Kinetics and inhibition studies of the L205R mutant of cAMP-dependent protein kinase involved in Cushing's syndrome.

Overproduction of cortisol by the hypothalamus-pituitary-adrenal hormone system results in the clinical disorder known as Cushing's syndrome. Genomics studies have identified a key mutation (L205R) in the α-isoform of the catalytic subunit of cAMP-dependent protein kinase (PKACα) in adrenal adenomas of patients with adrenocorticotropic hormone-independent Cushing's syndrome. Here， we conducted kinetics and inhibition studies on the L205R-PKACα mutant. We have found that the L205R mutation affects the kinetics of both Kemptide and ATP as substrates， decreasing the catalytic efficiency (/) for each substrate by 12-fold and 4.5-fold， respectively. We have also determined the IC and for the peptide substrate-competitive inhibitor PKI(5-24) and the ATP-competitive inhibitor H89. The L205R mutation had no effect on the potency of H89， but causes a > 250-fold loss in potency for PKI(5-24). Collectively， these data provide insights for the development of L205R-PKACα inhibitors as potential therapeutics.