Localization and subcellular distribution of cellular ras gene products in rat brain

Mizoguchi, Akira; Ueda, Takashi; Ikeda, Keisuke; Shiku, Hiroshi; Mizoguti, Humio; Takai, Yoshimi

doi:10.1016/0169-328x(89)90015-6

Cited by 53 publications

(24 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Subceilular fractionation of rat cerebrum was carried out by the method of Ueda et al (31), which was a slight modification of the method of Cotman and Taylor (3) and that of Whittaker et al (32). The detailed procedures have been described previously (19). Electron microscopic analysis of all subcellular fractions indicated that the ultrastructural characteristics of these fractions obtained by our procedures were similar to those reported previously by Ueda et al (31).…”

Section: Methodssupporting

confidence: 56%

“…smg p21B is abundant in human platelets, and its amount is at least 10 times higher than that described for ras p21s (22 These results suggest that smg p2ls are present in synaptic plasma membranes, synaptic vesicles, and intrasynaptosomal mitochondria and in the components of the neuronal cell bodies such as endoplasmic reticulum and mitochondria. We have previously shown that ras p2is are found in the synaptic plasma membrane fraction but not in the vesicle, mitochondrial, or cytosol fraction (19). Moreover, we have found that smg p25A is abundant in the synaptosome fraction (13; Mizoguchi et al, submitted).…”

Section: Discussionmentioning

confidence: 70%

See 1 more Smart Citation

Tissue and subcellular distributions of the smg-21/rap1/Krev-1 proteins which are partly distinct from those of c-ras p21s.

Kim¹,

Mizoguchi²,

Kikuchi³

et al. 1990

Mol. Cell. Biol.

View full text Add to dashboard Cite

We have made a specific antiserum recognizing both smg p21A (the raplAlKrev-l protein) and -B (the raplB protein), ras p21-like GTP-binding proteins having the same putative effector domain as ras p2ls and have used this antiserum to study the tissue and subcellular distributions of smg p2ls by immunoblot and inmunocytochemical analyses. By immunoblot analysis, smg p2ls were detected in various rat tissues and at the highest level in brain. By light microscopic immunocytochemical analysis, smg p2ls were also detected in various rat tissues. Particularly, smg p2ls in brain were found abundantly in the cytoplasmic region of most types of neuronal cell bodies and moderately in neuropil, whereas c-ras p2ls were found more abundantly in neuropil than in the cytoplasmic region of most types of neuronal cell bodies. smg p2ls in testis were found in spermatogenic cells, in which c-ras p2ls were not significantly detected. By subcellular fractionation analysis of cerebrum, smg p2ls were detected in all of the particulate fractions but not in the cytosol fraction. Among the particulate fractions, approximately 70% of smg p2ls was recovered with the highest specific content in the fraction containing mainly synaptosomes, mitochondria, and myelin. In further fractionation of this fraction, approximately 40% of smg p2ls was recovered in each of the synaptosome fraction and the mitochondrial fraction. This subceilular distribution of smg p2ls in cerebrum was partly distinct from that of c-ras p21s, which were mainly recovered in the synaptosome and microsome fractions but present at very low levels in the mitochondrial fraction. In the synaptosome fraction, smg p2ls were recovered mainly in the synaptic plasma membrane fraction and partly in the synaptic vesicle and mitochondrial fractions with similar specific contents but not in the synaptic cytosol fraction. This intrasynaptosomal distribution of smg p2ls was also partly distinct from that of c-ras p2ls, which were recovered in the synaptic plasma membrane fraction but not in the synaptic vesicle or mitochondrial fraction. These tissue and subcellular distributions of smg p2ls together with the fact that smg p2ls have the same putative effector domain as ras p2ls suggest that smg p2ls exert their own specific actions in addition to the actions similar or antagonistic to those of c-ras p2ls.

show abstract

Section: Methodssupporting

confidence: 56%

Section: Discussionmentioning

confidence: 70%

Tissue and subcellular distributions of the smg-21/rap1/Krev-1 proteins which are partly distinct from those of c-ras p21s.

Kim¹,

Mizoguchi²,

Kikuchi³

et al. 1990

Mol. Cell. Biol.

View full text Add to dashboard Cite

show abstract

“…Primary cultured rat hippocampal neurons were prepared as described previously (14). CSV of rat brain was prepared as described previously (15).…”

mentioning

confidence: 99%

“…Miscellaneous Procedures-SDS-PAGE (16), subcellular fractionation of rat brain (15), immunofluorescence microscopy of frozen sections (17) and cultured cells (18), and immunoelectron microscopy (19) were performed as described. Protein concentrations were determined with bovine serum albumin as a reference protein as described previously (20).…”

mentioning

confidence: 99%

Rabconnectin-3, a Novel Protein That Binds Both GDP/GTP Exchange Protein and GTPase-activating Protein for Rab3 Small G Protein Family

Nagano¹,

Kawabe²,

Nakanishi³

et al. 2002

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Rab3A, a member of the Rab3 small G protein family, regulates Ca2؉ -dependent exocytosis of neurotransmitter. The cyclical activation and inactivation of Rab3A are essential for the Rab3A action in exocytosis. GDPRab3A is activated to GTP-Rab3A by Rab3 GDP/GTP exchange protein (Rab3 GEP), and GTP-Rab3A is inactivated to GDP-Rab3A by Rab3 GTPase-activating protein (Rab3 GAP). It remains unknown how or in which step of the multiple exocytosis steps these regulators are activated and inactivated. We isolated here a novel protein that was co-immunoprecipitated with Rab3 GEP and GAP by their respective antibodies from the crude synaptic vesicle fraction of rat brain. The protein, named rabconnectin-3, bound both Rab3 GEP and GAP. The cDNA of rabconnectin-3 was cloned from a human cDNA library and its primary structure was determined. Human rabconnectin-3 consisted of 3,036 amino acids and showed a calculated M r of 339,753. It had 12 WD domains. Tissue and subcellular distribution analyses in rat indicated that rabconnectin-3 was abundantly expressed in the brain where it was enriched in the synaptic vesicle fraction. Immunofluorescence and immunoelectron microscopy revealed that rabconnectin-3 was concentrated on synaptic vesicles at synapses.These results indicate that rabconnectin-3 serves as a scaffold molecule for both Rab3 GEP and GAP on synaptic vesicles.Rab3A is a member of the Rab3 family, along with Rab3B, -3C, and -3D, and plays a key regulatory role in Ca 2ϩ -dependent exocytosis of neurotransmitter (for reviews, see Refs. 1-3). The process of Ca 2ϩ -dependent exocytosis of neurotransmitter consists of several steps: translocation of synaptic vesicles from the reserve pool to the active zone of the presynaptic plasma membrane where a Ca 2ϩ channel localizes, docking of the vesicles to the active zone, transition from the docking to the priming of the vesicles in the readily releasable pool, and fusion of the vesicles with the membrane induced by Ca 2ϩ influx (1-3). The Rab3A gene knockout mouse analysis has revealed two actions of Rab3A: it facilitates the translocation and docking of synaptic vesicles to the presynaptic plasma membrane (4), and it prevents Ca 2ϩ -triggered fusion of the vesicles with the plasma membrane (5).The Rab3 family members are regulated by three regulators: Rab GDI 1 and Rab3 GEP and GAP (1-3). Rab3 GEP and GAP are specific for the Rab3 family members, but Rab GDI is active on all the Rab family members. The cyclical activation and inactivation of Rab3A by the action of these regulators are essential for Ca 2ϩ -dependent exocytosis of neurotransmitter. A current model for the mode of action of these regulators is as follows: GDP-Rab3A is kept in the cytosol in a complex with Rab GDI. This complex is recruited to synaptic vesicles where GDP-Rab3A is activated to GTP-Rab3A by the action of Rab3 GEP with the help of another unidentified molecule, such as GDI displacement factor (6) or Rab recycling factor (7) proposed for other vesicle trafficking systems. GTP-Rab3A binds to its two...

show abstract

“…Several of these proteins have been isolated from bovine brain membrane preparations (27,28), and of particular interest was the presence of the G proteins c-ras p21, sing p25/Rab-3, and smg p21/Rap-i in synaptic areas (29)(30)(31). These three proteins were enriched in synaptic plasma membranes, and the latter two, which had been purified from brain membranes and cloned from brain cDNA libraries (27,28,32), were also enriched in synaptic vesicles (29)(30)(31) (4); the concentrations of purified CaM kinases II and Gr were about 100 ,ug/ml and 25 ,ug/ml, respectively. Protein kinase C (PKC) was purified by modification of a method using ion-exchange and hydrophobic affinity chromatography (33).…”

mentioning

confidence: 99%

Phosphorylation of a Ras-related GTP-binding protein, Rap-1b, by a neuronal Ca2+/calmodulin-dependent protein kinase, CaM kinase Gr.

Sahyoun

McDonald

Farrell

et al. 1991

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

View full text Add to dashboard Cite

A neuron-specific Ca2_/calmodulin-dependent protein kinase, CaM kinase Gr, phosphorylates selectively a Ras-related GTP-binding protein (Rap-lb) (27,28), and of particular interest was the presence of the G proteins c-ras p21, sing p25/Rab-3, and smg p21/Rap-i in synaptic areas (29-31). These three proteins were enriched in synaptic plasma membranes, and the latter two, which had been purified from brain membranes and cloned from brain cDNA libraries (27,28,32), were also enriched in synaptic vesicles (29-31). Accordingly, these three members of the Ras superfamily may be implicated in important synaptic functions such as signal transduction and/or neurotransmitter release.Here we show that the neuronal CaM kinase Gr selectively and stoichiometrically phosphorylates the small G protein Rap-lb, thus potentially establishing a link between a neuronal Ca2+/calmodulin-dependent signaling mechanism and a low molecular weight G-protein signal-transduction pathway. The data are discussed in the context of other relevant neuronal protein kinases, their substrates, synaptic localization, and regulation by autophosphorylation. MATERIALS AND METHODS[y-32P]ATP (t30 Ci/mmol; 1 Ci -37 GBq) was purchased from Du Pont/NEN. Synthetic peptides la and lb were supplied by Bio-synthesis (Denton, TX). Syntide-2 was provided by J. McDermed (Wellcome Research Laboratories), and Kemptide was bought from Sigma. Catalytic subunit of cAMP-dependent protein kinase (PKA), phosphatidylserine, and 4a-phorbol 12,13-dibutyrate (PDBu) were also from Sigma. CaM kinase II and CaM kinase Gr were purified from the forebrain and cerebellum, respectively, of adult male Sprague-Dawley rats by ion-exchange, gel-permeation, and calmodulin-agarose chromatography (4); the concentrations of purified CaM kinases II and Gr were about 100 ,ug/ml and 25 ,ug/ml, respectively. Protein kinase C (PKC) was purified by modification of a method using ion-exchange and hydrophobic affinity chromatography (33). Recombinant 2643The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

show abstract

Localization and subcellular distribution of cellular ras gene products in rat brain

Cited by 53 publications

References 45 publications

Tissue and subcellular distributions of the smg-21/rap1/Krev-1 proteins which are partly distinct from those of c-ras p21s.

Tissue and subcellular distributions of the smg-21/rap1/Krev-1 proteins which are partly distinct from those of c-ras p21s.

Rabconnectin-3, a Novel Protein That Binds Both GDP/GTP Exchange Protein and GTPase-activating Protein for Rab3 Small G Protein Family

Phosphorylation of a Ras-related GTP-binding protein, Rap-1b, by a neuronal Ca2+/calmodulin-dependent protein kinase, CaM kinase Gr.

Contact Info

Product

Resources

About