Gld2 activity is regulated by phosphorylation in the N-terminal domain

ABSTRACT

The de-regulation of microRNAs (miRNAs) is associated with multiple human diseases, yet cellular mechanisms governing miRNA abundance remain largely elusive. Human miR-122 is required for Hepatitis C proliferation, and low miR-122 abundance is associated with hepatic cancer. The adenylyltransferase Gld2 catalyses the post-transcriptional addition of a single adenine residue (A + 1) to the 3ʹ-end of miR-122, enhancing its stability. Gld2 activity is inhibited by binding to the Hepatitis C virus core protein during HepC infection, but no other mechanisms of Gld2 regulation are known. We found that Gld2 activity is regulated by site-specific phosphorylation in its disordered N-terminal domain. We identified two phosphorylation sites (S62, S110) where phosphomimetic substitutions increased Gld2 activity and one site (S116) that markedly reduced activity. Using mass spectrometry, we confirmed that HEK 293 cells readily phosphorylate the N-terminus of Gld2. We identified protein kinase A (PKA) and protein kinase B (Akt1) as the kinases that site-specifically phosphorylate Gld2 at S116, abolishing Gld2-mediated nucleotide addition. The data demonstrate a novel phosphorylation-dependent mechanism to regulate Gld2 activity, revealing tumour suppressor miRNAs as a previously unknown target of Akt1-dependent signalling.

KEYWORDS:

• Phosphorylation
• enzyme kinetics
• post-translational modification
• RNA editing
• microRNA

Acknowledgments

We are thankful to Dr David W. Litchfield, Dr Laszlo Gyenis, and Teresa Nuñez de Villavicencio-Diaz for their advice on kinases and their gift of CK2α, CK2 holoenzyme, Abl, PKA, and CDK5 kinases and to Matthew Turk and Anish Engineer for advice and discussion.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary Data

Supplementary data for this article can be accessed on publisher’s website.

Additional information

Funding

This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada [04776-2014 to I.U.H.and 04282-2014 to P.O.]; J.P. Bickell Foundation to I.U.H.; Canada Foundation for Innovation (229917 to P.O.), the Ontario Research Fund (229917 to P.O.); Canada Research Chairs Program (950-229917 to P.O.); Canadian Cancer Society Research Institute (704324 to P.O.); Ontario Graduate Scholarship to C.Z.C.; Alexander Graham Bell Canada Graduate Scholarship- Doctoral from the Natural Sciences and Engineering Research Council of Canada to C.Z.C.; Queen Elizabeth II Scholarship in Science and Technology to N.B.; a scholarship from the Saudi Arabian Cultural Bureau to E.M.