6 research outputs found
AMP-Activated Protein Kinase Regulates the Cell Surface Proteome and Integrin Membrane Traffic
<div><p>The cell surface proteome controls numerous cellular functions including cell migration and adhesion, intercellular communication and nutrient uptake. Cell surface proteins are controlled by acute changes in protein abundance at the plasma membrane through regulation of endocytosis and recycling (endomembrane traffic). Many cellular signals regulate endomembrane traffic, including metabolic signaling; however, the extent to which the cell surface proteome is controlled by acute regulation of endomembrane traffic under various conditions remains incompletely understood. AMP-activated protein kinase (AMPK) is a key metabolic sensor that is activated upon reduced cellular energy availability. AMPK activation alters the endomembrane traffic of a few specific proteins, as part of an adaptive response to increase energy intake and reduce energy expenditure. How increased AMPK activity during energy stress may globally regulate the cell surface proteome is not well understood. To study how AMPK may regulate the cell surface proteome, we used cell-impermeable biotinylation to selectively purify cell surface proteins under various conditions. Using ESI-MS/MS, we found that acute (90 min) treatment with the AMPK activator A-769662 elicits broad control of the cell surface abundance of diverse proteins. In particular, A-769662 treatment depleted from the cell surface proteins with functions in cell migration and adhesion. To complement our mass spectrometry results, we used other methods to show that A-769662 treatment results in impaired cell migration. Further, A-769662 treatment reduced the cell surface abundance of β1-integrin, a key cell migration protein, and AMPK gene silencing prevented this effect. While the control of the cell surface abundance of various proteins by A-769662 treatment was broad, it was also selective, as this treatment did not change the cell surface abundance of the transferrin receptor. Hence, the cell surface proteome is subject to acute regulation by treatment with A-769662, at least some of which is mediated by the metabolic sensor AMPK.</p></div
Inhibition of AMPK by siRNA gene silencing or by compound C prevents the reduction in cell surface β1-integrin elicited by A-769662 treatment.
<p>(<b><i>A-C)</i></b> RPE cells were transfected with siRNA targeting AMPK α1/2 or non-targeting (NT, control) siRNA. (<b><i>A</i></b>) Whole cell lysates were prepared and resolved by immunoblotting and probed with anti-AMPK α1/2 or anti-actin antibodies. Shown are immunoblots representative of at least 3 independent experiments. (<b><i>B</i></b>) Following siRNA transfection, cells were treated with 100 μM A-769662 for 60 min as indicated. Intact cells were labeled with an antibody specific for an exofacial epitope on β1-integrin. Shown are representative fluorescence micrographs depicting cell surface β1-integrin fluorescence. Scale = 5 μm (<b><i>C)</i></b> Cell surface β1-integrin levels obtained by fluorescence microscopy were quantified. Shown are the cell surface β1-integrin measurements in individual cells (diamonds) as well as the median ± interquartile range of these values in each treatment condition (n = 3 independent experiments). (<b><i>D</i></b>) RPE cells were treated with 100 μM A-769662 or 40 μM compound C, alone or in combination, for 60 min as indicated. Intact cells were labeled with an antibody specific for an exofacial epitope on β1-integrin. Shown are representative fluorescence micrographs depicting cell surface β1-integrin fluorescence. Scale = 5 μm (<b><i>E)</i></b> Cell surface β1-integrin levels obtained by fluorescence microscopy as in (D) were quantified. Shown are the cell surface β1-integrin measurements in individual cells (diamonds) as well as the median ± interquartile range of these values in each treatment condition (n = 3 independent experiments).</p
Proteins with cell adhesion and migration GO classification depleted from the cell surface upon A-769662 treatment.
<p>Shown are the 32 proteins with Cell Adhesion and Migration GO classification detected in the cell surface fraction of control but not A-769662 treated cells. Shown for each are the detected peptide counts in each of the treatment conditions.</p><p>Proteins with cell adhesion and migration GO classification depleted from the cell surface upon A-769662 treatment.</p
AMPK activation and mass spectrometry analysis of cell surface proteins.
<p>(<b><i>A</i></b>) RPE cells were stimulated with 100 μM A-769662 in media containing 0.1% FBS for indicated times. Shown are representative immunoblots using antibodies as indicated. (<b><i>B</i></b>) Shown is a diagram depicting cell stimulation, surface biotinylation, purification of biotinylated proteins, mass spectrometry and peptide identification. We thus identified a total of 838 proteins within all cell surface fractions, of which 653 exhibited reduced detection in the cell surface fraction of cells treated with A-769662, 93 proteins exhibited increased cell surface abundance in cells treated with A-769662, and a further 92 were classified as exhibiting largely unaltered detection in the cell surface fraction upon AMPK activation. A complete list of identified proteins can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128013#pone.0128013.s001" target="_blank">S1 Table</a>.</p
Treatment with A-769662 reduces cell surface β1-integrin levels.
<p>(<b><i>A</i></b>) RPE cells were stimulated with 100 μM A-769662 for 90 min or left unstimulated (basal). Intact cells were labeled with an antibody specific for an exofacial epitope on β1-integrin. Shown are representative fluorescence micrographs depicting cell surface β1-integrin fluorescence. Scale = 5 μm (<b><i>B</i></b>) Cell surface β1-integrin levels obtained by fluorescence microscopy were quantified as described in <i>Materials and Methods</i> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128013#pone.0128013.s009" target="_blank">S5B Fig</a>. Shown are the cell surface β1-integrin measurements in individual cells (diamonds) as well as the median ± interquartile range of these values in each treatment condition (n = 4 independent experiments). (<b><i>C</i></b>) RPE cells were stimulated with 2 mM AICAR for 90 min or left unstimulated (basal), followed by cell-surface biotinylation, purification of biotinylated proteins and immunoblotting of fractions with an antibody specific to β1-integrin. Shown is an immunoblot of cell surface β1-intergin (<i>top panel</i>, corresponding to the streptavidin pull-down), and of the corresponding intracellular β1-integrin (<i>bottom panel</i>, corresponding to the above supernatant), representative of 4 independent experiments. (<b><i>D</i></b>) Shown are representative immunoblots of whole-cell lysates prepared from cells stimulated with either 100 μM A-769662, 2 mM AICAR, 40 μM compound C (each for 90 min) or left unstimulated (control), probed with antibodies to detect total cellular β1-integrin or actin (load).</p
Treatment with A-769662 does not change cell surface TfR levels.
<p>(<b><i>A</i></b>) RPE cells were stimulated with 100 μM A-769662 for 90 min or left unstimulated (basal). Intact cells were labeled with an antibody specific for an exofacial epitope on Transferrin Receptor (TfR). Shown are representative fluorescence micrographs depicting cell surface TfR fluorescence. Scale = 5 μm (<b><i>B</i></b>) Cell surface TfR levels obtained by fluorescence microscopy were quantified. Shown are the cell surface TfR measurements in individual cells (diamonds) as well as the median ± interquartile range of these values in each treatment condition (n = 3 independent experiments).</p