Posttranslationally Processed Structure of the Human Platelet Protein smg p21B: Evidence for Geranylgeranylation and Carboxyl Methylation of the C-Terminal Cysteine

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 87; no. 22; pp. 8960 - 8964
• Main Authors: Kawata, Masahito, Farnsworth, Christopher C., Yoshida, Yasuhisa, Gelb, Michael H., Glomset, John A., Takai, Yoshimi
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.11.1990; National Acad Sciences
• Subjects: Amino acids; Application programming interfaces; Biochemistry; Biological and medical sciences; Blood Platelets - metabolism; Cysteine - metabolism; Fundamental and applied biological sciences. Psychology; Gas Chromatography-Mass Spectrometry; Gels; GTP-Binding Proteins - chemistry; GTP-Binding Proteins - metabolism; guanine nucleotide-binding protein; Humans; In Vitro Techniques; Methylation; Mevalonic Acid - analogs & derivatives; Mevalonic Acid - chemistry; Molecular and cellular biology; Molecular genetics; Pentanes; Platelets; Polyisoprenyl Phosphates - metabolism; Protein Processing, Post-Translational; Proteins; rap GTP-Binding Proteins; smg p21B protein; Terpenoids; Translation. Translation factors. Protein processing; Ungulates; Human; Binding protein; GTP; Translation; Molecular processing
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.87.22.8960

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 a3H-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.