Phosphorylation and activation of beta-adrenergic receptor kinase by protein kinase C.
The aim of this study was to test the possible modification of beta-adrenergic receptor kinase (beta ARK) activity by second messengers and/or their downstream components. Using human mononuclear leukocytes (MNL), we found that calcium ionophores could elevate beta ARK activity by about 80% in a protein kinase C (PKC)-dependent manner. This was confirmed by the ability of the PKC activator phorbol 12-myristate 13-acetate (PMA) to produce a similar effect, suggesting a PKC-dependent modulation of beta ARK activity. In vitro experiments with purified proteins showed that PKC could directly phosphorylate beta ARK1 with an apparent Km for beta ARK1 of 6 nM. The ability of beta ARK1 to phosphorylate rhodopsin was 61% greater when it was phosphorylated by PKC. The level of phosphorylation of beta ARK1 immunoprecipitated from MNL and Sf9 cells overexpressing this kinase was enhanced by about 2-3-fold after PMA treatment. Functional significance of PKC-dependent increase in beta ARK activity ws demonstrated by beta-adrenergic receptor (beta AR) homologous desensitization experiments in MNL. beta AR desensitization, as induced by exposure to 10 microM isoproterenol (5 min at 37 degrees C), was increased from 42 +/- 10% in control to 68 +/- 8% in PMA-pretreated MNL. beta ARK inhibitor heparin (160 micrograms/ml) prevented the augmenting effect of PMA on beta AR desensitization. These results show that beta ARK activity can be increased through phosphorylation by PKC, thus indicating that beta ARK can be preconditioned to modulate the subsequent cellular responsiveness to receptor activation, providing the cell with a mechanism by which specific homologous desensitization can be regulated heterologously.
Chuang
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Consorzio Mario Negri Sud, Istituto di Ricerche Farmacologiche Mario Negri, Santa Maria Imbaro, Italy.
LeVine
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H
De Blasi
A
A
D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D015151:Immunoblotting; D007963:Leukocytes, Mononuclear / Q000201:enzymology / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism*; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism; D015290:Second Messenger Systems; D018411:Spodoptera / Q000166:cytology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D051552:beta-Adrenergic Receptor Kinases
D011943:Receptors, Adrenergic, beta; D011994:Recombinant Proteins; D012243:Rhodopsin; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D051552:beta-Adrenergic Receptor Kinases; D013755:Tetradecanoylphorbol Acetate