GTPase stimulation in shrimp Ras(Q(61)K) with geranylgeranyl pyrophosphatebut not mammalian GAP

BALB/3T3 cells were transformed by transfection with DNA encoding the mutat ed ras(Q(61)K) from shrimp Penaeus japonicus (Huang et al., 2000). The GTPa se-activating protein (GAP) in the cytosol fraction was significantly expre ssed and degraded, compared to untransformed cells on the western blot. To understand this in more detail, the interaction of the bacterially expresse d shrimp Ras (S-Ras) with GAP was investigated using GAP purified from mous e brains. SDS-polyacrylamide gel electrophoresis revealed the monomers of t he purified GAP to have a relative mass of 65,000. Since the purified GAP w as bound to the Ras conjugated affinity sepharose column with high affinity and its GTP hydolysis activity upon binding with tubulin was suppressed, t he purified enzyme was concluded to be neurofibromin-like. The purified GAP enhanced the intrinsic GTPase activity of the S-Ras, to convert it into th e 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-hy drolysis 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 geran ylgeranyl pyrophosphate at mug 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 geranylgeran yl pyrophosphate at carboxyl terminal CAAX assists GTP hydrolysis to Ras pr oteins probably in a manner similar to the substrate assisted catalysis in GTPase mechanism. J. Exp. Zool. 290:642-651, 2001. (C) 2001 Wiley-Liss, Inc .