Alternative AKT2 splicing produces protein lacking the hydrophobic motif regulatory region.

Guido Plotz,Stefan Zeuzem,Ricardo M Biondi,Angela Brieger,Laura A Lopez-Garcia,Emanuele Buratti

doi:10.1371/journal.pone.0242819

Abstract

Three AKT serine/threonine kinase isoforms (AKT1/AKT2/AKT3) mediate proliferation, metabolism, differentiation and anti-apoptotic signals. AKT isoforms are activated downstream of PI3-kinase and also by PI3-kinase independent mechanisms. Mutations in the lipid phosphatase PTEN and PI3-kinase that increase PIP3 levels increase AKT signaling in a large proportion of human cancers. AKT and other AGC kinases possess a regulatory mechanism that relies on a conserved hydrophobic motif (HM) C-terminal to the catalytic core. In AKT, the HM is contiguous to the serine 473 and two other newly discovered (serine 477 and tyrosine 479) regulatory phosphorylation sites. In AKT genes, this regulatory HM region is encoded in the final exon. We identified a splice variant of AKT2 (AKT2-13a), which contains an alternative final exon and lacks the HM regulatory site. We validated the presence of mRNA for this AKT2-13a splice variant in different tissues, and the presence of AKT2-13a protein in extracts from HEK293 cells. When overexpressed in HEK293 cells, AKT2-13a is phosphorylated at the activation loop and at the zipper/turn motif phosphorylation sites but has reduced specific activity. Analysis of the human transcriptome corresponding to other AGC kinases revealed that all three AKT isoforms express alternative transcripts lacking the HM regulatory motif, which was not the case for SGK1-3, S6K1-2, and classical, novel and atypical PKC isoforms. The transcripts of splice variants of Akt1-3 excluding the HM regulatory region could lead to expression of deregulated forms of AKT.

Highlights

AKT isoforms are activated downstream of PI3-kinase signaling and play both redundant as well as specific roles in signaling [1]
While attempting to clone AKT2 by PCR from a cDNA library from cancer-tissue, we unexpectedly identified a splice variant of AKT2 that lacked the hydrophobic motif (HM) regulatory site
The quality of the splice sites, as determined by similarity to consensus splice sites, is below the average of the other AKT2 exons (S2 Fig). They support inclusion in the mRNA, as has been found for another alternatively spliced gene before [19]. This finding prompted us to analyze the C-terminal splicing of AKT and other kinases containing HM regulatory sites in databases that assemble experimental data for splice variants in different tissues (Fig 2A and S3 Fig)

Summary

Introduction

AKT isoforms are activated downstream of PI3-kinase signaling and play both redundant as well as specific roles in signaling [1]. The phosphatidylinositol-3,4,5-triphosphate (PIP3) second messenger at the cell membrane triggers the recruitment of AKT to the membrane through its N-terminal PH-domain and enables its subsequent activation by its upstream kinases, PDK1 Alternative AKT2 isoform lacking hydrophobic motif analysis, decision to publish, or preparation of the manuscript

Methods

Results

Conclusion