AMP-Activated Protein Kinase Regulates the Cell Surface Proteome and Integrin Membrane Traffic

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.

Download Full-text

AMP-Activated Protein Kinase Regulates the Cell Surface Proteome and Integrin Membrane Traffic

10.32920/14668971.v1 ◽

2021 ◽

Author(s):

Antonescu Costin ◽

Eden Ross ◽

Rehman Ata ◽

Thanusi Thavarajah ◽

Sergei Medvedev ◽

...

Keyword(s):

Cell Migration ◽

Protein Kinase ◽

Cell Surface ◽

Surface Proteins ◽

Cell Surface Proteins ◽

Metabolic Sensor ◽

Acute Changes ◽

Surface Proteome ◽

Amp Activated Protein Kinase ◽

Cell Surface Proteome

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.

Download Full-text

The regulation of the cell surface proteome by amp-activated protein kinase

10.32920/ryerson.14662851 ◽

2021 ◽

Author(s):

Eden Ross

Keyword(s):

Protein Kinase ◽

Cell Surface ◽

Metabolic Regulation ◽

Cultured Cells ◽

Cellular Adhesion ◽

Ampk Activation ◽

And Migration ◽

Surface Proteome ◽

Amp Activated Protein Kinase ◽

Cell Surface Proteome

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.

Download Full-text

AMP-Activated Protein Kinase Regulates the Cell Surface Proteome and Integrin Membrane Traffic

10.32920/14668971 ◽

2021 ◽

Author(s):

Antonescu Costin ◽

Eden Ross ◽

Rehman Ata ◽

Thanusi Thavarajah ◽

Sergei Medvedev ◽

...

Keyword(s):

Cell Migration ◽

Protein Kinase ◽

Cell Surface ◽

Surface Proteins ◽

Cell Surface Proteins ◽

Metabolic Sensor ◽

Acute Changes ◽

Surface Proteome ◽

Amp Activated Protein Kinase ◽

Cell Surface Proteome

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.

Download Full-text

Regulation of β1 Integrin Traffic and ARF6 Gtpase by Amp-Activated Protein Kinase

10.32920/ryerson.14645772.v1 ◽

2021 ◽

Author(s):

Rehman Ata

Keyword(s):

Protein Kinase ◽

Cell Surface ◽

Cancer Progression ◽

Membrane Traffic ◽

Β1 Integrin ◽

Ampk Activation ◽

Reciprocal Regulation ◽

Surface Receptors ◽

Energy Stress ◽

Amp Activated Protein Kinase

Integrins are cell surface receptors that physically bridge the extracellular matrix to the cytoskeleton and responsible for adhesion, migration, and signaling. Integrin function is intimately controlled by their membrane traffic. For example, integrins are dynamically internalized from the cell posterior and recycled to the cell anterior during cell migration. Misregulation of integrins is intimately linked with cancer progression, including metastasis and cell proliferation and survival. We have recently uncovered that integrin membrane traffic is controlled by AMP-activated protein kinase (AMPK), an energy stress sensing kinase within cells at becomes activated upon energy stress such as by an increase in cell AMP:ATP ratio. I confirmed that AMPK activation resulted in a reduction of cell surface β1-integrin. Using assays that selectively measure integrin exocytosis and endocytosis, I found that AMPK activation regulates β1-integrin recycling and possibly endocytosis. I demonstrated that AMPK regulates Arf6 localization, a key protein which regulates β1-integrin membrane traffic. I confirmed that Arf6 and clathrin are involved in reciprocal regulation, thus highlighting the possible pathway for β1-integrin regulation by AMPK.

Download Full-text

Regulation of β1 Integrin Traffic and ARF6 Gtpase by Amp-Activated Protein Kinase

10.32920/ryerson.14645772 ◽

2021 ◽

Author(s):

Rehman Ata

Keyword(s):

Protein Kinase ◽

Cell Surface ◽

Cancer Progression ◽

Membrane Traffic ◽

Β1 Integrin ◽

Ampk Activation ◽

Reciprocal Regulation ◽

Surface Receptors ◽

Energy Stress ◽

Amp Activated Protein Kinase

Integrins are cell surface receptors that physically bridge the extracellular matrix to the cytoskeleton and responsible for adhesion, migration, and signaling. Integrin function is intimately controlled by their membrane traffic. For example, integrins are dynamically internalized from the cell posterior and recycled to the cell anterior during cell migration. Misregulation of integrins is intimately linked with cancer progression, including metastasis and cell proliferation and survival. We have recently uncovered that integrin membrane traffic is controlled by AMP-activated protein kinase (AMPK), an energy stress sensing kinase within cells at becomes activated upon energy stress such as by an increase in cell AMP:ATP ratio. I confirmed that AMPK activation resulted in a reduction of cell surface β1-integrin. Using assays that selectively measure integrin exocytosis and endocytosis, I found that AMPK activation regulates β1-integrin recycling and possibly endocytosis. I demonstrated that AMPK regulates Arf6 localization, a key protein which regulates β1-integrin membrane traffic. I confirmed that Arf6 and clathrin are involved in reciprocal regulation, thus highlighting the possible pathway for β1-integrin regulation by AMPK.

Download Full-text

CIRFESS: An interactive resource for querying the set of theoretically detectable peptides for cell surface and extracellular enrichment proteomic studies

10.1101/2020.01.22.916148 ◽

2020 ◽

Author(s):

Matthew Waas ◽

Jack Littrell ◽

Rebekah L. Gundry

Keyword(s):

Cell Surface ◽

Critical Role ◽

Affinity Reagents ◽

Extracellular Proteome ◽

Online Resource ◽

Health And Disease ◽

Limited Mass ◽

Multiple Prediction ◽

Surface Proteome ◽

Cell Surface Proteome

AbstractCell surface transmembrane, extracellular, and secreted proteins are high value targets for immunophenotyping, drug development, and studies related to intercellular communication in health and disease. As the number of specific and validated affinity reagents that target this subproteome are limited, mass spectrometry (MS)-based approaches will continue to play a critical role in enabling discovery and quantitation of these molecules. Given the technical considerations that make MS-based cell surface proteome studies uniquely challenging, it can be difficult to select an appropriate experimental approach. To this end, we have integrated multiple prediction strategies and annotations into a single online resource, Compiled Interactive Resource for Extracellular and Surface Studies (CIRFESS). CIRFESS enables rapid interrogation of the human proteome to reveal the cell surface proteome theoretically detectable by current approaches and highlights where current prediction strategies provide concordant and discordant information. We applied CIRFESS to identify the percentage of various subsets of the proteome which are expected to be captured by targeted enrichment strategies, including two established methods and one that is possible but not yet demonstrated. These results will inform the selection of available proteomic strategies and development of new strategies to enhance coverage of the cell surface and extracellular proteome. CIRFESS is available at www.cellsurfer.net/cirfess.

Download Full-text