Poster abstracts

Poster number 74 submitted by Christina Chung

MicroRNA stability is regulated by oncogenic protein kinase Akt1

Christina Z. Chung (Biochemistry, The University of Western Ontario), Emad Manni, Nileeka Balasuriya, Xuguang Liu (Biochemistry, The University of Western Ontario), Shawn S. C. Li (Biochemistry, Oncology and Child Health Research Institute, The University of Western Ontario), Patrick ODonoghue (Biochemistry, Chemistry, The University of Western Ontario), Ilka U. Heinemann (Biochemistry, The University of Western Ontario)

Abstract:
The deregulation of microRNAs (miRNAs) is associated with multiple human diseases, yet cellular mechanisms governing miRNA abundance remain largely elusive. We elucidated the first link between oncogenic kinase activity and the regulation of miRNA stability. Human miR-122 is required for Hepatitis C proliferation and low miR-122 abundance is associated with hepatic cancer. The adenylyltransferase Gld2 catalyzes the post-transcriptional addition of a single adenine residue (A+1) to the 3’-end of miR-122, enhancing its stability. 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 corresponding kinases that site-specifically phosphorylate Gld2 at S116, abolishing Gld2-mediated nucleotide addition. Transfection of Gld2 and fully activated ppAkt1, but not inactive Akt1 into HEK 293 cells, destabilizes miR-122 in living cells. The data demonstrate a novel phosphorylation-dependent mechanism to regulate Gld2 activity and miRNA stability, revealing tumor suppressor miRNAs as a previously unknown target of Akt1-dependent oncogenic signaling.

Keywords: miRNA regulation, site-specific phosphorylation, Akt1