The COOH-terminal domain of the Rap1A (Krev-1) protein is isoprenylated and supports transformation by an H-Ras:Rap1A chimeric protein.
Although the Rap1A protein resembles the oncogenic Ras proteins both structurally and biochemically, Rap1A exhibits no oncogenic properties. Rather, overexpression of Rap1A can reverse Ras-induced transformation of NIH 3T3 cells. Because the greatest divergence in amino acid sequence between Ras and Rap1A occurs at the COOH terminus, the role of this domain in the opposing biological activities of these proteins was examined. COOH-terminal processing and membrane association of Rap1A were studied by constructing and expressing a chimeric protein (composed of residues 1 to 110 of an H-Ras activated by a Leu-61 mutation attached to residues 111 to 184 of Rap1A) in NIH 3T3 cells and a full-length human Rap1A protein in a baculovirus-Sf9 insect cell system. Both the chimeric protein and the full-length protein were synthesized as a 23-kDa cytosolic precursor that rapidly bound to membranes and was converted into a 22-kDa form that incorporated label derived from [3H]mevalonate. The mature 22-kDa form also contained a COOH-terminal methyl group. Full-length Rap1A, expressed in insect cells, was modified by a C20 (geranylgeranyl) isoprenoid. In contrast, H-Ras, expressed in either Sf9 insect or NIH 3T3 mouse cells contained a C15 (farnesyl) group. This suggests that the Rap1A COOH terminus is modified by a prenyl transferase that is distinct from the farnesyl transferase that modifies Ras proteins. Nevertheless, in NIH 3T3 cells the chimeric Ras:Rap1A protein retained the transforming activity conferred by the NH2-terminal Ras61L domain. This demonstrates that the modifications and localization signals of the COOH terminus of Rap1A can support the interactions between H-Ras and membranes that are required for transformation.
Buss
J E
JE
La Jolla Cancer Research Foundation, California 92037.
Quilliam
L A
LA
Kato
K
K
Casey
P J
PJ
Solski
P A
PA
Wong
G
G
Clark
R
R
McCormick
F
F
Bokoch
G M
GM
Der
C J
CJ
D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D019204:GTP-Binding Proteins / Q000502:physiology*; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000378:metabolism*; D008745:Methylation; D009036:Moths; D010168:Palmitates / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D016283:Proto-Oncogene Proteins p21(ras) / Q000502:physiology*; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D013729:Terpenes / Q000378:metabolism; D020668:rap GTP-Binding Proteins
D008565:Membrane Proteins; D010168:Palmitates; D011993:Recombinant Fusion Proteins; D013729:Terpenes; D019204:GTP-Binding Proteins; D016283:Proto-Oncogene Proteins p21(ras); D020668:rap GTP-Binding Proteins