Subcellular distribution in cerebral cortex of two proteins phosphorylated by a cAMP-dependent protein kinase.

Ueda, Tetsufumi; Greengard, Paul; Berzins, Klavs; Cohen, Rochelle S.; Blomberg, F.; Grab, Dennis J.; Siekevitz, Philip

doi:10.1083/jcb.83.2.308

Cited by 198 publications

(112 citation statements)

References 28 publications

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“…For example, synapsin I, a principle substrate of the kinase, is primarily associated with vesicles (25,53). Moreover, highly enriched vesicle fractions contain an endogenous protein kinase activity that phosphorylates synapsin I at the sites that are phosphorylated by purified synapsin I kinase (54,55).…”

Section: Discussionmentioning

confidence: 99%

“…It has been proposed that they are primarily structural, serving to anchor membrane or cytosolic proteins in the region of the postsynaptic membrane (21). The fraction is enriched in cyclic nucleotide phosphodiesterase activity (22,23), calmodulin (24), and both-cAMP- (25)(26)(27) and calmodulin-dependent protein kinase activities (27)(28)(29). Immunocytochemical evidence suggests that a calmodulin-dependent protein phosphatase, calcineurin, may also be located in PSDs in situ (30,31).…”

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confidence: 99%

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Biochemical and immunochemical evidence that the "major postsynaptic density protein" is a subunit of a calmodulin-dependent protein kinase.

Kennedy¹,

Bennett²,

Erondu³

1983

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

515

261

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By three criteria, two biochemical and one immunochemical, the major postsynaptic density protein (mPSDp) is indistinguishable from the 50-kilodalton (kDa) a subunit of a brain calmodulin-dependent protein kinase. First, the two proteins comigrate on NaDodSO4/polyacrylamide gels. Second, iodinated tryptic peptide maps of the two -are identical. Finally, a monoclonal antibody (6G9) that was raised against the protein kinase binds on immunoblots to a single 50 kDa band in crude brain homogenates and to both the a subunit of the purified kinase and the mPSDp from postsynaptic density fractions. The purified kinase holoenzyme also contains a 60-kDa subunit termed 13. A comparison of the peptide map of .8 with the maps of 60-kDa proteins from the postsynaptic density fraction suggests that ,B is present there but is not the only protein present in this molecular weight range. These results indicate that the calmodulin-dependent protein kinase is a major constituent of the postsynaptic density fraction and thus may be a component of type I postsynaptic densities.Many synaptic junctions in the central nervous system contain a prominent specialized structure called the "postsynaptic density" (PSD) (1-5). When viewed by electron microscopy, it is a fibrous, electron-opaque thickening lying opposite the presynaptic terminal, on the cytoplasmic side of the postsynaptic membrane. The morphology of postsynaptic densities is variable. Some are thick [20-60 nm (5)] and appear to cover the entire postsynaptic surface area, whereas others are thin, often discontinuous, patches (1, 6). The former have been termed type I PSDs, while the latter are called-type II (1). Some investigators have suggested that different types of densities are associated with synapses of particular types. For example, type I PSDs most often occur in synapses that are thought to be excitatory (1,7,8), whereas type II PSDs are seen in synapses thought to be inhibitory (9, 10). This hypothesis suggests.that morphologically distinct PSDs may also contain distinct proteins that serve specialized functions associated with their particular transmitter type.In order to understand the structure and function of PSDs, various research groups have developed subcellular fractionation methods to isolate highly enriched preparations of PSDlike material (11-13). These procedures involve osmotic lysis of a subcellular fraction enriched in synaptosomes, followed by purification of junctional membrane complexes and extraction of membrane components by treatment with detergent. The detergent-insoluble residue, purified by density gradient fractionation, consists of electron-opaque, fibrous, disk-shaped structures, 20-60 nm thick and 200-500 nm in diameter (11-15). These structures are -similar in appearance and staining characteristics to type I PSDs in intact fixed tissue, and they are not produced by detergent treatment of other subcellular organelles such as mitochondria or myelin (11). Thus, this subcellular fraction is considered to be highly enriched in type...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Biochemical and immunochemical evidence that the "major postsynaptic density protein" is a subunit of a calmodulin-dependent protein kinase.

Kennedy¹,

Bennett²,

Erondu³

1983

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

515

261

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“…Subcellular Fractionation of Mouse Brain-Subcellular fractionation of the mouse brain was performed as described previously (19,20). Briefly, adult mouse brains were homogenized in an ice-cold solution containing 0.32 M sucrose, 1 mM HEPES-KOH, pH 7.4, 1 mM NaHCO 3 , 1 mM MgCl 2 , 0.1 mM phenylmethanesulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM NaF, and 1 mM Na 3 VO 4 , and spun at 1,000 ϫ g and then 13,800 ϫ g, each for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Interaction with Protocadherin-γ Regulates the Cell Surface Expression of Protocadherin-α

Murata

Hamada

Morishita

et al. 2004

Journal of Biological Chemistry

102

106

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“…The subcellular fractions of bovine cerebrum were prepared as described previously (36). Synaptosomes were prepared from cerebra of male Sprague-Dawley rats (150 -200 g) and purified through the Percoll gradient centrifugation step, as described by Dunkley et al (37).…”

Section: Materials-[␥-mentioning

confidence: 99%

Glycolysis and Glutamate Accumulation into Synaptic Vesicles

Ikemoto

Bole

Ueda

2003

Journal of Biological Chemistry

173

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Glucose is the major source of brain energy and is essential for maintaining normal brain and neuronal function. Hypoglycemia causes impaired synaptic transmission. This occurs even before significant reduction in global cellular ATP concentration, and relationships among glycolysis, ATP supply, and synaptic transmission are not well understood. We demonstrate that the glycolytic enzymes glyceraldehyde phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate kinase (3-PGK) are enriched in synaptic vesicles, forming a functional complex, and that synaptic vesicles are capable of accumulating the excitatory neurotransmitter glutamate by harnessing ATP produced by vesiclebound GAPDH/3-PGK at the expense of their substrates. The GAPDH inhibitor iodoacetate suppressed GAPDH/ 3-PGK-dependent, but not exogenous ATP-dependent, Glycolysis plays a vital role in maintaining normal brain function. Glucose is known to serve as the major substrate for cerebral energy under normal conditions (1). Recent evidence suggests a direct correlation between glucose utilization and cognitive function (2). Reduction of glucose levels results in pathophysiological states and abnormal electrophysiological activity; however, this occurs long before significant alteration in tissue ATP levels is detected (3-7). Substitution of pyruvate for glucose does not support normal evoked neuronal activity, although tissue ATP level returns to normal (8 -10). Abnormal synaptic transmission caused by hypoglycemia occurs in part if not entirely by a presynaptic mechanism (7,11,12). Fleck et al. (7) have shown that substantial reduction of extracellular glucose results in a decrease in stimulus-evoked Glu release, with no changes in ATP levels. These studies together suggest that glycolysis or glycolytic intermediate(s) are necessary for normal synaptic transmission independent of global cellular ATP levels.In an attempt to reveal the underlying mechanism of hypoglycemia-induced aberrant synaptic transmission, we previously explored the possibility that glycolytic intermediates could modify proteins localized in the nerve ending (13,14). 3-Phosphoglycerate (3-PG) 1 was demonstrated to stimulate phosphorylation of 155-and 72-kDa proteins. The latter was identified as glucose-1,6-bisphosphate synthetase, and 1,3-bisphosphoglycerate (1,3-BPG) was found to serve as the direct substrate for phosphorylation of this enzyme, by donating 1-phosphate. Both of these phosphorylated proteins are enriched in the synaptosomal (nerve ending preparation) as well as cell body soluble fractions, but the significance of these modifications in synaptic transmission remains unclear.In this paper, we show that the glycolytic intermediate 1,3-BPG forms an acyl-enzyme intermediate with vesicle-bound glyceraldehyde phosphate dehydrogenase (GAPDH), that vesicle-bound GAPDH exists in a complex with 3-phosphoglycerate kinase (3-PGK), and that activation of vesicle-associated GAPDH and 3-PGK is sufficient to support vesicular uptake of Glu. Glutamate is now recognized as the major ex...

show abstract

Subcellular distribution in cerebral cortex of two proteins phosphorylated by a cAMP-dependent protein kinase.

Cited by 198 publications

References 28 publications

Biochemical and immunochemical evidence that the "major postsynaptic density protein" is a subunit of a calmodulin-dependent protein kinase.

Biochemical and immunochemical evidence that the "major postsynaptic density protein" is a subunit of a calmodulin-dependent protein kinase.

Interaction with Protocadherin-γ Regulates the Cell Surface Expression of Protocadherin-α

Glycolysis and Glutamate Accumulation into Synaptic Vesicles

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