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Identification of a major autophosphorylation site on postsynaptic density-associated Ca2+/calmodulin-dependent protein kinase
Year:
1994
Source of publication :
Journal of Biological Chemistry
Authors :
Gollop, Natan
;
.
Volume :
269
Co-Authors:
Dosemeci, A., Laboratory of Neurobiology, National Institutes of Health, Bethesda, MD 20892, United States, Laboratory of Neurobiology, Bldg. 36, National Institutes of Health, Bethesda, MD 20892, United States
Gollop, N., Laboratory of Biochemical Genetics, NHLBI, National Institutes of Health, Bethesda, MD 20892, United States
Jaffe, H., Laboratory of Neurochemistry, NINDS, National Institutes of Health, Bethesda, MD 20892, United States
Facilitators :
From page:
31330
To page:
31333
(
Total pages:
4
)
Abstract:
One of the most abundant proteins in postsynaptic densities is identical or very similar to the α-subunit of the Ca2+/calmodulin-dependent protein kinase II. Auto-phosphorylation of this protein in isolated postsynaptic densities was studied under various conditions, following inhibition of endogenous phosphatase activity with microcystin-LR. Phosphorylation accompanied by a shift in the enzyme's electrophoretic mobility was observed upon incubation with Ca2+ and calmodulin at 37°C. Brief incubation with Ca2+ and calmodulin at O°C resulted in a low level of phosphorylation and no change in mobility. Following this limited Ca2+-dependent phosphorylation, however, a high level of phosphorylation could be achieved in the absence of Ca2+, upon incubation at 37°C. Comparison of reverse-phase HPLC phosphopeptide elution profiles obtained following phosphorylation at 37°C, in the presence and absence of Ca2+, as described above, showed differences, suggesting that certain distinct sites may be phosphorylated under each condition. A major phosphopeptide peak, however, with the amino acid sequence Met-Leu-Thr(P)-Ile-Asn-Pro-Ser-Lys was identified under both conditions. This sequence is identical to the predicted sequence containing Thr-253 of the Ca2+/calmodulin-dependent protein kinase II. The results suggest that phosphorylation at Thr-253 requires an initial Ca2+-dependent phosphorylation, which may be at a different site, but does not depend on the continued presence of Ca2+ to proceed. The observed mode of regulation of autophosphorylation at Thr-253 appears to be unique to the postsynaptic density-associated enzyme.
Note:
Related Files :
adenosine triphosphate
alpha chain
Animal
animal tissue
In vitro
Synapses
threonine
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More details
DOI :
Article number:
Affiliations:
Database:
Scopus
Publication Type:
article
;
.
Language:
English
Editors' remarks:
ID:
19316
Last updated date:
02/03/2022 17:27
Creation date:
16/04/2018 23:27
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Scientific Publication
Identification of a major autophosphorylation site on postsynaptic density-associated Ca2+/calmodulin-dependent protein kinase
269
Dosemeci, A., Laboratory of Neurobiology, National Institutes of Health, Bethesda, MD 20892, United States, Laboratory of Neurobiology, Bldg. 36, National Institutes of Health, Bethesda, MD 20892, United States
Gollop, N., Laboratory of Biochemical Genetics, NHLBI, National Institutes of Health, Bethesda, MD 20892, United States
Jaffe, H., Laboratory of Neurochemistry, NINDS, National Institutes of Health, Bethesda, MD 20892, United States
Identification of a major autophosphorylation site on postsynaptic density-associated Ca2+/calmodulin-dependent protein kinase
One of the most abundant proteins in postsynaptic densities is identical or very similar to the α-subunit of the Ca2+/calmodulin-dependent protein kinase II. Auto-phosphorylation of this protein in isolated postsynaptic densities was studied under various conditions, following inhibition of endogenous phosphatase activity with microcystin-LR. Phosphorylation accompanied by a shift in the enzyme's electrophoretic mobility was observed upon incubation with Ca2+ and calmodulin at 37°C. Brief incubation with Ca2+ and calmodulin at O°C resulted in a low level of phosphorylation and no change in mobility. Following this limited Ca2+-dependent phosphorylation, however, a high level of phosphorylation could be achieved in the absence of Ca2+, upon incubation at 37°C. Comparison of reverse-phase HPLC phosphopeptide elution profiles obtained following phosphorylation at 37°C, in the presence and absence of Ca2+, as described above, showed differences, suggesting that certain distinct sites may be phosphorylated under each condition. A major phosphopeptide peak, however, with the amino acid sequence Met-Leu-Thr(P)-Ile-Asn-Pro-Ser-Lys was identified under both conditions. This sequence is identical to the predicted sequence containing Thr-253 of the Ca2+/calmodulin-dependent protein kinase II. The results suggest that phosphorylation at Thr-253 requires an initial Ca2+-dependent phosphorylation, which may be at a different site, but does not depend on the continued presence of Ca2+ to proceed. The observed mode of regulation of autophosphorylation at Thr-253 appears to be unique to the postsynaptic density-associated enzyme.
Scientific Publication
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