rho GAP of 28 kDa (GAP2), but not of 190 kDa (p190), requires Asp65 and Asp67 of rho GTPase for its activation.
Two distinct GTPase-activating proteins (GAPs), i.e. rho GAPs of 28 kDa (GAP2) and of 190 kDa (p190), stimulate the intrinsic GTPase activity of the rho protein. The rho GAP activity of p190 resides in its C-terminal domain (p190C). Neither GAP2 nor p190C activates the ras GTPase. We replaced Asp65 and Asp67 residues of rho GTPase with the corresponding ras residues and examined whether the domain containing them is involved in its activation by rho GAPs. Mutation of either Asp65 to Glu or Asp67 to Ser did not change the Kd value for GTP gamma S of the rho protein. The Ser67 mutation reduced the intrinsic GTPase activity of the rho protein, while no change was observed with the Glu65 mutation. Both mutations abolished activation of rho GTPase by GAP2. The GAP2-dependent activation of rho GTPase was inhibited by the addition of GTP gamma S-bound wild type rho but not by either GTP gamma S-bound Glu65- or Ser67-rho, indicating that both Asp65 and Asp67 are essential for interaction of rho protein with GAP2. On the contrary, p190C activated both Glu65- and Ser67-rho GTPases to the extent and in a dose dependence to those seen in the wild GTPase. These results suggest that GAP2 and p190 interact with different residues or domains of the rho GTPase for their activation.
Morii
N
N
Department of Pharmacology, Kyoto University Faculty of Medicine, Japan.
Kumagai
N
N
Nur-E-Kamal
M S
MS
Narumiya
S
S
Maruta
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism*; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D012234:Rho Factor / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D005971:Glutamates; D011506:Proteins; D012234:Rho Factor; D020703:ras GTPase-Activating Proteins; D001224:Aspartic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D018698:Glutamic Acid; D012694:Serine; D020558:GTP Phosphohydrolases