PTTH-stimulated ecdysone secretion is dependent upon tyrosine phosphorylation in the prothoracic glands of Manduca sexta

PTTH stimulates ecdysteroid secretion by the insect prothoracic glands. The peptide activates cAMP synthesis in a calcium-dependent manner, ultimately enhancing ecdysteroid synthesis. We have found that PTTH stimulates a rapid increase in tyrosine phosphorylation of at least four proteins in the prothoracic glands of larval Manduca sexta, as seen on Western blots of glandular lysates probed with antibody directed against phosphotyrosine. PTTH-stimulated tyrosine phosphorylation is blocked by an inhibitor of Src family tyrosine kinases, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo3,4-d]-pyrimidine (PP1). The inhibitor also blocks PTTH-stimulated ecdysone secretion, as well as PTTH-stimulated cAMP synthesis. Direct activation of the catalytic subunit of adenylyl cyclase by forskolin is not affected by PP1. In addition, ecdysteroid secretion stimulated by the cAMP analog dbcAMP is not blocked by PP1. These findings point to an important role for a Src-family tyrosine kinase at a very early step in the PTTH signaling pathway, prior to the activation of adenylyl cyclase.