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Published on: August 4, 2019
Expression of Human PTEN-L in a Yeast Heterologous Model Unveils Specific N-Terminal Motifs Controlling PTEN-L
Teresa Fernández-Acero1, Eleonora Bertalmio1, Sandra Luna2
1Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad Complutense de Madrid (UCM) & Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS). Plaza de Ramón y Cajal s/n, 28040 Madrid, Spain.
Abstract:
The tumour suppressor PTEN is frequently downregulated, mutated or lost in several types of tumours and congenital disorders including PHTS (PTEN Hamartoma Tumour Syndrome) and ASD (Autism Spectrum Disorder). PTEN is a lipid phosphatase whose activity over the lipid messenger PIP3 counteracts the stimulation of the oncogenic phosphatidylinositol 3-kinase (PI3K) pathway. Recently, several extended versions of PTEN produced in the cell by alternative translation initiation have been described, among which, PTEN-L and PTEN-M represent the longest isoforms. We previously developed a humanized yeast model in which the expression of PI3K in Saccharomyces cerevisiae led to growth inhibition that could be suppressed by co-expression of PTEN. Here, we show that the expression of PTEN-L and PTEN-M in yeast results in robust counteracting of PI3K-dependent growth inhibition. N-terminally tagged GFP-PTEN-L was sharply localized at the yeast plasma membrane. Point mutations of a putative membrane-binding helix located at the PTEN-L extension or its deletion shifted localization to nuclear. Also, a shift from plasma membrane to nucleus was observed in mutants at basic amino acid clusters at the PIP2-binding motif, and at the Cα2 and CBR3 loops at the C2 domain. In contrast, C-terminally tagged PTEN-L-GFP displayed mitochondrial localization in yeast, which was shifted to plasma membrane by removing the first 22 PTEN-L residues. Our results suggest an important role of the N-terminal extension of alternative PTEN isoforms on their spatial and functional regulation.
Insights
The tumor suppressor PTEN-L and PTEN-M isoforms counteract phosphatidylinositol 3-kinase (PI3K) pathway activation. Their N-terminal extensions are crucial for PTEN localization and function.
Area of Science:
- Molecular biology
- Cell biology
- Biochemistry
Background:
- The tumor suppressor PTEN is frequently altered in various cancers and disorders like PTEN Hamartoma Tumor Syndrome (PHTS) and Autism Spectrum Disorder (ASD).
- PTEN functions as a lipid phosphatase, opposing the oncogenic phosphatidylinositol 3-kinase (PI3K) pathway by regulating the PIP3 lipid messenger.
- Alternative translation initiation produces extended PTEN isoforms, including PTEN-L and PTEN-M, whose roles are less understood.
Purpose of the Study:
- To investigate the function of extended PTEN isoforms, PTEN-L and PTEN-M, in counteracting PI3K pathway activity.
- To determine the role of the N-terminal extension of PTEN-L in its subcellular localization and function.
- To identify specific domains and motifs within PTEN-L that regulate its localization.
Main Methods:
- Utilized a humanized yeast model engineered to express PI3K, causing growth inhibition.
- Co-expressed PTEN, PTEN-L, and PTEN-M in yeast to assess their ability to suppress PI3K-dependent growth inhibition.
- Employed N-terminal and C-terminal GFP tagging of PTEN-L to study its localization.
- Introduced point mutations and deletions in PTEN-L to investigate the role of specific regions (N-terminal extension, membrane-binding helix, PIP2-binding motif, C2 domain loops) in localization.
Main Results:
- PTEN-L and PTEN-M robustly suppressed PI3K-dependent growth inhibition in yeast.
- N-terminally tagged GFP-PTEN-L localized to the yeast plasma membrane.
- Mutations or deletion of the N-terminal membrane-binding helix or alterations in PIP2-binding motifs and C2 domain loops caused nuclear mislocalization.
- C-terminally tagged PTEN-L-GFP localized to mitochondria, but removal of the first 22 residues shifted localization to the plasma membrane.
Conclusions:
- The N-terminal extension of alternative PTEN isoforms plays a critical role in their spatial distribution and functional regulation.
- PTEN-L and PTEN-M exhibit potent tumor suppressor activity by inhibiting the PI3K pathway.
- Specific structural elements within PTEN-L dictate its subcellular localization, impacting its biological activity.
