[Comparison of the mode of action of ras p 21 with those of protein kinase A and C in the gene expression].
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Animals↗
Biomedical subjects
K Kaibuchi
Publications and source records attributed to K Kaibuchi.
Transforming and c-fos promoter/enhancer-stimulating activities of a GDP/GTP exchange protein for small GTP-binding proteins.
There is a superfamily of ras p21/ras p21-like small GTP-binding proteins (G proteins). Small G proteins have GDP-bound inactive and GTP-bound active forms which are interconvertible by GDP/GTP exchange and GTPase reactions. The former and latter reactions are regulated by GDP/GTP exchange proteins (GEPs) and GTPase activating proteins (GAPs), respectively. We have isolated two types of GEP: one is a stimulatory type called GDP dissociation stimulator (GDS) and the other is an inhibitory type called GDP dissociation inhibitor (GDI). Among the GEPs thus far isolated, only smg GDS is active on Ki-ras p21. This GEP is also active on smg p21 and rho p21, but is inactive on Ha-ras p21. For the action of smg GDS, the post-translational processing of the C-terminal region of its substrate small G proteins is essential. Point-mutated Ki-ras p21 strongly transforms NIH/3T3 cells and markedly stimulates the c-fos promoter/enhancer in this cell line, whereas normal Ki-ras p21 is almost inactive in these activities. smg GDS induces the Ki-ras p21 activation which eventually leads to the transformation of NIH/3T3 cells and to the stimulation of the c-fos promoter/enhancer in this cell line. Thus, smg GDS plays an important role in regulating the activity of Ki-ras p21 as well as of other small G proteins.
3T3 Cells↗
Post-translational modifications of the C-terminal region of the rho protein are important for its interaction with membranes and the stimulatory and inhibitory GDP/GTP exchange proteins.
We have recently found, by use of the rhoA p21 purified from bovine aortic smooth muscle, that it is similarly post-translationally processed as described for ras p21s: it is first geranylgeranylated at the cysteine residue in the C-terminal region followed by removal of the three C-terminal amino acids and the subsequent carboxyl methylation of the revealed C-terminal cysteine residue. In the present study, we investigated the function(s) of these post-translational modifications of the C-terminal region of rhoA p21 by use of the rhoA p21s purified from bovine aortic smooth muscle and rhoA p21-overexpressing Escherichia coli since the bacterial protein was not modified with a geranylgeranyl moiety. Bovine rhoA p21 bound to plasma membranes and phosphatidylserine-linked Affigel, but bacterial rhoA p21 did not bind to them. The inhibitory GDP/GTP exchange protein for rhoA p21, named GDP dissociation inhibitor (GDI), made a complex with the GDP-bound form of bovine rhoA p21 and thereby inhibited the dissociation of GDP from and the subsequent binding of GTP to it. However, rho GDI neither made a complex with the GDP-bound form of bacterial rhoA p21 nor affected these reactions of the bacterial protein. The stimulatory GDP/GTP exchange protein for rhoA p21, named GDP dissociation stimulator (GDS), stimulated the dissociation of GDP from bovine rhoA p21, but was inactive for the bacterial protein. In contrast, the GTPase activating protein for rhoA p21 is active not only for bovine rhoA p21 but also for the bacterial protein. These results suggest that the post-translational modifications of the C-terminal region of bovine rhoA p21, most presumably the geranylgeranylation, which are absent in bacterial rhoA p21, play important roles in its interaction with membranes and the stimulatory and inhibitory GDP/GTP exchange proteins but not with the GAP.
Animals↗
Purification and characterization from bovine brain cytosol of proteins that regulate the GDP/GTP exchange reaction of smg p21s, ras p21-like GTP-binding proteins.
Novel regulatory proteins for smg p21A and -B, ras p21-like GTP-binding proteins (G proteins) having the same putative effector domain as ras p21s, were purified to near homogeneity from bovine brain cytosol and characterized. These regulatory proteins, designated as GDP dissociation stimulator (GDS) 1 and -2, stimulated the dissociation of both [3H]GDP and [35S] guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) from smg p21s to the same extent. smg p21 GDS1 and -2 also stimulated the binding of [35S]GTP gamma S to the GDP-bound form of smg p21s but not that to the guanine nucleotide-free form. These actions of smg p21 GDS1 and -2 were specific for smg p21s and inactive for other ras p21/ras p21-like G proteins including c-Ha-ras p21, rhoB p20, and smg p25A. Neither smg p21 GDS1 nor -2 stimulated the GTPase activity of smg p21s and by itself showed [35S]GTP gamma S-binding or GTPase activity. smg p21 GDS1 and -2 showed very similar physical and kinetic properties and were indistinguishable by peptide map analysis. The Mr values of smg p21 GDS1 and -2 were estimated to be about 53,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and from the S values, indicating that smg p21 GDS1 and -2 are composed of a single polypeptide without a subunit structure. smg p21 GDS1 and -2 were distinguishable from GTPase activating proteins (GAPs) for the ras and rho proteins, and smg p21B, and GDP dissociation inhibitors for smg p25A and the rho proteins previously identified in bovine brain cytosol. These results indicate that bovine brain contains regulatory proteins for smg p21s that stimulate the dissociation of GDP from and thereby the subsequent binding of GTP to smg p21s in addition to smg p21 GAP. It is likely that the conversion from the GDP-bound inactive form of smg p21s to the GTP-bound active form is regulated by smg p21 GDS and that its reverse reaction is regulated by smg p21 GAP.
Amino Acid Sequence↗
Comparison of the mode of action of ras p21 with those of protein kinases A and C in the stimulation of gene expression in NIH/3T3 cells.
To compare the mode of action of ras p21 with those of protein kinases A and C in the regulation of gene expression in NIH/3T3 cells, we investigated the transcriptional activity of various enhancer/promoters and enhancer motifs in the cells transfected with the c-Ha-rasva112 complementary DNA (cDNA). The results indicate that the c-Ha-rasva112 protein stimulates the enhancer/promoters of the c-fos gene, the metallothionein IIA gene, the simian virus 40 (SV40) virus genome and the Rous sarcoma (RS) virus genome, and the serum-response element and the 12-O-tetradecanoylphorbol-13-acetate (TPA)-response element in a manner independent of protein kinases A and C in NIH/3T3 cells.
Animals↗
Partial purification and characterization of GDP dissociation stimulator (GDS) for the rho proteins from bovine brain cytosol.
A novel type of regulatory proteins for the rho proteins (rhoA p21 and rhoB p20), ras p21-like small GTP-binding proteins (G proteins), are partially purified from bovine brain cytosol. These regulatory proteins, named rho GDP dissociation stimulator (GDS) 1 and -2, stimulate the dissociation of GDP from rhoA p21 and rhoB p20. rho GDS1 and -2 are inactive for other ras p21/ras p21-like small G proteins including c-Ha-ras p21, smg p21B, and smg p25A. Since we have previously shown that the rate limiting step for the GDP/GTP exchange reaction of the rho proteins is the dissociation of GDP from these proteins, the present results suggest that rho GDS1 and -2 stimulate the GDP/GTP exchange reaction of the rho proteins. rho GDS1 and -2 are distinct from the GAP- and GDI-types of regulatory proteins for the rho proteins previously purified from bovine brain cytosol. rho GAP stimulates the GTPase activity of the rho proteins and rho GDI inhibits the GDP/GTP exchange reaction of the rho proteins. The present results together with these earlier observations indicate that the rho proteins are regulated by at least three different types of regulatory proteins, GDS, GDI, and GAP.
Animals↗
Activation of the c-fos serum-response element by the activated c-Ha-ras protein in a manner independent of protein kinase C and cAMP-dependent protein kinase.
12-O-Tetradecanoylphorbol-13-acetate (TPA) activated the c-fos gene enhancer linked to the chloramphenicol acetyltransferase or luciferase reporter gene in the wild type PC-12 cells but not in the variant PC-12 cells that originated from the wild type cells. Transfection of the c-Ha-rasval12 complementary DNA (cDNA) or addition of dibutyryl cAMP to the wild type PC-12 cells as well as to the variant PC-12 cells activated the c-fos gene enhancer. Prolonged treatment of the wild type PC-12 cells with phorbol-12,13-dibutyrate caused down-regulation of protein kinase C. In these cells, TPA did not stimulate the c-fos gene enhancer any more, but transfection of the c-Ha-rasval12 cDNA still stimulated the c-fos gene enhancer to the same extent as induced in the control cells. Transfection of the c-Ha-rasval12 cDNA or addition of TPA to the wild type PC-12 cells stimulated the serum-response element but not the cAMP-response element. Dibutyryl cAMP stimulated both the serum-response element and the cAMP-response element in the wild type PC-12 cells. These results indicate that the c-Ha-rasval12 protein activates the serum-response element, but not the cAMP-response element in the c-fos gene enhancer, and that the signal pathway from the c-Ha-rasval12 protein to the c-fos serum-response element is independent of protein kinase C and cAMP-dependent protein kinase.
Blotting, Western↗
Activation of the serum-response and TPA-response elements by expression of the v-abl protein: comparison of the mode of action of the v-abl protein with those of protein kinase C, cyclic AMP-dependent protein kinase, and the activated c-raf protein.
In the present study, we have compared the mode of action of the v-abl protein in the regulation of gene expression with those of serine/threonine protein kinases such as protein kinase C, cyclic AMP-dependent protein kinase, and the activated c-raf protein, by measuring the transcriptional activity of the serum-response element, the 12-O-tetradecanoylphorbol-13-acetate (TPA)-response element, and the cyclic AMP-response element in NIH3T3 cells transfected with the v-abl gene. The results indicate that the v-abl protein stimulates the serum-response element and the TPA-response element, but not the cyclic AMP-response element, in a manner similar to that of the activated c-raf protein, but different from those of protein kinase C and cyclic AMP-dependent protein kinase.
Blood Physiological Phenomena↗
Molecular cloning and characterization of a novel type of regulatory protein (GDI) for the rho proteins, ras p21-like small GTP-binding proteins.
We have recently purified to near homogeneity a novel type of regulatory protein for the rho proteins, ras p21-like small GTP-binding proteins, from bovine brain cytosol. This regulatory protein, named GDP dissociation inhibitor for the rho proteins (rho GDI), regulates the GDP/GTP exchange reaction of the rho proteins by inhibiting the dissociation of GDP from them, and the subsequent binding of GTP to them. In the present studies, we have isolated the cDNA of rho GDI from a bovine brain cDNA library using oligonucleotide probes designed from the partial amino acid sequences of the purified rho GDI and determined its complete nucleotide and deduced amino acid sequences. The cDNA contains an open reading frame encoding a protein of 204 amino acids with a calculated Mr value of 23,421. This Mr value is similar to those of the purified rho GDI estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultra-centrifugation, both of which are about 27,000. The rho GDI cDNA is expressed in Escherichia coli and COS7 cells and the encoded protein exhibits rho GDI activity. The 1.9-kilobase rho GDI mRNA corresponding to the isolated cDNA is detected in various rat tissues by Northern blot analysis. Hydropathy analysis indicates that rho GDI is overall hydrophilic except for one hydrophobic region. Computer homology search has revealed that rho GDI is a novel protein that does not share a high amino acid sequence homology with any known protein.
Amino Acid Sequence↗
Activation of the serum response element and 12-O-tetradecanoylphorbol-13-acetate response element by the activated c-raf-1 protein in a manner independent of protein kinase C.
Transfection of the cDNA encoding the activated c-raf-1 protein or addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) or dibutyryl cAMP to NIH/3T3 cells activated the c-fos gene enhancer linked to the chloramphenicol acetyltransferase or luciferase reporter gene. Prolonged treatment of NIH/3T3 cells with phorbol 12,13-dibutyrate caused down-regulation of protein kinase C. In these cells, addition of TPA did not stimulate the c-fos gene enhancer any more, but transfection of the c-raf-1 cDNA or addition of dibutyryl cAMP still stimulated the c-fos gene enhancer to the same extent as those induced in the control cells. Transfection of the c-raf-1 cDNA or addition of TPA to NIH/3T3 cells stimulated the serum response element and TPA response element but not the cAMP response element. In contrast, addition of dibutyryl cAMP to NIH/3T3 cells stimulated the cAMP response element but not the serum response element or TPA response element. These results indicate that the activated c-raf-1 protein stimulates the serum response element and TPA response element in a manner independent of protein kinase C and cAMP-dependent protein kinase. Since the c-fos gene enhancer has been shown to contain the serum response element and cAMP response element, it is most likely that the c-raf-1 protein is involved in the regulation of c-fos gene expression through the serum response element.
Animals↗
Molecular genetic analysis of the regulatory and catalytic domains of protein kinase C.
We have constructed the expression plasmids harboring protein kinase C (PKC) mutant cDNAs with a series of deletions in the PKC coding region. These plasmids were transfected into COS7 cells to characterize the PKC mutants. Immunoblot analysis using the anti-PKC antibody identified proteins with the Mr values expected from the PKC mutant cDNAs in the extracts from COS7 cells. The wild-type PKC, when expressed in COS7 cells, conferred increased phorbol ester binding activity on intact cells; but the PKC mutants with the deletion around the C1 region did not show this activity. The wild-type PKC showed protein kinase activity dependent on phospholipid, Ca2+, and phorbol ester, whereas these PKC mutants exhibited protein kinase activity independent of the activators in a cell-free system. A PKC mutant cDNA with the deletion in the C2 region gave increased phorbol ester binding activity. Protein kinase activity of this mutant was much less dependent on Ca2+ compared with the wild-type PKC. A PKC mutant cDNA with the deletion in the C3 region conferred increased phorbol ester binding activity, but neither activator-dependent nor -independent protein kinase activity. These results indicate that elimination of the C1 region of PKC gives rise to constitutively active PKC independent of phospholipid, Ca2+, and phorbol ester and that the C1-C3 regions play distinct roles in the regulatory and catalytic function of PKC. In another series of experiments, transfection of some PKC mutant cDNAs with the deletions around the C1 region into Chinese hamster ovary and Jurkat cells activated the activator protein-1-binding element or the c-fos gene enhancer linked to the chloramphenicol acetyltransferase reporter gene in the absence of phorbol ester. Microinjection of these constructs into Xenopus oocytes induced initiation of germinal vesicle breakdown, indicating that they stimulated the PKC pathway in vivo. Thus, the phorbol ester-independent PKC mutant cDNAs could be a powerful tool to investigate the transmembrane signaling pathway mediated by PKC.
Amino Acid Sequence↗
A protein kinase C cDNA without the regulatory domain is active after transfection in vivo in the absence of phorbol ester.
We constructed mutant protein kinase C (PKC) cDNAs which expressed PKC activity in vivo in the absence of phorbol ester activation. A hybrid PKC gene, PKAC, was constructed by substituting the coding region for the N-terminal 253 amino acids of PKC alpha with the N-terminal 17 amino acids of the cyclic AMP-dependent protein kinase catalytic subunit (PKA). A truncated PKC gene, delta PKC beta, lacking the coding region for amino acid positions 6 to 159 of PKC beta was also constructed. These mutant kinase genes expressed under the control of the SR alpha promoter activated the c-fos gene enhancer in Jurkat cells and initiated maturation of Xenopus laevis oocytes. Phorbol ester binding activity was absent in both constructs but was preserved in another hybrid gene, PKCA, which was composed of the coding region for 1 to 253 amino acids of PKC alpha at the N-terminal side and the coding region for 18 to 350 amino acids of PKA at the C-terminal side. These results indicate that elimination of the regulatory domain of PKC produces constitutively active PKC that can bypass activation by the phorbol ester. delta PKC beta, in synergy with a calcium ionophore, was capable of activating the interleukin 2 promoter, indicating that cooperation of PKC-dependent and calcium-dependent pathways is necessary for activation of the interleukin 2 gene.
Animals↗
[Modes of action of protein kinase C and growth factors].
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Animals↗
Isolation of a second yeast Saccharomyces cerevisiae gene (GPA2) coding for guanine nucleotide-binding regulatory protein: studies on its structure and possible functions.
In a previous paper, we demonstrated that a gene coding for a protein homologous to the alpha subunit of mammalian guanine nucleotide-binding regulatory (G) proteins occurs in Saccharomyces cerevisiae. The gene, designated GPA1, encodes a protein (GP1 alpha) of 472 amino acids with a calculated Mr of 54,075. Here we report the isolation of another G-protein-homologous gene, GPA2, which encodes an amino acid sequence of 449 amino acid residues with a Mr of 50,516. The predicted primary structure of the GPA2-encoded protein (GP2 alpha) is homologous to mammalian G proteins [inhibitory and stimulatory G proteins (Gi and Gs, respectively), a G protein of unknown function (Go), and transducins (Gt)] as well as yeast GP1 alpha. When aligned with the alpha subunit of Gi (Gi alpha) to obtain maximal homology, GP2 alpha was found to contain a stretch of 83 additional amino acid residues near the NH2 terminus. The gene was mapped in chromosome V, close to the centromere. Haploid cells carrying a disrupted GPA2 gene are viable. Cells carrying a high copy number of plasmid GPA2 (YEpGPA2) had markedly elevated levels of cAMP and could suppress a temperature-sensitive mutation of RAS2. These results suggest that GPA2 may be involved in the regulation of cAMP levels in S. cerevisiae.
Amino Acid Sequence↗
The role of G proteins in yeast signal transduction.
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Amino Acid Sequence↗
GPA1, a haploid-specific essential gene, encodes a yeast homolog of mammalian G protein which may be involved in mating factor signal transduction.
GPA1 protein of Saccharomyces cerevisiae is homologous to the alpha subunit of mammalian G protein. GPA1 transcript was found in haploid cells but was not detected in diploid cells. Disruption of GPA1 resulted in a haploid-specific lethal phenotype, indicating that GPA1 is a haploid-specific essential gene for cell growth. Upon regulation of expression of GPA1 by the galactose-inducible GAL1 promoter, the loss of GPA1 function was found to lead to cell-cycle arrest at the late G1 phase. Mutants that suppress the lethality of the gpa1::HIS3 mutation showed a sterile phenotype that was not cell-type-specific. These results suggest that GPA1 protein may control the signal for mating-factor-mediated cell-cycle arrest.
Cell Cycle↗
Direct evidence for involvement of a guanine nucleotide-binding protein in chemotactic peptide-stimulated formation of inositol bisphosphate and trisphosphate in differentiated human leukemic (HL-60) cells. Reconstitution with Gi or Go of the plasma membranes ADP-ribosylated by pertussis toxin.
fMet-Leu-Phe (fMLP) stimulated the formation of inositol bis- and trisphosphate in the [3H]inositol-labeled plasma membranes from the human leukemic (HL-60) cells differentiated to neutrophil-like cells by dibutyryl cyclic AMP. The stimulatory effect of fMLP was completely dependent on the simultaneous presence of GTP and Ca2+. The fMLP-stimulated formation of the phosphorylated inositols was markedly reduced by the prior ADP-ribosylation of the membranes with pertussis toxin. This toxin ADP-ribosylated a Mr approximately 40,000 protein, presumably the alpha subunit of Gi and/or Go, in the membranes. Reconstitution of the membranes ADP-ribosylated by pertussis toxin with Gi or Go purified from rat brain restored the fMLP-stimulated formation of the phosphorylated inositols. The efficiency of the rat brain Gi and Go in this capacity was roughly equal. The rat brain Gi or Go ADP-ribosylated beforehand by pertussis toxin was inactive in this reconstitution. These results indicate that both rat brain Gi and Go have the potency to couple functionally the fMLP receptor to the phospholipase C-mediated polyphosphoinositide hydrolysis and suggest that Gi or Go may be involved in the mechanism of signal transduction from the fMLP receptor to this reaction in the differentiated HL-60 cells.
Adenosine Diphosphate Ribose↗
Activation of protein kinase C by the action of 9,11-epithio-11,12-methano-thromboxane A2 (STA2), a stable analogue of thromboxane A2, in human platelets.
Incubation of human washed platelets with 9,11-epithio-11, 12-methano-thromboxane A2 (STA2), a stable analogue of thromboxane A2, caused the activation of protein kinase C and myosin light chain (MLC) kinase to the same extents as those induced by thrombin as judged by measuring the phosphorylation of a 40-kilodalton protein and MLC, respectively. However, STA2 stimulated much less phosphoinositide turnover than thrombin. Furthermore, the doses of STA2 necessary for protein kinase C activation and phosphoinositide turnover were higher than those necessary for MLC kinase activation, although the doses of thrombin necessary for these three reactions were nearly the same. These results suggest that protein kinase C may be activated at the Ca2+ concentrations higher than those required for MLC kinase activation by the action of STA2, presumably due to the inability of this agonist to produce diacylglycerol in an amount enough to increase the affinity of the enzyme for Ca2+.
Blood Platelets↗