Identification of a platelet Mr 22,000 GTP-binding protein as the novel smg-21 gene product having the same putative effector domain as the ras gene products

Ohmori, Toshihiko; Kikuchi, Akira; Yamamoto, Katsuhiko; Kawata, Masahito; Kondo, Jun; Takai, Yoshimi

doi:10.1016/s0006-291x(88)80302-4

Cited by 52 publications

(12 citation statements)

References 18 publications

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“…The presence of small-molecular-mass GTP-binding proteins (s mg) has been demonstrated in human platelets [15][16][17][18][19][20][21][22][23][24]. One of these is phosphorylated in vivo in response to a PGE,-induced increase in the cAMP concentration in intact platelets, and in vitro by the cAMP-dependent protein kinase [24].…”

Section: Introductionmentioning

confidence: 99%

Evidence for a role of rap1 protein in the regulation of human platelet Ca2+ fluxes

Corvazier

Enouf

Papp

et al. 1992

Biochemical Journal

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The relationship between the 22-24 kDa cyclic AMP (cAMP)-dependent phosphoprotein previously described as being involved in the regulation of human platelet membrane Ca2+ transport and a GTP-binding protein of low molecular mass (ras-like protein) was investigated. After isolation of plasma membranes and intracellular membranes, it was found that guanosine 5'-[y-thio]triphosphate (GTP[S]) bound to plasma membrane proteins ranging in molecular mass from 22 to 29 kDa, but not to intracellular membranes. The major GTP-binding protein appeared as a 24 kDa protein under reduced conditions and a 22 kDa protein under non-reduced conditions. A similar membrane location and electrophoretic mobility were found for both the cAMP phosphoprotein and the protein recognized by a specific anti-rapI antibody. The identity between the cAMP phosphoprotein and the rapl GTP-binding protein was further examined by studying the functional effect of GTP on plasma membrane Ca2+ transport. A maximal GTP[S] concentration of 40 /,M was found to:(1) inhibit to the same degree (40%) both Ca2+-ATPase activity and the Ca2+ transport function mediated by the Ca2+-ATPase; (2) inhibit the phosphorylation of the 22-24 kDa protein by the catalytic subunit of the cAMP-dependent protein kinase (C.Sub.); and (3) abolish the stimulation of Ca2+ uptake induced by C.Sub. It is concluded that the platelet cAMP phosphoprotein is indeed the rapl GTP-binding protein, and that it regulates plasma membrane Ca2+ transport, thus providing evidence for a new role of a ras-related protein.

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Section: Introductionmentioning

confidence: 99%

Evidence for a role of rap1 protein in the regulation of human platelet Ca2+ fluxes

Corvazier

Enouf

Papp

et al. 1992

Biochemical Journal

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“…They are found in most mammalian tissues, though their tissue and subcellular distributions differ somewhat from those of ras p21 proteins (11). smg p21 proteins have consensus amino acid sequences for GDP/GTP-binding and GTPase activities and exhibit these activities (3)(4)(5)(6)(7)(8)(9)(10). The GTP-bound form of smg p21 proteins is active whereas the GDP-bound form is inactive, and the two forms can be interconverted by a specific GDP/GTP exchange protein, named GDP dissociation stimulator (12), and by a specific GTPase-activating protein (13,14).…”

mentioning

confidence: 99%

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

Kawata

Farnsworth

Yoshida

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

146

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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.

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“…However, this protein had an apparent molecular mass of 21 kDa and did not bind [u-~~P]GTP on nitrocellulose blots under the conditions used [l]. The presence of the raplA gene product in platelets has been documented by others [22,24]. The above results suggested that G,27 might be a ral gene product.…”

Section: Resultsmentioning

confidence: 57%

“…Our results also provide further evidence that the Gn-proteins, as defined by the methodology we have used [l], represent a distinct group of high-affinity GTP-binding proteins. Thus, raplA protein, though present at high concentrations in platelets [22,24], was not readily detected on nitrocellulose blots. Similarly, platelet ras proteins [25] and G25K [35] were not easily detected by this technique.…”

Section: Discussionmentioning

confidence: 99%

“…The rho proteins are substrates for ADP-ribosylation by botulinum exoenzyme C3 [ 18,191. In addition, a 21 kDa protein purified from bovine brain is coded for by the c-K-rt-zs gene 1207 and a protein (termed smg ~21) purified from bovine brain [21] and human platelets [22] is coded for by the FapfA gene [23,24]. The protein products of some of the other Fas-related genes (e.g.…”

Section: Introductionmentioning

confidence: 99%

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Identification of multiple ral gene products in human platelets that account for some but not all of the platelet G_n‐proteins

1990

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Polyclonal antibodies raised against specific recombinant low molecular mass GTP‐binding proteins were tested for their ability to recognize partially purified human platelet membrane Gn‐proteins (i.e. proteins that bind [α‐32PlGTP on nitrocellulose blots of SDS/polyacrylamide gels). An antiserum against simian ralA protein recognized a 27 kDa human platelet protein with the same apparent molecular mass as the major platelet Gn‐protein (Gn27). In further analysis by two‐dimensional polyacrylamide gel electrophoresis, the isoelectric focusing step permitted resolution of 12 major Gn‐protein forms, seven of 27 kDa (Gn27a‐g), one of 26 kDa (Gn26) and four of 24 kDa (Gn24a‐d). The ralA antibody reacted strongly with the five most basic Gn27 species (a‐e), weakly with Gn26 and not at all with Gn27f, Gn27g or Gn24a‐d. We conclude that ral gene products account for some but probably not for all of the platelet Gn‐proteins.

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Identification of a platelet Mr 22,000 GTP-binding protein as the novel smg-21 gene product having the same putative effector domain as the ras gene products

Cited by 52 publications

References 18 publications

Evidence for a role of rap1 protein in the regulation of human platelet Ca2+ fluxes

Evidence for a role of rap1 protein in the regulation of human platelet Ca2+ fluxes

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

Identification of multiple ral gene products in human platelets that account for some but not all of the platelet G_n‐proteins

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Identification of a platelet Mr 22,000 GTP-binding protein as the novel smg-21 gene product having the same putative effector domain as the ras gene products

Cited by 52 publications

References 18 publications

Evidence for a role of rap1 protein in the regulation of human platelet Ca2+ fluxes

Evidence for a role of rap1 protein in the regulation of human platelet Ca2+ fluxes

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

Identification of multiple ral gene products in human platelets that account for some but not all of the platelet Gn‐proteins

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Identification of multiple ral gene products in human platelets that account for some but not all of the platelet G_n‐proteins