP domain but not by expressing the myr 5 myosin domain. Expression of myr 5 in mammalian normal rat kidney (NRK) or HtTA-1 HeLa cells induced a loss of actin stress fibers and focal contacts with concomitant morphological changes and rounding up of the cells. Similar morphological changes were observed in HtTA-1 HeLa cells expressing just the myr 5 Rho-GAP domain but not in cells expressing myr 5 M1695. These morphological changes induced by myr 5 were inhibited by coexpression of RhoV14, which is defective in GTP hydrolysis, but not by RhoI117. myr 5 was localized in dynamic regions of the cell periphery, in the perinuclear region in the Golgi area, along stress fibers, and in the cytosol. These results demonstrate that myr 5 has in vitro and in vivo Rho-GAP activity. No evidence for a Rho effector function of the myr 5 myosin domain was obtained.
myr 5是一种来自大鼠的非常规肌球蛋白（IX类），它含有一个Rho家族GTP酶激活蛋白（GAP）结构域。在此，我们研究了myr 5 GAP活性的特异性、GAP激活GTP水解的分子机制、myr 5在活细胞中过表达的后果及其亚细胞定位。myr 5 GAP活性对Rho表现出高度特异性。为了使RhoA、Cdc42Hs和Rac1达到相似的GTP酶激活速率，与RhoA相比，Cdc42Hs或Rac1分别需要高100倍或1000倍浓度的重组myr 5 GAP结构域。用编码myr 5的重组杆状病毒感染的Sf9昆虫细胞的细胞裂解物对RhoA的GAP活性增加，但对Cdc42Hs或Rac1没有增加。对可能有助于加速GTP水解的保守精氨酸残基的Rho家族GAP结构域序列分析揭示了一个单一的保守精氨酸残基。myr 5 GAP结构域中相应的精氨酸残基突变为甲硫氨酸（M1695）实际上消除了Rho - GAP活性。myr 5在Sf9昆虫细胞中的表达诱导了许多含有偶尔膨大部的长而细的突起的形成。这种形态变化依赖于myr 5 Rho - GAP活性，因为它们是由myr 5尾部或仅myr 5 Rho - GAP结构域的表达诱导的，而不是由myr 5肌球蛋白结构域的表达诱导的。myr 5在哺乳动物正常大鼠肾（NRK）或HtTA - 1 HeLa细胞中的表达导致肌动蛋白应力纤维和粘着斑的丧失，同时伴有形态变化和细胞变圆。在仅表达myr 5 Rho - GAP结构域的HtTA - 1 HeLa细胞中观察到类似的形态变化，但在表达myr 5 M1695的细胞中未观察到。myr 5诱导的这些形态变化被GTP水解缺陷的RhoV14的共表达所抑制，但未被RhoI117抑制。myr 5定位于细胞周边的动态区域、高尔基体区域的核周区域、沿着应力纤维以及细胞质中。这些结果表明myr 5在体外和体内都具有Rho - GAP活性。未获得myr 5肌球蛋白结构域具有Rho效应器功能的证据。
