Rasp21 sequences opposite the nucleotide binding pocket are required for GRF-mediated nucleotide release.

TY - JOUR

T1 - Rasp21 sequences opposite the nucleotide binding pocket are required for GRF-mediated nucleotide release.

AU - Leonardsen, L

AU - DeClue, J E

AU - Lybaek, H

AU - Lowy, D R

AU - Willumsen, B M

N1 - Keywords: Amino Acid Sequence; Animals; Escherichia coli; GTPase-Activating Proteins; Guanine Nucleotide Exchange Factors; Guanosine Diphosphate; Humans; Mice; Molecular Sequence Data; Mutagenesis, Insertional; Point Mutation; Protein Structure, Tertiary; Proteins; Proto-Oncogene Proteins p21(ras); Sequence Deletion; Substrate Specificity; ras GTPase-Activating Proteins; ras Guanine Nucleotide Exchange Factors; ras-GRF1

PY - 1996

Y1 - 1996

N2 - The substrate requirements for the catalytic activity of the mouse Cdc25 homolog Guanine nucleotide Release Factor, GRF, were determined using the catalytic domain of GRF expressed in insect cells and E. coli expressed H-Ras mutants. We found a requirement for the loop 7 residues in Ras (amino acids 102 to 105) for stimulation of guanine nucleotide release. The dependence on the switch I and II regions of Rasp21 (encompassing the residues that shift position in the GTP- versus GDP-bound protein), which had been seen with Sdc25-mediated exchange, was also found for GRF. In addition, the sensitivity of H-Ras to GRF was abolished when residues 130-139 were replaced by proline-aspartic acid-glutamine, whereas substitution of the entire loop 8 (residues 123-130 replaced by leucine-isoleucine-arginine) had no effect on the stimulation of guanine nucleotide release by GRF. Substrate activity of Ras proteins were independent of their post-translational processing, GDP release was stimulated threefold more effectively by GRF than was GTP release, and no major differences were found between the mammalian N-, H- and K-Ras proteins. Examining the responsiveness of the Ras protein from S. pombe and the human Ras like proteins RhoA, Rap1A, Rac1 and G25K revealed a strict Ras specificity; of these only S. pombe Ras was GRF sensitive.

AB - The substrate requirements for the catalytic activity of the mouse Cdc25 homolog Guanine nucleotide Release Factor, GRF, were determined using the catalytic domain of GRF expressed in insect cells and E. coli expressed H-Ras mutants. We found a requirement for the loop 7 residues in Ras (amino acids 102 to 105) for stimulation of guanine nucleotide release. The dependence on the switch I and II regions of Rasp21 (encompassing the residues that shift position in the GTP- versus GDP-bound protein), which had been seen with Sdc25-mediated exchange, was also found for GRF. In addition, the sensitivity of H-Ras to GRF was abolished when residues 130-139 were replaced by proline-aspartic acid-glutamine, whereas substitution of the entire loop 8 (residues 123-130 replaced by leucine-isoleucine-arginine) had no effect on the stimulation of guanine nucleotide release by GRF. Substrate activity of Ras proteins were independent of their post-translational processing, GDP release was stimulated threefold more effectively by GRF than was GTP release, and no major differences were found between the mammalian N-, H- and K-Ras proteins. Examining the responsiveness of the Ras protein from S. pombe and the human Ras like proteins RhoA, Rap1A, Rac1 and G25K revealed a strict Ras specificity; of these only S. pombe Ras was GRF sensitive.

M3 - Journal article

C2 - 8950985

SN - 0950-9232

VL - 13

SP - 2177

EP - 2187

JO - Oncogene

JF - Oncogene

IS - 10

ER -