PMA induces GCMa phosphorylation and alters its stability via the PKC- and ERK-dependent pathway.

The glial cells missing a (GCMa) transcription factor plays a pivotal role in the placental development by regulating the expression of several genes in the placenta that are responsible for the proper formation of the syncytiotrophoblast. It is well known that the function of GCMa is regulated at both transcriptional and post-translational levels by the cyclic AMP (cAMP)/protein kinase A (PKA)-dependent pathway, the activation of which increases the GCMa protein level and leads to trophoblast differentiation into the syncytiotrophoblast. However, little is known about the regulatory control of GCMa by PKC-dependent signaling mechanism(s). To investigate whether GCMa is regulated by PKC-dependent pathway, we treated the human choriocarcinoma JEG-3 cells with phorbol 12-myristate 13-acetate (PMA) and studied its effect on the GCMa protein using a monoclonal anti-GCMa antibody we prepared. PMA caused a transient decrease in the endogenous GCMa protein level in JEG-3 cells that was accompanied by an increase in GCMa phosphorylation. The phosphorylation and degradation of GCMa by PMA treatment was effectively reduced by pretreatment with protein kinase C (PKC) inhibitors and a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor, indicating a PKC- and MEK-dependent mechanism. Furthermore, we identified the serine residues 328, 378 and 383 to be the phosphorylation sites on GCMa that are involved in the PMA-induced degradation of GCMa. Our data demonstrate for the first time that GCMa is phosphorylated by the PKC- and MEK/extracellular signal-regulated kinase (ERK)-dependent mechanism, and that this phosphorylation is involved in its degradation process.