Roles of phosphoinositide-dependent kinase-1 in α1B-adrenoceptor phosphorylation and desensitization.

The role of phosphoinositide-dependent protein kinase-1 (PDK-1) activity on α(1B)-adrenoceptor phosphorylation and function was explored using pharmacological inhibitors and expression of a dominant-negative mutant of this enzyme. Noradrenaline-, phorbol myristate acetate-, lysophosphatidic acid- and epidermal growth factor-mediated α(1B)-adrenoceptor phosphorylation were markedly reduced by the two inhibitors used: UCN-01 [(7-hydroxystaurosporine; (3R*,8S*, 9R*, 10R*,12R*)-2,3,9,10,11,12-hexahydro-3-hydroxy-9-methoxy-8-methyl-10-(methylamino)-8,12-epoxy-1H, 8H-2,7b,12a-triazadibenzo[a,g]-cyclonona[cde]triden-1-one)] and OSU-03012 [(2-amino-N-[4-[5-(2-phenanthrenyl)-3-trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-acetamide)]. A similar effect was observed in cells expressing a PDK-1 dominant-negative mutant. Phosphorylated PDK-1 (S241) and protein kinase C α (T497) were associated with cell membranes in the basal state which increased in response to the hormonal stimuli mentioned previously. UCN-01 essentially abolished phospho-PDK-1 membrane-association and markedly attenuated that of protein kinase C α. Consistent with the findings, UCN-01 reduced lysophosphatidic acid- and epidermal growth factor-induced α(1B-)adrenoceptor desensitization. Our data suggest that PDK-1 plays a permissive role in α(1B)-adrenoceptor desensitization and phosphorylation and participates in the formation of signaling complexes, which delicately modulate receptor function and regulation.