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Biomedical subjects

K Kaibuchi

Publications and source records attributed to K Kaibuchi.

At least 163 records · Page 9Linked to original sources
PubMed1993

Rab3A GTPase-activating protein-inhibiting activity of Rabphilin-3A, a putative Rab3A target protein.

Rabphilin-3A is a putative target protein for Rab3A, a member of the small G protein superfamily that is implicated in regulated secretion, particularly in neurotransmitter release. Rabphilin-3A contains at least two functionally different domains: the N-terminal Rab3A-binding domain and the C-terminal C2 domain, which interacts with both Ca2+ and phospholipid. Because Rabphilin-3A interacts preferentially with GTP-Rab3A rather than with GDP-Rab3A, we have examined here whether Rabphilin-3A affects the GTPase activity of Rab3A. Rabphilin-3A and its N-terminal fragment, but not its C-terminal fragment, very weakly stimulated the basal GTPase activity of Rab3A. However, Rabphilin-3A and its N-terminal fragment strongly inhibited the Rab3A GAP-stimulated GTPase activity of Rab3A. Ca2+ and phospholipid showed no effect on these activities of Rabphilin-3A. The physiological significance of the GAP activity of Rabphilin-3A is obscure, but it is likely that Rabphilin-3A inhibits Rab3A GAP activity and keeps Rab3A in the GTP-bound active form during its action as a target molecule for Rab3A.

Adaptor Proteins, Signal Transducing↗
PubMed1993

Interaction of Sec4 with GDI proteins from bovine brain, Drosophila melanogaster and Saccharomyces cerevisiae. Conservation of GDI membrane dissociation activity.

Rab GDP dissociation inhibitor (Rab GDI), will induce the dissociation of GDP-bound rab3A from synaptic membranes and will inhibit GDP dissociation from Sec4, a member of the Rab subgroup of the Ras GTPase superfamily which is required for exocytosis in Saccharomyces cerevisiae. We report that Rab GDI releases GDP-bound Sec4 from yeast membranes. dGDI, a Drosophila homologue can similarly inhibit GDP dissociation from Sec4 and release GDP-bound Sec4 from yeast membranes. An activity partially purified from yeast cytosol dissociates GDP-bound Sec4 from yeast membranes, suggesting that yeast also possess a GDI protein that functions to recycle Sec4 from its target membrane.

Animals↗
PubMed1993

Combination of arachidonic acid and guanosine 5'-O-(3-thiotriphosphate) induce translocation of rac p21s to membrane and activation of NADPH oxidase in a cell-free system.

The superoxide-generating NADPH oxidase system in phagocytes consists of membrane-associated cytochrome b558 and three cytosolic components named p67-phox, p47-phox, and rac p21s. In a cell-free system consisting of membrane and cytosol, the oxidase can be activated with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and an unsaturated fatty acid such as arachidonic acid (AA). Incubation of cytosol and membrane with AA alone caused clear translocation of p47-phox and p67-phox to the membrane, but only slight translocation of rac p21s. GTP gamma S alone did not significantly induce the translocation of rac p21s. However, GTP gamma S in combination with AA markedly augmented rac p21s translocation to the membrane. The translocation of rac p21s is not induced by GDP or GDP beta S. These results indicate that the GTP-bound active form of rac p21s is the entity that is translocated to the membrane by the action of AA.

Amino Acid Sequence↗
PubMed1993

Rab GDP dissociation inhibitor as a general regulator for the membrane association of rab proteins.

Rab proteins comprise a family of small GTPases that serve a regulatory role in membrane traffic. These proteins are in part cytosolic and in part associated with the membranes of specific exocytic and endocytic organelles. Smg p25A/rab3A GDI, a cytosolic protein which inhibits the dissociation of GDP from smg p25A/rab3A, Sec4p, and rab11, has also been found to prevent association of rab3A with the membrane. In this study, we have used Madin-Darby canine kidney cells permeabilized with the bacterial toxin streptolysin O to test the general activity of rab3A GDI in modulating the membrane association of various small GTP-binding proteins. Rab3A GDP dissociation inhibitor (GDI) removed from the membrane all rab proteins we have tested and inhibited the membrane binding of in vitro translated rab proteins. However, rab3A GDI had a limited effect on the membrane association of a mutant rab5 protein which contained a farnesylated cysteine motif. Finally, we found that, although rab3A GDI resides primarily in the cytosol, it is also associated with compartments of the exocytic and endocytic pathways. Since rab3A GDI can modulate the membrane association of various rab proteins, we propose to rename it rab GDI.

Amino Acid Sequence↗
PubMed1993

Translocation of Ki-ras p21 between membrane and cytoplasm by smg GDS.

We have previously shown that smg GDS forms a complex with Ki-ras 4B p21 and thereby stimulates its GDP/GTP exchange reaction. We have shown here that smg GDS inhibits the binding of Ki-ras 4B p21 to membranes and induces the dissociation of prebound Ki-ras 4B p21 from membranes. Moreover, we have found that cotransfection of the smg GDS and Ki-ras 4B p21 cDNAs into COS7 cells increases the ratio of the cytosolic form of ras p21 to the membrane-bound form and the ratio of the GTP-bound form of ras p21 to the GDP-bound form. These results indicate that smg GDS regulates not only the GDP/GTP exchange reaction of Ki-ras 4B p21 but also its translocation between membrane and cytoplasm.

Animals↗
PubMed1993

The post-translational processing of ras p21 is critical for its stimulation of mitogen-activated protein kinase.

The point-mutated active form of ras p21 is known to activate mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) in intact mammalian cells and Xenopus oocytes, although the direct target molecule of ras p21 remains to be identified. To elucidate the role of the post-translational processing of ras p21 for the MAP kinase activation, we established the cell-free system in which ras p21 activated MAP kinase. The guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) bound form of post-translationally processed Ki-ras 4B p21 activated MAP kinase in the cytosol fraction of Xenopus oocytes, but the GTP gamma S bound form of post-translationally unprocessed Ki-ras 4B p21 or the GDP bound form of processed or unprocessed Ki-ras 4B p21 was far less effective. The GTP gamma S bound form of processed Ki-ras 4B p21 activated recombinant ERK2 in the presence of the cytosol fraction of Xenopus oocytes, but the unprocessed protein was far less effective. These results provide a complete biochemical assay for ras p21 to activate MAP kinase in a cell-free system and indicate that all the elements downstream of ras p21 necessary for the MAP kinase activation are cytosolic and that the post-translational processing of ras p21 is important for the MAP kinase activation.

Animals↗
PubMed1993

A protein factor for ras p21-dependent activation of mitogen-activated protein (MAP) kinase through MAP kinase kinase.

To identify the direct target molecule of ras p21 in higher eukaryotes, we have recently developed the cell-free system in which ras p21 activates mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK). In this cell-free system, the guanosine 5'-[gamma-thio]triphosphate- bound form of Ki-ras p21, but not the GDP-bound form, activates endogenous Xenopus MAP kinase as well as recombinant ERK2 in the presence of the cytosol fraction of Xenopus oocytes. We separated two protein factors from the cytosol fraction of Xenopus oocytes by column chromatography: one was the inactive form of MAP kinase kinase and the other was a factor tentatively named ras p21-dependent ERK-kinase stimulator (REKS). The former and latter showed M(r) values of approximately 45,000 and 150,000-200,000, respectively, as estimated by gel filtration. Both factors were necessary for Ki-ras p21-dependent activation of MAP kinase/ERK2. These results indicate that an additional protein factor (REKS) is essential for Ki-ras p21 to activate MAP kinase through MAP kinase kinase.

Animals↗
PubMed1993

Regulators of small GTPases.

Small GTPases are converted from the GDP-bound inactive form to the GTP-bound active form by a GDP/GTP exchange reaction which is regulated by GDP/GTP exchange proteins (GEPs). We have found both stimulatory and inhibitory GEPs, which we have named GDP dissociation stimulators (GDSs) and GDP dissociation inhibitors (GDIs) respectively. We have isolated Smg GDS, Rho GDI and Rab GDI, cloned them, and determined their primary structures. These GEPs are active on a group of small GTPases: Smg GDS on at least K-Ras, Rap1/Smg21, Rho and Rac; Rho GDI on at least Rho, Rac and Cdc42; Rab GDI on most of the Rab family members. These GEPs have an additional function, regulating the translocation of their substrate small GTPases between the membrane and the cytosol. The GEPs interact only with the post-translationally modified form of their substrate small GTPases.

Animals↗
PubMed1993

Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin.

In a previous study (H. Shirataki, K. Kaibuchi, T. Yamaguchi, K. Wada, H. Horiuchi, and Y. Takai, J. Biol. Chem. 267:10946-10949, 1992), we highly purified from bovine brain crude membranes the putative target protein for smg p25A/rab3A p25, a ras p21-related small GTP-binding protein implicated in neurotransmitter release. In this study, we have isolated and sequenced the cDNA of this protein from a bovine brain cDNA library. The cDNA had an open reading frame encoding a protein of 704 amino acids with a calculated M(r) of 77,976. We tentatively refer to this protein as rabphilin-3A. Structural analysis of rabphilin-3A revealed the existence of two copies of an internal repeat that were homologous to the C2 domain of protein kinase C as described for synaptotagmin, which is known to be localized in the membrane of the synaptic vesicle and to bind to membrane phospholipid in a Ca(2+)-dependent manner. The isolated cDNA was expressed in COS7 cells, and the encoded protein was recognized with an anti-rabphilin-3A polyclonal antibody and was identical in size with rabphilin-3A purified from bovine brain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, both rabphilin-3A purified from bovine brain and recombinant rabphilin-3A made a complex with the GTP gamma S-bound form of rab3A p25 but not with the GDP-bound form of rab3A p25. Immunoblot and Northern (RNA) blot analyses showed that rabphilin-3A was highly expressed in bovine and rat brains. These results indicate that rabphilin-3A is a novel protein that has C2 domains and selectively interacts with the GTP-bound form of rab3A p25.

Adaptor Proteins, Signal Transducing↗
PubMed1992

Post-translational processing of rac p21s is important both for their interaction with the GDP/GTP exchange proteins and for their activation of NADPH oxidase.

rac1 and rac2 p21s are ras p21-like small GTP-binding proteins which are implicated in the NADPH oxidase-catalyzed superoxide generation in phagocytes. rac1 and rac2 p21s have a Cys-A-A-Leu (A = aliphatic amino acid) structure in their C-terminal region which may undergo post-translational processing including prenylation, proteolysis, and carboxyl methylation. We studied the function of this post-translational processing of rac p21s in their interaction with the stimulatory and inhibitory GDP/GTP exchange proteins for rac p21s, named smg GDS and rho GDI, and in their NADPH oxidase activation. We produced human recombinant rac1 and rac2 p21s in insect cells and purified them from the membrane and soluble fractions as the post-translationally processed and unprocessed forms, respectively. Post-translationally processed rac1 and rac2 p21s were sensitive to both smg GDS and rho GDI, but post-translationally unprocessed rac1 and rac2 p21s were insensitive to them. The GTP gamma S (guanosine 5'-(3-O-thio)triphosphate)-bound form of post-translationally processed rac1 and rac2 p21s stimulated the NADPH oxidase activity, but post-translationally unprocessed rac1 and rac2 p21s were far less effective. These results indicate that both rac1 and rac2 p21s stimulate the NADPH oxidase activity and that their post-translational processing is important not only for their interaction with smg GDS and rho GDI but also for their NADPH oxidase activation.

Amino Acid Sequence↗
PubMed1992

Protein kinase C mutants in the auto-inhibitory region exhibit two distinct properties.

To define the role of the auto-inhibitory region of protein kinase C (PKC), Arg22-Lys23-Gly24-Ala25-Leu26-Arg27, site-directed mutations were introduced into the basic residues. Three mutants, PKCAla22,23, PKCAla27, and PKCAla22,23,27, apparently fell into two distinct types with regard to their biochemical properties and biological activities, as judged by the enhancement of a c-fos promoter in Jurkat cells and by the initiation of germinal vesicle breakdown (GVBD) in Xenopus laevis oocytes. (i) PKCAla22,23 and PKCAla27 had activators independent in vitro kinase activity, high phosphorylation levels in vivo, and localized in both cytosolic and particulate fractions. These mutants were not fully biologically active. (ii) PKCAla22,23,27 had a low phosphorylation level in vivo, was found predominantly in the particulate fraction and was the most biologically active. These results suggest that basic residues in the auto-inhibitory domain account for the regulation of kinase activity and the cytosolic retention of PKC. The particulate association or the cytosolic clearance of PKC may facilitate signal transduction in the cell.

Amino Acid Sequence↗
PubMed1992

A possible target protein for smg-25A/rab3A small GTP-binding protein.

The smg-25A/rab3A protein (smg p25A) is a small GTP-binding protein implicated in intracellular vesicle traffic, particularly in neurotransmitter release from the presynapse. In the present study, we attempted to identify a target protein in bovine brain crude membranes that might be interacted with the GTP-bound form of smg p25A. When the guanosine-5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of radioiodinated smg p25A and the crude membrane fraction of bovine brain were incubated with a cross-linker, disuccinimidyl suberate, and the sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography, one radioactive band with a M(r) of about 110,000 was detected. This radioactive band appeared to be composed of radioiodinated smg p25A and a molecule with a M(r) of about 86,000. This molecule, tentatively termed here smg p25A target, was extracted from the membranes by a detergent and highly purified by column chromatographies and sucrose density gradient ultracentrifugation. The purified smg p25A target was sensitive to heat boiling and tryptic digestion, indicating that smg p25A target is a protein molecule. The M(r) of the purified smg p25A target was estimated to be about 85,000-86,000 from SDS-PAGE and to be about 100,000 from the S value. The cross-linking of radioiodinated smg p25A with the purified smg p25A target was inhibited by the GTP gamma S-bound form of non-radioactive smg p25A with an IC50 of about 8 nM. The GDP-bound form of smg p25A was much less effective. Other small GTP-binding proteins, such as c-Ki-ras p21, rhoA p21, smg p21B, and rab11 p24 were ineffective. These results indicate that a protein with a M(r) of about 85,000-100,000 is a target for smg p25A.

Animals↗
PubMed1992

Regulation of the superoxide-generating NADPH oxidase by a small GTP-binding protein and its stimulatory and inhibitory GDP/GTP exchange proteins.

The superoxide-generating NADPH oxidase system in phagocytes consists of at least membrane-associated cytochrome b558 and three cytosolic components named SOCI/NCF-3/sigma 1/C1, SOCII/NCF-1/p47-phox, and SO-CIII/NCF-2/p67-phox. p47-phox and p67-phox were isolated, and their primary structures were determined, but SOCI has not been well characterized. In the present study, we first purified SOCI to homogeneity from the cytosol fraction of the differentiated HL-60 cells. The purified SOCI was a small GTP-binding protein (G protein) with a M(r) of about 22,000. The guanosine 5'-(3-O-thio)triphosphate-bound form, but not the GDP-bound form, of this small G protein showed the SOCI activity. The partial amino acid sequence of SOCI thus far determined was identical to the amino acid sequence deduced from the cDNA encoding rac2 p21. None of the purified small G proteins, including Ki-ras p21, smg p21B/rap1B p21, rhoA p21, and rac1 p21, showed the SOCI activity. These results indicate that SOCI is a small G protein very similar, if not identical, to rac2 p21. The GDP/GTP exchange reaction of SOCI was stimulated and inhibited by stimulatory and inhibitory GDP/GTP exchange proteins for small G proteins, named smg GDS and rho GDI, respectively. The NADPH oxidase activity was also stimulated and inhibited by smg GDS and rho GDI, respectively. These results indicate that the superoxide-generating NADPH oxidase system is regulated by both smg GDS and rho GDI through rac2 p21 or the rac2-related small G protein in phagocytes.

Amino Acid Sequence↗
PubMed1992

Involvement of rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction.

In the rabbit mesenteric arterial smooth muscle skinned by saponin, Ca2+ induced contraction in a concentration-dependent manner. Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), a non-hydrolyzable GTP analogue, lowered the Ca2+ concentrations required for this contraction and increased the Ca2+ sensitivity of the skinned smooth muscle contraction. GTP gamma S alone did not induce the contraction in the absence of Ca2+. This GTP gamma S-enhanced Ca2+ sensitivity was completely abolished by an exoenzyme of Staphylococcus aureus, named EDIN, and an exoenzyme of Clostridium botulinum, named C3, both of which are known to ADP-ribosylate the rho p21 family that belongs to the ras p21-like small GTP-binding protein superfamily. The GTP gamma S-bound form of rhoA p21 overcame the inhibitory action of EDIN. smg p21B, another small GTP-binding protein, was inactive. EDIN ADP-ribosylated a protein, which was most likely to be rho p21, in the skinned smooth muscle. The GTP gamma S-bound form of rhoA p21, but not the GDP-bound form, substituted for GTP gamma S and enhanced the Ca2+ sensitivity of the skinned smooth muscle contraction. smg p21B was inactive. These results indicate that rhoA p21 is involved in the GTP gamma S-enhanced Ca2+ sensitivity of the smooth muscle contraction.

Adenosine Diphosphate Ribose↗
PubMed1992

Phosphorylation of smg p21B/rap1B p21 by cyclic GMP-dependent protein kinase.

smg p21B/rap1B p21, a member of ras p21-like small GTP-binding protein superfamily, has been shown to be phosphorylated by cyclic AMP-dependent protein kinase (protein kinase A). We show here that this protein was also phosphorylated by cyclic GMP-dependent protein kinase (protein kinase G) in a cell-free system. The same serine residue (Ser179) in the C-terminal region was phosphorylated by both protein kinases G and A. The Km and Vmax values of smg p21B for protein kinase G were 5 x 10(-7) M and 4 x 10(-9) mol/min/mg, and those values for protein kinase A were 1 x 10(-7) M and 3 x 10(-8) mol/min/mg.

Autoradiography↗
PubMed1992

Both stimulatory and inhibitory GDP/GTP exchange proteins, smg GDS and rho GDI, are active on multiple small GTP-binding proteins.

Six peaks of small GTP-binding proteins (G proteins) were separated by column chromatographies from the cytosol fraction of the differentiated HL-60 cells: two peaks of rho p21, one peak of smg/rap1 p21, two peaks of rac1 p21, and one peak of an unidentified small G protein with a Mr of about 20,000 (20 KG). smg GDS, previously thought to be a stimulatory GDP/GTP exchange protein for smg p21, Ki-ras p21, and rho p21, but not for Ha-ras p21 or smg p25A, was also active on rac1 p21. rho GDI, previously thought to be an inhibitory GDP/GTP exchange protein specific for rho p21, was also active on rac1 p21. These results indicate that both smg GDS and rho GDI are active on multiple small G proteins.

Cell Differentiation↗
PubMed1992

Transforming and c-fos promoter/enhancer-stimulating activities of a stimulatory GDP/GTP exchange protein for small GTP-binding proteins.

smg GDP dissociation stimulator (GDS) is a stimulatory GDP/GTP exchange protein for a group of ras p21-like small GTP-binding proteins (G proteins) including c-Ki-ras p21, smg p21A, smg p21B, and rhoA p21. smg GDS converts the GDP-bound inactive form to the GTP-bound active form of each small G protein by stimulating their GDP/GTP exchange reaction in a cell-free system. The point-mutated c-Ki-ras p21 (c-Ki-rasval12 p21) is known to strongly transform NIH/3T3 cells and to markedly stimulate the c-fos promoter/enhancer in this cell line, whereas the normal c-Ki-ras p21 is weak in these activities. In the present study, we examined the effect of smg GDS on these activities to explore its physiological function. Overexpression of both smg GDS and c-Ki-ras p21 strongly transformed NIH/3T3 cells, whereas overexpression of either smg GDS or c-Ki-ras p21 alone weakly transformed the cells. Furthermore, overexpression of both smg GDS and c-Ki-ras p21 markedly stimulated the c-fos promoter/enhancer in NIH/3T3 cells, whereas overexpression of either smg GDS or c-Ki-ras p21 alone weakly stimulated it. These results indicate that smg GDS transforms NIH/3T3 cells and stimulates the c-fos promoter/enhancer in this cell line in cooperation with c-Ki-ras p21.

3T3 Cells↗