Phosphorylation of Activation Transcription Factor-2 at Serine 121 by Protein Kinase C Controls c-Jun-mediated...

Area of Science:

• Molecular Biology
• Cellular Signaling
• Gene Regulation

Background:

• Activation transcription factor-2 (ATF-2) is a transcription factor regulated by phosphorylation by various kinases.
• The specific role of protein kinase C (PKC) mediated phosphorylation of ATF-2 in transcriptional control of AP-1/cyclic AMP-response element genes in vivo is not well understood.

Purpose of the Study:

• To investigate the role of PKC-mediated phosphorylation of ATF-2 at Serine-121 in transcriptional regulation.
• To determine the temporal dynamics and subcellular localization of ATF-2 phosphorylation in response to TPA.

Main Methods:

• Utilized antibodies specific for phosphorylated serine residues of ATF-2.
• Employed HeLa cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) to study phosphorylation dynamics.
• Performed site-directed mutagenesis (Serine-to-Alanine) to assess the functional impact of phosphorylation at Serine-121.
• Assessed reporter gene activity (AP-1/cyclic AMP-response element and Gal4-reporter genes).

Main Results:

• PKC phosphorylates ATF-2 at Serine-121 (ATF-2pS121), with detectable levels appearing late in the TPA response.
• Phosphorylation at Serine-121, unlike other sites, increases in the nucleus of TPA-treated HeLa cells.
• Mutation of Serine-121 to Alanine represses c-Jun-dependent transcription of AP-1/cyclic AMP-response element reporter genes and p300-mediated activation of a Gal4-reporter gene.

Conclusions:

• Phosphorylation of ATF-2 at Serine-121 by PKC is a critical event in the late phase of TPA-induced transcriptional activation.
• ATF-2 phosphorylation at Serine-121 plays a significant role in c-Jun-mediated gene transcription, particularly for AP-1/cyclic AMP-response element containing genes.