PMA-induced GCMa phosphorylation stimulates its transcriptional activity and degradation

Abstract

Glial cells missing Drosophila homolog a (GCMa) is a member of the GCM transcription factor family and plays critical roles in trophoblast differentiation and placental functions. It is well established that the cyclic AMP (cAMP)-dependent pathway induces the expression and transcriptional activity of GCMa by regulating post-translational modifications of GCMa, which results in enhancement of trophoblast differentiation. We previously observed that phorbol 12-myristate 13-acetate (PMA) stimulates phosphorylation of GCMa on serines 328, 378 and 383 through the protein kinase C (PKC)-and mitogen-activated protein kinase kinase (MEK)/extracellular signalregulated kinase (ERK)-dependent pathway, which decreases the protein stability of GCMa. Here we report that PMA increases the ubiquitination level of GCMa, dependent on the phosphorylation of GCMa on serines 328, 378 and 383. We found that this phosphorylation also stimulates the transcriptional activity of GCMa. Our data indicate that the PMA-induced PKC-and MEK/ERKdependent pathway enhances the degradation as well as the transcriptional activity of GCMa. We also examined the impact of this signaling pathway on trophoblasts and the results suggest that the PKC-and MEK/ERK-dependent pathway is involved in the regulation of trophoblast differentiation.

The hypoparathyroidism-associated mutation in Drosophila Gcm compromises protein stability and glial cell formation

“…We also show that a higher portion of Gcm R59L proteins remain possibly phosphorylated upon CIP treatment and PKC overexpression, suggesting an involvement of PKC in altering Gcm R59L phosphorylation. Interestingly, previous studies have similarly implicated a role for PKC kinase in mammalian Gcma phosphorylation2627. Our results are consistent with the notion that PKC plays a major role in the phosphorylation of Gcm proteins across species.…”

“…Since Gcm R59L interacts with the F-box protein Slimb and the prerequisite for the recognition is Gcm phosphorylation, it is possible that a change in the Gcm phosphorylation status occurs, affecting Gcm R59L interaction with Slimb and its ubiquitination levels. Based on the speculation and results from previous studies that implicated Protein Kinase C (PKC) as a kinase for human Gcma (hGcma) phosphorylation2627, we first tested whether Gcm proteins interact with PKC in Drosophila . With readily access to tools for one of the six Drosophila PKCs, PKC53E , we cloned PKC53E into the pUAST expression vector carrying a N-terminal HA epitope tag in three tandem repeats (3xHA, henceforth denoted as PKC in the text).…”

“…Previous studies have indicated that Gcm proteins exhibit differences in stability and are under tight regulation via post-translational modification24252627. Moreover, Gcm proteins have been shown to undergo regulated degradation via the ubiquitination-proteasome system (UPS), a widely used mechanism to control protein turnover2829.…”

Holothurian Glycosaminoglycan Inhibits Metastasis and Thrombosis via Targeting of Nuclear Factor-κB/Tissue Factor/Factor Xa Pathway in Melanoma B16F10 Cells

“…Addition of hGAG at >0.5 µM reverses the activated p-p38 induced TF expression ( p <0.001). Similarly, considering that PMA could induce activation of protein kinase C (PKC)/mitogen-activated protein kinase kinase (MEK)/ERK pathway [50], [51], the B16F10 tumor cells were treated with PMA at 10 µM alone or in combination with hGAG at the indicated concentration for 24 h/37°C and the TF expression was analyzed. Likewise, cells treated with PMA alone displayed an enhanced expression of TF, and hGAG treatment at >0.5 µM significantly attenuated TF expression facilitated by ERK activation.…”

New insights in the clockwork mechanism regulating lineage specification: Lessons from the Drosophila nervous system

“…In addition to mRNA and protein stability, Gcm is also regulated by post‐translational modifications such as phosphorylation and acetylation. Phosphorylation per se has not been demonstrated for Drosophila Gcm, but in humans, GCM1 is phosphorylated by protein kinase C (PKC) at serines 328, 378, and 383, which leads to increased transactivation potential (Yasui et al, ,b). Since serines 378 and 383 are conserved in Drosophila Gcm, it would be interesting to test whether PKC regulation of Gcm is conserved during Drosophila nervous system development.…”