Regulation of mammalian poly(A) polymerase through ERK signaling pathway

Abstract

Primary mRNA transcript is processed to mature form by a series of coordinated events (capping, splicing, cleavage and poly(A) addition) in nucleus. 3’ end formation defines the end of RNA synthesis via endonucleotic cleavage and poly(A) addition at 3’ of mRNA. Poly(A) polymerase(PAP) is the enzyme responsible for the synthesis poly(A) tails. Poly(A) tail plays an essential role in mRNA stability, transport and translation. Regulations of polyadenylation are required in diverse cellular processes. It is well known that poly(A) polymerase is phosphorylated at C-terminal domain by cyclin dependent kinase during the cell cycle. In this study, we could detect unexpected phosphorylation on C-terminal domain of poly(A) polymerase in the condition that growth-arrest cells is stimulated by serum induction. Serum-related phosphorylation increased within 10min after stimulation and was specifically inhibited by MAPK inhibitor, PD98059. So, instead of cdks, MAPK pathway seems to be involved in the phosphorylation of PAP. After all, we could find that poly(A) polymerase was directly phosphorylated in vitro by ERK. Furthermore, by Q-TOF MS/MS and analysis site directed mutagenesis, Ser537 was identified as unique phosphorylation site. By using anti-phospho Ser537 specific antibody, it was confirmed that phosphorylation status of Ser537 was increased by serum induction or mitogen phorbol 12-myristate 13-acetate(PMA) treatment in vivo. Activities of S537A mutant shows increased activity than wt, when they are prepared from unsynchronized cell. Phosphorylation of dephosphorylated PAP in vitro by ERK reduced the activity of PAP. Activity of GST-PAP(S537D), that mimics the phosphorylated-537Serine, is also decreased than wt. From these results, the activity of PAP is seems to be regulated through phosphorylation of 537Serine. These data suggest that ERK is a novel regulatory kinase for PAP and further, that PAP activity could be regulated by extracellular stimuli through a...