Alternative AKT2 splicing produces protein lacking the hydrophobic motif regulatory region

Area of Science:

• Molecular Biology
• Cancer Biology
• Signal Transduction

Background:

• Three AKT serine/threonine kinase isoforms (AKT1/AKT2/AKT3) are crucial for cell proliferation, metabolism, differentiation, and apoptosis.
• AKT signaling is frequently dysregulated in human cancers due to mutations in upstream regulators like PI3-kinase and PTEN.
• AKT kinases, like other AGC kinases, are regulated by a conserved hydrophobic motif (HM) located C-terminal to the catalytic core, near key phosphorylation sites.

Purpose of the Study:

• To identify and characterize novel AKT splice variants.
• To investigate the functional consequences of lacking the hydrophobic motif (HM) regulatory site in AKT isoforms.
• To explore the prevalence of HM-lacking transcripts across different AGC kinase families.

Main Methods:

• Identification and validation of the AKT2-13a splice variant using mRNA and protein analysis in various tissues and cell lines (HEK293).
• Functional characterization of AKT2-13a by assessing its phosphorylation status and specific kinase activity upon overexpression in HEK293 cells.
• Bioinformatic analysis of the human transcriptome to identify alternative transcripts lacking the HM regulatory region in AKT isoforms and other AGC kinases.

Main Results:

• A novel AKT2 splice variant, AKT2-13a, was identified, characterized, and confirmed to be present in human tissues and cell lines.
• Overexpression of AKT2-13a in HEK293 cells resulted in phosphorylation at activation loop and zipper/turn motif sites but exhibited reduced specific activity.
• Analysis revealed that all three AKT isoforms (AKT1, AKT2, AKT3) express alternative transcripts lacking the HM regulatory motif, unlike other AGC kinases (e.g., SGK, S6K, PKC).

Conclusions:

• The identified AKT2-13a splice variant, and similar variants in AKT1 and AKT3, lack the critical hydrophobic motif (HM) regulatory site.
• These HM-deficient AKT splice variants may lead to the expression of deregulated AKT forms.
• The existence of these alternative transcripts suggests a novel mechanism for AKT pathway dysregulation in cancer.