Disrupting the geranylgeranylation at the C-termini of the shrimp Ras by depriving guanine nucleotide binding at the N-terminal

In order to assess the effects of guanine nucleotide binding on the geranylgeranylation at the CAAX box of the shrimp Ras, we experimented with the shrimp Penaeus japonicus Ras (S-Ras) which is geranylgeranylated at the C-termini, shares 85% homology with mammalian K(B)-Ras protein and demonstrates identity in the guanine nucleotide binding domains (Huang C-F, Chuang N-N. 1999. J Exp Zool 283:510-521). Several point mutations in the S-ras gene were generated at codons 12 (G12V), 61 (Q61K), and 116 (N116I). The bacterially expressed mutant S-Ras proteins, G12V and Q61K, were bound with GTP without hydrolysis. In contrast, the mutant S-Ras N116I was defective in its ability to bind any guanine nucleotides. Autoradiography studies showed that the purified shrimp protein geranylgeranyltransferase I (Lin R-S, Chuang N-N. 1998. J Exp Zool 281:565-573) was unable to catalyze the transfer of [(3)H]-geranylgeranylpyrophosphate to this mutant N116I but very competently caused the geranylgeranylation of GTP-locked mutants, G12V and Q61K. These results demonstrate that the geranylgeranylation at the CAAX box of the shrimp Ras protein requires the proper binding of guanine nucleotide at its N-terminal region. J. Exp. Zool. 286:441-449, 2000.