Supplementary MaterialsS1 Table: Shown is the complete list of proteins identified as (i) depleted from your cell surface upon A-769662 treatment, (ii) enriched at the cell surface by A-769662 treatment and (iii) largely unchanged at the cell surface by A-769662. fragment ion intensities were very similar between control and A-769662 treated cells, showing that the differences Prkwnk1 in detection of specific proteins between conditions was unlikely to be due to sampling error.(PDF) pone.0128013.s003.pdf (59K) GUID:?D320A0E3-FDB8-43A8-9F25-5AA1B7766E4E S4 Table: Shown are sample mass spectrometry measurements for any subset of peptides corresponding to integrin -11. Shown are the following for parent Tartaric acid ions (first tab): of parent ion, of each fragment and fragment intensity (shown in attached. xls document).(XLSX) pone.0128013.s004.xlsx (257K) GUID:?4CA0B377-C7C8-4FCA-8654-A2691D01A814 S1 Fig: Cell surface biotinylation allows selective purification of integral and membrane-associated cell surface proteins. RPE cells were subjected to surface biotinylated by treatment with sulfo-NHS-SS-biotin or left untreated (background), following by purification of cell surface proteins by streptavidin bead pull-down. (phosphorylation of acetyl CoA carboxylase [8], controls aerobic glycolysis the activation of HIF-1 [9], controls the formation of tight junctions [10], microtubule dynamics [11], and controls the cell cycle p53 phosphorylation [12]. Activated AMPK also limits energy rigorous processes and increases nutrient intake by regulation of cell surface membrane Tartaric acid traffic [1]. AMPK Tartaric acid activation impairs the internalization of the facilitative glucose transporters GLUT4 in skeletal muscle mass cells [13] and cardiomyocytes [14], and GLUT1 in a variety of cell types [15]. The producing increase in cell surface GLUTs increases the rate of glucose uptake, Tartaric acid which facilitates the maintenance of ATP homeostasis [16]. AMPK activation increases the internalization of the Na/K-ATPase [1] and also controls the cell-surface membrane Tartaric acid traffic of the tight junction protein occulin [17], of the fatty acid transporter CD36 [18] and of the Na+/H+ exchanger NHE5 [19]. The extent of the control of the cell surface proteome by AMPK beyond this small but growing quantity of proteins is usually unknown. AMPK might be expected to preferentially exert control over cell surface abundance of proteins that contribute to energy-demanding processes. Cell migration is an energy demanding process, as it requires actin remodeling and coordinated cell surface and endomembrane traffic. As such, cell migration might be tightly controlled, such that the extent of cell migration may match energy availability. Indeed hypoxia-mediated activation of AMPK reduces cell adhesion in endothelial cells [20] and brokers that elicit AMPK activation regulate cell adhesion and migration: berberine [21], AICAR and phenformin [22] or metformin [23] alter cell migration. As many of these brokers and treatments have cellular effects additional to the activation of AMPK [24], the possible regulation of cell adhesion and migration by AMPK activation requires further study. Cell adhesion and migration are controlled by the regulated membrane traffic of integrins, a family of transmembrane proteins that actually bridge the actin cytoskeleton to the extracellular matrix. Integrins are heterodimers comprised of one – and one -subunit [25]. 1-integrin is the principal binding partner of many -integrins and as such is usually a key cell adhesion and migration molecule [25]. The leading edge of the lamellipodium of migrating cells is usually a zone of dynamic actin remodeling, which generates pushing forces around the membrane, in part as a result of the conversation of integrins with actin filaments [26]. Cell migration requires dynamic integrin membrane traffic [27]. Integrins undergo internalization both clathrin-dependent and-independent mechanisms [28], and are recycled back to the plasma membrane via Rab4, Rab11 and/or Rab21 endosomes [27,29C31]. Hence, the control of integrin membrane traffic regulates cell migration [27]. Whether AMPK may broadly and acutely control the cell surface proteome in order to limit energy expenditure is usually poorly understood. Recently, strategies have already been developed to review the cell surface area proteome systematically. Several studies possess used cell-impermeable lysine- or glycan-reactive biotinylation reagents to label surface-exposed proteins, purification.