Effect of inhibitors of mitogen-activated protein kinase kinase on α1B-adrenoceptor phosphorylation

Abstract

1 Mitogen-activated protein kinases mediate hormone/neurotransmitter action on proliferation and differentiation and participate in receptor regulation. The effect of inhibitors of mitogen-activated kinase kinase (MEK) on α_1B-adrenoceptor phosphorylation state and function was studied using different cell lines. It was observed that at nanomolar concentrations the MEK inhibitors, PD98059 (2′-amino-3′-methoxyflavone) and UO126 [1,4-(diamino-2,3-dicyano/1,4-bis-(2-aminophenylthio)-butadiene], increased α_1B-adrenoceptor phosphorylation and diminished the functional response of this receptor to noradrenaline. These agents did not alter the action of lysophosphatidic acid.

2 Staurosporine (IC₅₀ ≈ 0.8 nm) (a general protein kinase inhibitor) and bis-indolyl-maleimide I (IC₅₀ ≈ 200 nm) (a selective protein kinase C inhibitor) inhibited PD98059-induced α_1B-adrenoceptor phosphorylation. In contrast, neither wortmannin (phosphoinositide 3-kinase inhibitor) nor genistein (protein tyrosine kinase inhibitor) had any effect. The data suggest the possibility that MEK might exert control on the activity of the enzymes that regulate receptor phosphorylation, such as G-protein-coupled receptor kinases, protein kinase C or serine/threonine protein phosphatases.

3 Coimmunoprecipitation studies showed a constant association of total extracellular signal-regulated kinase 2 (ERK2) with α_1B-adrenoceptors. Association of phospho-ERK 1/2 to α_1B-adrenoceptors increased not only in response to agonist but also in response to agents that increase α_1B-adrenoceptor and ERK1/2 phosphorylation [such as endothelin-1, phorbol 12-myristate-13-acetate (PMA) and epidermal growth factor (EGF)]; not surprisingly, PD98059 decreased this effect.

4 Our data show that blockade of MEK activity results in increased α_1B-adrenoceptor phosphorylation, diminished adrenoceptor function and perturbation of receptor–ERK1/2 interaction.