Inhibition of tyrosine phosphorylation blocks hormone-stimulated calcium influx in an insect steroidogenic gland.

In the tobacco hornworm Manduca sexta (M. sexta) as in other insects, ecdysone synthesis occurs in the prothoracic glands and is stimulated by the brain neuropeptide prothoracicotropic hormone (PTTH). PTTH activates the prothoracic glands through the second messenger cAMP, the synthesis of which is stimulated by calcium. We previously found that the Src kinase inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-D]-pyrimidine (PP1) inhibits PTTH-stimulated cAMP synthesis and ecdysone secretion. In the present study, we show that by contrast, PP1 does not block cAMP synthesis stimulated by the calcium ionophore A23187, and that PP1 augments A23187-stimulated ecdysone secretion. Hence, once glandular levels of calcium are elevated, Src family kinase activity is no longer needed for, and may actually inhibit, steroidogenesis. PP1 blocks calcium influx in PTTH-stimulated prothoracic glands, indicating that tyrosine phosphorylation by a member of the Src kinase family is required for calcium influx. These results suggest that prothoracic gland calcium channels are regulated either directly or indirectly by tyrosine phosphorylation.

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)pyrazolo[3,4-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.