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

Abstract

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.

PKCβII inhibits the ubiquitination of β‐arrestin2 in an autophosphorylation‐dependent manner

“…These results are opposite to the previously reported roles of PKCs on the homologous regulatory processes of GPCRs [8,10]. We conducted more systemic subsequent studies to outline a novel pathway that is involved in the PKC-mediated inhibition of homologous regulatory pathways of GPCRs.…”

“…For example, the enzymatic activities of GRK2 were shown to be increased through phosphorylation on the 29th serine residue by PKCa, c, and d in vitro [8]. Subsequent studies suggested that PKC activates GRK2 by enhancing its translocation to the plasma membrane [9,10]. On the other hand, some studies have shown that PKCs phosphorylate Raf kinase inhibitor protein (RKIP), which in turn inhibits GRK2 activities [11,12].…”

Trafficking of the HIV Coreceptor CXCR4

“…However, involvement of arrestins in GPCR internalization in the absence of receptor phosphorylation has been reported (63,64). In the case of CXCR4, phosphorylation could occur by several mechanisms, such as SDF-1␣-mediated receptor activation and phosphorylation by GRKs, direct phosphorylation of receptor by protein kinase C as a result of phorbol ester stimulation, or phosphorylation by GRK2 as a result of its activation via phorbol ester-mediated activation of protein kinase C (54,55). The serines at positions 344, 346, 347, and 348 are potentially good candidates for phosphorylation by GRK2, since they are Cterminal to an acidic residue (65).…”

“…GRK2 (54,55). We cotransfected HEK-293 cells with CXCR4 and dynamin-K44A, two different arrestin-3 dominant negative mutants and a dominant negative mutant of GRK2.…”

Ligand-gated Ion Channels

“…Protein kinase C-mediated phosphorylation increases membrane association (Chuang et al, 1995;Winstel et al, 1996) GRK3 bARK2 ENSG00000100077 b-Adrenergic receptor kinase 2, ADRBK2 -…”