PROTHORACICOTROPIC HORMONE REGULATES THE PHOSPHORYLATION OF A SPECIFIC PROTEIN IN THE PROTHORACIC GLANDS OF THE TOBACCO HORNWORM, MANDUCA-SEXTA

Particulate (membrane) fractions were obtained from homogenates of prothoracic glands from staged larvae and pupae of the tobacco hornworm, Manduca sexta. The addition of cAMP to these fractions stimulated the in vitro phosphorylation of at least three phosphoproteins. This cAMP-mediated phosphorylation was blocked by the addition of the specific inhibitor of the cAMP-dependent protein kinase. When intact prothoracic glands were preincubated with ([P]O4)-P-32 to label endogenous ATP, one of these proteins (34 kDa) was phosphorylated in response to dibutyryl cAMP, forskolin, as well as the brain neurohormone, big prothoracicotropic hormone (PTTH). Neither cyclic GMP nor small PTTH stimulated 34 kDa protein phosphorylation, although the latter elicited increased ecdysteroid synthesis by larval glands. Further, injection of big PTTH into intact larvae previously loaded with ([P]O4)-P-32 also resulted in enhanced phosphorylation of the same prothoracic gland protein. In prothoracic glands from animals at all developmental stages examined except diapausing pupae the protein was phosphorylated in vitro, although glands activated at the time of dissection showed a reduced phosphorylation response in vitro. The intensity of phosphorylation in intact glands was big PTTH dose-dependent, and the dose-response curve of phosphorylation closely followed the curve for PTTH-stimulated ecdysteroid secretion. The time course of phosphorylation is consistent with a role for this phosphoprotein as an intermediate between PTTH stimulation of adenylate cyclase and ecdysteroid biosynthesis.