Summary
Incubation of plasma membranes isolated from bovine aorta with either 0.5 mM CaCl2 or with a phorbol ester (1 µM phorbol 12,13-dibutyrate) and phosphatidylserine in an EGTA-containing buffer resulted in the phosphorylation of 10 proteins (Mr of 158, 105, 75, 62, 44, 39, 33, 22, 15 and 9 kDa), presumably due to activation of endogenous protein kinase C (PKC). After heat treatment of the aortic plasma membranes at 80°C for 5 min in order to inactivate all endogenous protein kinase, phosphatase and ATPase activities, membrane phosphorylation was absolutely-dependent upon the addition of an exogenous, partially-purified PKC preparation from bovine aorta. Under these conditions, a total of 17 phosphoproteins could be detected (Mr of 158, 105, 75, 44, 39, 33, 30, 29, 27, 25, 22, 17.5, 16, 15, 11, 10 and 9kDa). The most prominent phosphoprotein band in native membranes had a molecular weight of 75 kDa (p75); several characteristics suggest that p75 might be autophosphorylated PKC. The phosphorylation of aortic plasma membranes by exogenous PKC required phosphatidylserine and was calcium-dependent (10−5 to 10−7 M Ca2+); the addition of diolein resulted in little or no enhancement of phosphorylation. Replacement of phosphatidylserine with oleic acid resulted in the same number of phosphoproteins, but the extent of phosphorylation was diminished. The phosphorylation pattern was altered slightly if the aortic plasma membranes were isolated in the presence of 1 mM Ca2+ instead of EGTA buffers as in the standard procedure. Experiments were performed to determine if the p39 substrate of PKC in aortic plasma membranes was calpactin II (lipocortin I). Immunoblotting established that calpactin II was present in aortic plasma membranes, but there was no corresponding phosphoprotein on the autoradiographs.
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Abbreviations
- DG:
-
Diacylglycerol
- PS:
-
Phosphatidylserine
- PMSF:
-
Phenylmethylsulfonyl Fluoride
- pNPP:
-
p-Nitrophenyl Phosphate
- PDBu:
-
phorbol 12,13-Dibutyrate
- OA:
-
OleicAcid
- DO:
-
Diolein
- PKC:
-
Protein Kinase C
- PKC19–36 :
-
Protein kinase C (19–36) peptide
- EGF:
-
Epidermal Growth Factor
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Zhao, D., Dell, K.R., Hollenberg, M.D. et al. Phosphorylation of aortic plasma membranes by protein kinase C. Mol Cell Biochem 106, 171–180 (1991). https://doi.org/10.1007/BF00230183
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DOI: https://doi.org/10.1007/BF00230183