Posttranslationally processed structure of the human platelet protein smg p21B: evidence for geranylgeranylation and carboxyl methylation of the C-terminal cysteine.

Abstract

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 sing 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-Leu'8", these results indicate that smg p2IB 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. smg p21A and -B, also referred to as the raplA or Krev-1 and raplB proteins, respectively, belong to a superfamily of ras p21/ras p21-like small GTP-binding proteins (G proteins) (refs.

A CAAX or a CAAL motif and a second signal are sufficient for plasma membrane targeting of ras proteins

“…Certain other CAAX containing proteins, including the ras-related proteins Krev 1/rap lA (Kawata et al, 1990;Buss et al, 1991) and G25K (Maltese and Sheridan, 1990), and the -y-subunits of brain G-proteins (Yamane et al, 1990;Mumby et al, 1990) have been shown to be geranylgeranylated. The CAAX motifs of these C20 modified proteins all terminate with a leucine residue indicating that the X amino acid determines whether a CAAX motif is a substrate for a farnesyl or geranylgeranyl transferase (Seabra et al, 1991;Finegold et al, 1991).…”

Generation of specific antibodies against the rap1A, rap1B and rap2 small GTP‐binding proteins

“…These modifications include attachment of a C15 (farnesyl) isoprenoid to the cysteine of the CAAX motif, removal of the final three amino acids and methylation of the newly exposed Ca group [lo]. More recently, it was shown that raplA protein expressed in insect cells [ll] and raplB protein purified from human platelets [12] are modified by a C20 (geranylgeranyl) isoprenoid.Little is known about the function of rap proteins in mammalian cells. Interest has focussed on the raplA (Krev-I) protein, whose cDNA was cloned first using the feature of its homology to ras proteins [4] and more recently by its ability…”

“…These modifications include attachment of a C15 (farnesyl) isoprenoid to the cysteine of the CAAX motif, removal of the final three amino acids and methylation of the newly exposed Ca group [lo]. More recently, it was shown that raplA protein expressed in insect cells [ll] and raplB protein purified from human platelets [12] are modified by a C20 (geranylgeranyl) isoprenoid.…”

Protein Farnesyltransferase in Plants (Molecular Cloning and Expression of a Homolog of the [beta] Subunit from the Garden Pea)

“…Vol. 101, 1993 residues (AAX) of the protein substrate for FTase and GGTase I are removed, and the a-carboxyl group of the terminal Cys is methylated, whereas the isoprenoid moiety is linked to the Cys by a thioether bond (Kawata et al, 1990;Hancock et al, 1991a;Yamane et al, 1991).…”