Abstract
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.
Highlights
Cells interact with their environment through the molecules present on their surface
Treatment of cells with sulfoNHS-SS-Biotin followed by this biotin purification strategy resulted in recovery of a large number of proteins with very few visible protein bands observed in samples from cells not treated with sulfo-NHS-SS-Biotin, as assessed by silver staining (S1A Fig)
We observed that the epidermal growth factor receptor (EGFR) is found virtually exclusively in the cell surface fraction of cells treated with sulfo-NHS-SS-Biotin
Summary
Cells interact with their environment through the molecules present on their surface. The cell surface proteome must be tightly regulated in order to ensure homeostasis under conditions of cellular and systemic challenges. Cell surface membrane proteins undergo dynamic traffic between the plasma membrane and intracellular endosomes [1]. The steady-state abundance of proteins at the cell surface is determined by the balance of the rate of endocytosis and exocytosis/recycling of each specific protein [1]. About half of the 590 human kinases control various stages of endomembrane traffic [2], suggesting that the cell surface proteome is the subject of extensive control by various cues; much of this regulation remains poorly understood [1]
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