Phosphorylation and Activation of β-Adrenergic Receptor Kinase by Protein Kinase C

The aim of this study was to test the possible modification of β-adrenergic receptor kinase (βARK) activity by second messengers and/or their downstream components. Using human mononuclear leukocytes (MNL), we found that calcium ionophores could elevate βARK 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 βARK activity. In vitro experiments with purified proteins showed that PKC could directly phosphorylate βARK1 with an apparent Km for βARK1 of 6 nM. The ability of βARK1 to phosphorylate rhodopsin was 61% greater when it was phosphorylated by PKC. The level of phosphorylation of βARK1 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 βARK activity was demonstrated by β-adrenergic receptor (βAR) homologous desensitization experiments in MNL. βAR desensitization, as induced by exposure to 10 μM isoproterenol (5 min at 37°C), was increased from 42 ± 10% in control to 68 ± 8% in PMA-pretreated MNL. βARK inhibitor heparin (160 μg/ml) prevented the augmenting effect of PMA on βAR desensitization. These results show that βARK activity can be increased through phosphorylation by PKC, thus indicating that βARK 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.