Role of the C-terminal region of smg p21, a ras p21-like small GTP-binding protein, in membrane and smg p21 GDP/GTP exchange protein interactions.
Limited proteolysis with trypsin of smg p21B, a ras p21-like small GTP-binding protein having the same putative effector domain as ras p21s, produced the N-terminal fragment and the C-terminal tail of Lys-Lys-Ser-Ser-geranylgeranyl-Cys methyl ester. The Mr values of the intact smg p21B, the N-terminal fragment, and the C-terminal tail were estimated to be about 22,000, 20,500, and less than 1,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both the GDP- and GTP-bound forms of the intact smg p21B bound to various membranes and phosphatidylserine-linked Affi-Gel. However, both the GDP- and GTP-bound forms of the N-terminal fragment failed to bind to membranes and phosphatidylserine-linked Affi-Gel. In contrast, the C-terminal tail bound to membranes and phosphatidylserine-linked Affi-Gel. The N-terminal fragment contained a GDP/GTP-binding and GTPase domain and exhibited these two activities, but the C-terminal tail did not show any such activity. A GTPase-activating protein for smg p21 stimulated the GTPase activity of both the intact smg p21B and the N-terminal fragment. In contrast, a GDP/GTP exchange protein for smg p21, named GDP dissociation stimulator, stimulated the GDP/GTP exchange reaction of the intact smg p21B but not that of the N-terminal fragment. These results indicate 1) that smg p21B is composed of at least two functionally different domains, the N-terminal GDP/GTP-binding and GTPase domain and the C-terminal membrane-binding domain, 2) that smg p21B binds to membranes through its C-terminal hydrophobic and basic domain, and 3) that this C-terminal domain is also essential for the smg p21 GDP dissociation stimulator action but not for the smg p21 GTPase-activating protein action.
Hiroyoshi
M
M
Department of Biochemistry, Kobe University School of Medicine, Japan.
Kaibuchi
K
K
Kawamura
S
S
Hata
Y
Y
Takai
Y
Y
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D013569:Synapses / Q000378:metabolism; D014357:Trypsin; D020668:rap GTP-Binding Proteins
D011518:Proto-Oncogene Proteins; D006153:Guanosine Diphosphate; D014357:Trypsin; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins