Biochemical characterization of baculovirus-expressed rap1A/Krev-1 and its regulation by GTPase-activating proteins.
Normal human rap1A and 35A rap1A (which encodes a protein with a Thr-35----Ala mutation) were cloned into a baculovirus transfer vector and expressed in Sf9 insect cells. The resulting proteins were purified, and their nucleotide binding, GTPase activities, and responsiveness to GTPase-activating proteins (GAPs) were characterized and compared with those of Rap1 purified from human neutrophils. Recombinant wild-type Rap1A bound GTP gamma S, GTP, and GDP with affinities similar to those observed for neutrophil Rap1 protein. The rate of exchange of GTP by Rap1 without Mg2+ was much slower than that by Ras. The basal GTPase activities by both recombinant proteins were lower than that observed with the neutrophil Rap1, but the GTPase activity of the neutrophil and wild-type recombinant Rap1 proteins could be stimulated to similar levels by Rap-GAP activity in neutrophil cytosol. In contrast to wild-type Rap1A, the GTPase activity of 35A Rap was unresponsive to Rap-GAP stimulation. Neither recombinant Rap1A nor neutrophil Rap1 protein GTPase activity could be stimulated by recombinant Ras-GAP at a concentration 25-fold higher than that required to hydrolyze 50% of H-Ras-bound GTP under similar conditions. These results suggest that the putative effector domains (amino acids 32 to 40) shared between Rap1 and Ras are functionally similar and interact with their respective GAPs. However, although Rap1 and Ras are identical in this region, secondary structure or additional regions must confer the ability to respond to GAPs.
Quilliam
L A
LA
Department of Immunology, Scripps Clinic & Research Foundation, La Jolla, California 92037.
Der
C J
CJ
Clark
R
R
O'Rourke
E C
EC
Zhang
K
K
McCormick
F
F
Bokoch
G M
GM
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D020558:GTP Phosphohydrolases / Q000097:blood / Q000235:genetics*; D019204:GTP-Binding Proteins / Q000097:blood / Q000235:genetics*; D020690:GTPase-Activating Proteins; D005786:Gene Expression Regulation; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007304:Insect Viruses / Q000235:genetics*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D009504:Neutrophils / Q000378:metabolism; D015345:Oligonucleotide Probes; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013873:Thionucleotides / Q000378:metabolism; D020668:rap GTP-Binding Proteins; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D015345:Oligonucleotide Probes; D011506:Proteins; D011994:Recombinant Proteins; D013873:Thionucleotides; D020703:ras GTPase-Activating Proteins; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D010744:Phosphoric Monoester Hydrolases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; D008274:Magnesium