SER/THR AND TYR PHOSPHATASES ARE OBLIGATORY INTERMEDIATES IN THE cAMP SIGNAL TRANSDUCTION PATHWY INVOLVED IN STEROIDOGENIC TISSUES

FABIANA CORNEJO MACIEL, CECILIA PODEROSO, ALEJANDRA GOROSTIZAGA, CRISTINA PAZ, ERNESTO PODESTÁ

Melbourne, Australia

Congreso; Second messenger and Phosphoproteins Conference; 2001

Transient phosphorylation of proteins represents one of the most important posttransational modifications, playing a central role in many cellular processes. Then, kinases and phosphatases are considered as crucial regulatory enzymes involved in signal transduction pathways. Luteinizing hormone (LH) and adrenocorticotropin (ACTH) activate steroid production in Leydig and adrenal zona fasciculata (ZF) cells, respectively, through a mechanism that involves PKA activation and protein phosphorylation. In this work, we present evidence that this mechanism also includes Ser/Thr and Tyr dephosphorylation. In order to study the role played by Ser/Thr and Tyr phosphatases in rat ZF and MA-10 cells (mouse Leydig tumor cell line), we evaluated the effects of phosphatases inhibitors on hormone- and cAMP activated steroid production. Caliculin A (CAL) (Ser/Thr phosphatases inhibitor), and phenylarsine oxide (PAO) (Tyr phosphatases inhibitor), produced a concentration-dependent inhibition of steroidogenesis in both systems. CAL 100 nM and PAO 2 mM reduced progesterone production by MA-10 cells: control 1.07+/-0.08, CAL 100 nM 1.0+/-0.1, PAO 2 mM 0.65+/-0.07, 8Br-cAMP 72+/-6, CAL 100 nM+8Br-cAMP 2.2+/-0.4, PAO 2 mM+8Br-cAMP 3.0+/-0.5 ng progesterone/ml. Other compounds were tested with similar effects. In this context, our findings show that the hormone and second messenger promote the activation of two proteins of molecular masses of 110 and 50 kDa, detected by an in-gel Tyr phosphatase assay, and the dephosphorylation of endogenous proteins in Tyr residues. Taken together, these results demonstrate that Ser/Thr and Tyr dephosphorylation are obligatory events in this PKA-mediated siganling pathway. Hormone action activates Tyr phosphatases, and probably Ser/Thr phosphatases, that would act on basely phosphorylated proteins that exert a negative action on steroid production and that have to be dephosphorylated in order to promote steroidogenesis.