[Frontiers in Bioscience E3, 1289-1299, June 1, 2011]

SNAT2 transceptor signalling via mTOR: A role in cell growth and proliferation?

Jorge Pinilla, Juan Carlos Aledo, Emma Cwiklinski, Russell Hyde, Peter M Taylor, Harinder S Hundal

Division of Molecular Physiology, James Black Centre, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, United Kingdom

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials
3.1. Materials
3.2. Cell Culture
3.3. Analysis of cell number, size and intracellular amino acids
3.4. SDS-PAGE and immunoblotting
3.5. Measurement of System A uptake
3.6. Proteomic analysis of TAP-tagged SNAT2 complexes
3.7. Statistical analysis
4. Results
4.1. MCF-7 cells express SNAT1, SNAT2 and exhibit functional System A transport
4.2. Effects of sustained System A inhibition on cell growth and proliferation
4.3. Chronic cell incubation with Me-AIB and intracellular amino acid levels
4.4. System A-induced modulation of mTOR signalling
4.5. Analysis of TAP- tagged SNAT2 protein complexes
5. Discussion
6. Acknowledgment
7. References

1. ABSTRACT

We have investigated the effect of chronic competitive inhibition of SNAT2 (System A) amino acid (AA) transport, induced by incubation with a saturating dose of a non-metabolisable System A amino acid analogue (Me-AIB), on growth and proliferation of MCF-7 human breast cancer cells in complete culture medium. These cells express Na+- and pH-dependent SNAT2 AA transport and a saturating concentration of Me-AIB (10 mM) competitively inhibits (>90%) AA uptake via SNAT2. Incubation with Me-AIB for up to 5 days progressively reduced cell proliferation (~2-fold) and depleted intracellular concentrations of not only SNAT2 AA substrates but of essential branched chain AAs (e.g. leucine). Surprisingly, total cellular protein was maintained and cells subjected to chronic Me-AIB incubation exhibited a detectable increase in cell size. Analysis of mTOR signalling revealed that, despite a substantial reduction in size of the intracellular AA pool, Me-AIB elevated mTOR-dependent p70S6K1 phosphorylation. Proteomic analysis of TAP-tag purified SNAT2 fusion proteins identified two novel SNAT2-interacting proteins that may potentially function in conjunction with the SNAT2 transceptor to regulate signalling pathways influencing protein turnover and cell growth.