GTPase stimulation in shrimp Ras(Q(61)K) with geranylgeranyl pyrophosphate but not mammalian GAP.
BALB/3T3 cells were transformed by transfection with DNA encoding the mutated ras(Q(61)K) from shrimp Penaeus japonicus (Huang et al., 2000). The GTPase-activating protein (GAP) in the cytosol fraction was significantly expressed and degraded, compared to untransformed cells on the western blot. To understand this in more detail, the interaction of the bacterially expressed shrimp Ras (S-Ras) with GAP was investigated using GAP purified from mouse brains. SDS-polyacrylamide gel electrophoresis revealed the monomers of the purified GAP to have a relative mass of 65,000. Since the purified GAP was bound to the Ras conjugated affinity sepharose column with high affinity and its GTP hydolysis activity upon binding with tubulin was suppressed, the purified enzyme was concluded to be neurofibromin-like. The purified GAP enhanced the intrinsic GTPase activity of the S-Ras, to convert it into the inactive GDP-bound form, in agreement with findings for GTP-bound K(B)-Ras in vitro. To compare the effects between isoprenoids and GAP on the GTP-hydrolysis of Ras, we applied the GTP-locked shrimp mutant S-Ras(Q(61)K) and GTP-locked rat mutant K(B)-ras(Q(61)K). Radioassay studies showed that geranylgeranyl pyrophosphate at microg level catalyzed the GTP hydrolysis of S-Ras(Q(61)K) and K(B)-ras(Q(61)K) competently, but not farnesyl pyrophosphate or the purified GAP. The present study provides the view that the geranylgeranyl pyrophosphate at carboxyl terminal CAAX assists GTP hydrolysis to Ras proteins probably in a manner similar to the substrate assisted catalysis in GTPase mechanism.
Chang
K C
KC
Division of Biochemistry and Molecular Sciences, Institute of Zoology, Academia Sinica, Nankang, Taipei, Taiwan 11529.
Chuang
N N
NN
D000818:Animals; D015153:Blotting, Western; D018929:Cell Culture Techniques; D033364:Decapoda / Q000502:physiology*; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism / Q000494:pharmacology*; D011905:Genes, ras / Q000235:genetics*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D008322:Mammals; D011106:Polyisoprenyl Phosphates / Q000378:metabolism / Q000494:pharmacology*; D012717:Sesquiterpenes; D014162:Transfection
D020690:GTPase-Activating Proteins; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C015234:geranyl pyrophosphate; C004808:farnesyl pyrophosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases