PHOSPHORYLATION IN COCKROACH ANTENNAL PROTEINS DURING TRANSDUCTION OF OLFACTORY MESSAGES

Marielle RENUCCI, Alain TIRARD*, Leam SRENG, Jacqueline KHLAT and Jean-Luc CLÉMENT

Laboratoire de Neurobiologie "Communication Chimique", CNRS-UPR 9024, 31 chemin Joseph-Aiguier, BP 71, 13402 Marseille cedex 20, France.

In insect antennal extracts, Schleicher et al.-1 showed that protein kinase C (PKC) inhibitors abolish the transience of pheromone-induced rapid inositol triphosphate (IP3) responses, suggesting that pheromonal signals act on specific protein phosphorylation. To further validate this hypothesis, we studied the effects of second messengers and pheromonal blend on phosphorylation of antennal proteins in the male cockroach Periplaneta americana.

1. Proteins from adult male antennae were phosphorylated in vitro in the presence of [y32P]-ATP, then separated by SDS-PAGE. Over twelve phosphopolypeptides were routinely observed when no modulator was added to the incubation medium.
2. Depending on the modulator used, a variety of patterns in phosphoryiation were observed:
   • Addition of Ca++/calmodulin to the incubation medium enhanced phosphorylation of polypeptides with molecular weights of 38, 48, 51, 54 and 58 kDa.
   • By stimulating PKC, Ca++ / Phosphatidyl-Serine (PS) / Phorbol Myristate Acetate (PMA) led to the appearance of three phosphopolypeptides of 36, 70 and 120 kDa.
   • In the presence of cAMP, two polypeptides of 46 and 42 kDa appeared. The 42 kDa polypeptide also appeared in the presence of cGMP.
3. Comparison of antennal polypeptide content with that in the cerci, leg, brain. and fat body showed that the 28, 36, and 48 kDa polypeptides were specific to antennae.
4. /n vitro phosphorylation of proteins obtained from antennae subjected to pheromonal stimulation for 16 seconds prior to homogenisation revealed inhibition of the 120, 70, 64, 38, 28, and 26/27 kDa polypeptides and strong stimulation of the 58, 54, 51, 48, and 32 kDa polypeptides. A 107 kDa polypeptide was observed only after pheromonal stimulation by Ca++/PS/PMA.

In insects, it is now clear that pheromone induces changes in production of second messengers (especially IP3). In addition, our findings-2 suggest that pheromonal response, signal amplification, and desensitization involve specific phosphopolypeptides. Further study is needed to identify the proteins affected by pheromones and to fully understand how their phospho/dephosphorylation is regulated.

1 - Schleicher, S., Boekhoff I., Konietzko, U., and Breer, H., J. Comp. Physiol. B 164 (1994) 76.
2 - Renucci, M, Tirard, A., Sreng, L, Khlat, J. and J L Clement. Experientia (1996) (in press)