Posttranslationally processed structure of the human platelet protein smg p21B: evidence for geranylgeranylation and carboxyl methylation of the C-terminal cysteine.
smg p21A and -B are small GTP-binding proteins that share putative effector and consensus C-terminal sequences with ras p21 proteins. In the present report, we showed that human platelet smg p21B became labeled when intact platelets were incubated with exogenous [3H]mevalonolactone and when a purified preparation of smg p21B was incubated with bovine brain membranes and S-adenosyl-L-[methyl-3H]methionine. In addition, we demonstrated by gas chromatography/mass spectrometry that treatment of smg p21B with Raney nickel released a geranylgeranyl moiety in a molar ratio of about 1:1. In contrast, treatment of smg p21B with NH2OH or KOH yielded no evidence for the presence of a palmitoyl thioester. Extensive digestion of smg p21B with Achromobacter protease I yielded two C-terminal tripeptides that contained serine and cysteine in a molar ratio of 2:1. Both peptides were modified by a thioether-linked geranylgeranyl group. One of the peptides comigrated with a 3H-labeled proteolytic product of methylated smg p21B on reverse-phase HPLC and this peptide appeared at the same retention time as that of the other peptide after being treated with KOH. Since the cDNA-predicted C-terminal sequence of smg p21B contains a unique Ser-Ser-Cys peptide within its C-terminal domain, -Lys-Lys-Ser-Ser-Cys-Gln-Leu-Leu184, these results indicate that smg p21B is posttranslationally modified by geranylgeranylation of Cys-181 and suggest that further modifications cause proteolytic removal of the three predicted C-terminal amino acids followed by partial methylation of the cysteinyl carboxyl group.
Kawata
M
M
Department of Biochemistry, Kobe University School of Medicine, Japan.
Farnsworth
C C
CC
Yoshida
Y
Y
Gelb
M H
MH
Glomset
J A
JA
Takai
Y
Y
D001792:Blood Platelets / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D066298:In Vitro Techniques; D008745:Methylation; D008798:Mevalonic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D020668:rap GTP-Binding Proteins
D011106:Polyisoprenyl Phosphates; C015367:mevalonolactone; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; D003545:Cysteine; D008798:Mevalonic Acid