The product of the rap2 gene, member of the ras superfamily. Biochemical characterization and site-directed mutagenesis.
The human rap2 gene encodes a 183 amino acid protein that shares 46% identity with the K-ras p21. Its cDNA was engineered and inserted into the bacterial expression vector ptac; this allowed the production of high levels of soluble recombinant protein in Escherichia coli that was purified to near homogeneity. The rap2 protein binds GTP and exhibits a low intrinsic GTPase activity (rate constant of 0.5 x 10(-2) min-1). It exchanges its bound GDP with a half-life of 18 min at 37 degrees C in the presence of 10 mM Mg2+. Under the same conditions, the dissociation of bound GTP was at least 25-fold slower showing that the rap2 protein has a much higher affinity for GTP than GDP. The contribution of individual domains of the protein to its biochemical activities was investigated by site-directed mutagenesis. Substitution of Val for Gly at position 12 results in a 2-fold decrease in the GDP dissociation rate constant and GTPase activity. Replacement of the Ser at position 17 by Asn severely impairs the GTP binding ability of the protein and points to an important role of this residue in the coordination of Mg2+. Mutation of Thr-35 to Ala results in a decreased affinity for GTP and a reduction (3-fold) of the GTPase activity. Finally, substitution of Thr-145 by Ile leads to an imperfect binding of guanyl nucleotides as exemplified by an increase in their dissociation rate constants and reduction of the GTPase activity of the protein. These properties of the normal and mutant rap2 proteins are compared with those of ras p21 carrying similar substitutions and are discussed in relation to the structural models proposed for ras p21.
Lerosey
I
I
Institut National de la Santé et de la Recherche Médicale U-248, Faculté de Médecine Lariboisiére-Saint Louis, Paris, France.
Chardin
P
P
de Gunzburg
J
J
Tavitian
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D013329:Structure-Activity Relationship; D020668:rap GTP-Binding Proteins
D009841:Oligonucleotides; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins