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.
The many proteins at the cell surface (collectively, the cell surface proteome) control numerous cellular functions. The amount of each protein at the cell surface is dynamically controlled by endocytosis and recycling. AMP‐activated protein kinase (AMPK) is an important metabolic regulator that responds to energetic stress, such as increased cellular AMP:ATP and certain hormones. Studies examining a small number of cell surface proteins suggest that AMPK may have an important role in regulating endomembrane traffic. How AMPK may more broadly regulate the cell surface proteome is not known. We have refined a method to isolate the cell surface proteome from cultured cells, involving biotinylation of surface‐exposed proteins in intact cells and purification of biotinylated proteins, which we coupled to quantitative mass spectrometry to identify novel AMPK‐regulated membrane traffic phenomena. We find that activation of AMPK results in a decrease in the cell surface abundance of the β1‐integrin, revealing a mechanism by which cellular metabolism may regulate cell adhesion and migration. Our results provide novel insight into the regulation of the cell surface proteome by metabolic stress signaling.
The cell-surface proteome controls numerous cellular functions and is dynamically controlled by endocytosis and recycling under different cellular conditions. Energy stress is a state in which a cell must engage adaptive responses to ensure survival, including remodelling of the cell-surface proteome. AMP-activated protein kinase (AMPK) is an important metabolic regulator in the cell. Recent studies suggest AMPK activation may alter the endocytosis of a few specific proteins. How increased AMPK activity globally regulates the cell surface proteome is not known. I have developed a method to isolate the cell surface proteome from cultured cells. Coupling this method to quantitative mass spectrometry has allowed systematic identification of changes in the cell-surface proteome upon metabolic regulation. I found that activation of AMPK results in robust changes in the cell surface proteome, including cell adhesion and migration proteins. I confirmed that AMPK activation elicits a decrease in the cell surface abundance of the adhesion and migration protein β1-integrin, and that this is correlated with altered function of the endocytosis protein Dab2. Thus, my research furthers our understanding of how AMPK regulates the cell surface proteome and the specific mechanism by which AMPK regulates cellular adhesion and migration.
The cell-surface proteome controls numerous cellular functions and is dynamically controlled by endocytosis and recycling under different cellular conditions. Energy stress is a state in which a cell must engage adaptive responses to ensure survival, including remodelling of the cell-surface proteome. AMP-activated protein kinase (AMPK) is an important metabolic regulator in the cell. Recent studies suggest AMPK activation may alter the endocytosis of a few specific proteins. How increased AMPK activity globally regulates the cell surface proteome is not known. I have developed a method to isolate the cell surface proteome from cultured cells. Coupling this method to quantitative mass spectrometry has allowed systematic identification of changes in the cell-surface proteome upon metabolic regulation. I found that activation of AMPK results in robust changes in the cell surface proteome, including cell adhesion and migration proteins. I confirmed that AMPK activation elicits a decrease in the cell surface abundance of the adhesion and migration protein β1-integrin, and that this is correlated with altered function of the endocytosis protein Dab2. Thus, my research furthers our understanding of how AMPK regulates the cell surface proteome and the specific mechanism by which AMPK regulates cellular adhesion and migration.
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.
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