The big and intricate dreams of little organelles: Embracing complexity in the study of membrane traffic

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138421/1/tra12497_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138421/2/tra12497-sup-0001-EditorialProcess.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138421/3/tra12497.pd

Akt-ing Up Just About Everywhere: Compartment-Specific Akt Activation and Function in Receptor Tyrosine Kinase Signaling

The serine/threonine kinase Akt is a master regulator of many diverse cellular functions, including survival, growth, metabolism, migration, and differentiation. Receptor tyrosine kinases are critical regulators of Akt, as a result of activation of phosphatidylinositol-3-kinase (PI3K) signaling leading to Akt activation upon receptor stimulation. The signaling axis formed by receptor tyrosine kinases, PI3K and Akt, as well as the vast range of downstream substrates is thus central to control of cell physiology in many different contexts and tissues. This axis must be tightly regulated, as disruption of PI3K-Akt signaling underlies the pathology of many diseases such as cancer and diabetes. This sophisticated regulation of PI3K-Akt signaling is due in part to the spatial and temporal compartmentalization of Akt activation and function, including in specific nanoscale domains of the plasma membrane as well as in specific intracellular membrane compartments. Here, we review the evidence for localized activation of PI3K-Akt signaling by receptor tyrosine kinases in various specific cellular compartments, as well as that of compartment-specific functions of Akt leading to control of several fundamental cellular processes. This spatial and temporal control of Akt activation and function occurs by a large number of parallel molecular mechanisms that are central to regulation of cell physiology

Loss of Tumor Suppressor TMEM127 Drives Ret-Mediated Transformation Through Disrupted Membrane Dynamics

Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTKs) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumor pheochromocytoma (PCC) can be caused by activating mutations of the rearranged during transfection (RET) receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumor suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin-coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation

Multiscale interactome analysis coupled with off-target drug predictions reveals drug repurposing candidates for human coronavirus disease

The COVID-19 pandemic has highlighted the urgent need for the identification of new antiviral drug therapies for a variety of diseases. COVID-19 is caused by infection with the human coronavirus SARS-CoV-2, while other related human coronaviruses cause diseases ranging from severe respiratory infections to the common cold. We developed a computational approach to identify new antiviral drug targets and repurpose clinically-relevant drug compounds for the treatment of a range of human coronavirus diseases. Our approach is based on graph convolutional networks (GCN) and involves multiscale host-virus interactome analysis coupled to off-target drug predictions. Cell-based experimental assessment reveals several clinically-relevant drug repurposing candidates predicted by the in silico analyses to have antiviral activity against human coronavirus infection. In particular, we identify the MET inhibitor capmatinib as having potent and broad antiviral activity against several coronaviruses in a MET-independent manner, as well as novel roles for host cell proteins such as IRAK1/4 in supporting human coronavirus infection, which can inform further drug discovery studies.We gratefully acknowledge funding that supported this research support from the Ryerson University Faculty of Science (CNA), as well as funding support in the form of a CIFAR Catalyst Grant (JPJ and CNA), an NSERC Alliance Grant (CNA) and the Ryerson COVID-19 SRC Response Fund award (CNA). BW is partly supported by CIFAR AI Chairs Program. This work was also supported by a Mitacs award (BW), the European Union’s Horizon 2020 research and innovation program under a Marie Sklodowska-Curie grant (ER), by the CIFAR Azrieli Global Scholar program (JPJ), by the Ontario Early Researcher Awards program (JPJ and CNA), and by the Canada Research Chairs program (JPJ). We also thank Dr. James Rini (University of Toronto) for the kind gift of the 9.8E12 antibody used to detect the 229E Spike protein, and Dr. Scott Gray-Owen (University of Toronto) for the kind gift of the NL63 human coronavirus.Peer reviewe

Regulation of GLUT4 Intrinsic Activity and Internalization in L6 Muscle Cells

GLUT4 is the principal insulin-responsive glucose transporter in skeletal muscle. Insulin stimulation leads to exocytosis of intracellular GLUT4-containing vesicles to the cell surface, thereby increasing glucose uptake. Muscle contraction also elevates cell surface GLUT4 by a less understood mechanism. Once at the cell surface, GLUT4 may be subject to additional regulation, such as by modulation of its internalization rate or its intrinsic activity. The objective of this thesis was to identify the mechanism of GLUT4 internalization in muscle cells and to determine whether it is regulated by insulin treatment or by the signals elicited by muscle contraction. Skeletal muscle cells in culture stably expressing myc-tagged GLUT4 were used. We found that GLUT4 internalizes simultaneously through a clathrin-dependent and a clathrin- and caveolae-independent and cholesterol- and dynamin-dependent pathway. Insulin did not regulate GLUT4 internalization. In contrast, mitochondrial uncoupling, which may mimic the heightened energy demand that occurs during muscle contraction, retarded GLUT4 internalization by inhibiting the clathrin-independent route. Activation of both AMP-dependent kinase (AMPK) and protein kinase C (PKC) was necessary and sufficient for this response. We further hypothesized that the intrinsic activity of GLUT4 may be regulated under some conditions, based on a discrepancy between the amount of cell surface transporters and the rate of glucose uptake. In particular, inhibitors of p38 mitogen-activated protein kinase (p38MAPK) lowered insulin-dependent glucose uptake without reducing the number of GLUT4 units at the surface. We found that p38MAPK is activated by insulin through TAB1-dependent autophosphorylation, yet p38MAPK was dispensable for insulin-stimulated glucose uptake. Mechanisms other than p38MAPK must be involved in the regulation of GLUT4 intrinsic activity. In conclusion, in addition to its exocytosis, the activity and endocytosis of GLUT4 are regulated by stimuli that increase the rate of glucose uptake into muscle.Ph

Integrins and Cell Metabolism: An Intimate Relationship Impacting Cancer

Integrins are important regulators of cell survival, proliferation, adhesion and migration. Once activated, integrins establish a regulated link between the extracellular matrix and the cytoskeleton. Integrins have well-established functions in cancer, such as in controlling cell survival by engagement of many specific intracellular signaling pathways and in facilitating metastasis. Integrins and associated proteins are regulated by control of transcription, membrane traffic, and degradation, as well as by a number of post-translational modifications including glycosylation, allowing integrin function to be modulated to conform to various cellular needs and environmental conditions. In this review, we examine the control of integrin function by cell metabolism, and the impact of this regulation in cancer. Within this context, nutrient sufficiency or deprivation is sensed by a number of metabolic signaling pathways such as AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR) and hypoxia-inducible factor (HIF) 1, which collectively control integrin function by a number of mechanisms. Moreover, metabolic flux through specific pathways also controls integrins, such as by control of integrin glycosylation, thus impacting integrin-dependent cell adhesion and migration. Integrins also control various metabolic signals and pathways, establishing the reciprocity of this regulation. As cancer cells exhibit substantial changes in metabolism, such as a shift to aerobic glycolysis, enhanced glucose utilization and a heightened dependence on specific amino acids, the reciprocal regulation of integrins and metabolism may provide important clues for more effective treatment of various cancers

Phosphatidic Acid Plays a Regulatory Role in Clathrin-mediated Endocytosis

We have manipulated the activities of PLD and DGK, enzymes that regulate PA biosynthesis, and directly measured their effects on cellular PA levels and on clathrin-mediated endocytosis (CME). We report a previously unappreciated complexity in PA regulation and show that PA selectively regulates CME of EGF but not transferrin

Similar requirement for clathrin in EGF- and HGF- stimulated Akt phosphorylation

Receptor tyrosine kinases, such as the epidermal growth factor (EGF) receptor (EGFR) and Met lead to activation of intracellular signals including Akt, a critical regulator of cell survival, metabolism and proliferation. Upon binding their respective ligands, each of these receptors is recruited into clathrin coated pits (CCPs) eventually leading to endocytosis. We have recently shown that phosphorylation of Gab1 and Akt following EGFR activation requires clathrin, but does not require receptor endocytosis. We examined whether clathrin regulates Akt signaling downstream of Met, as it does for EGFR signaling. Stimulation with the Met ligand Hepatocyte Growth Factor (HGF) leads to enrichment of phosphorylated Gab1 (pGab1) within CCPs in ARPE-19 cells. Perturbation of clathrin using the inhibitor pitstop2 decreases HGF-stimulated Akt phosphorylation. These results indicate that clathrin may regulate Met signaling leading to Akt phosphorylation similarly as it does for EGFR signaling

Ready, set, internalize: mechanisms and regulation of GLUT4 endocytosis

The facilitative glucose transporter GLUT4, a recycling membrane protein, is required for dietary glucose uptake into muscle and fat cells. GLUT4 is also responsible for the increased glucose uptake by myofibres during muscle contraction. Defects in GLUT4 membrane traffic contribute to loss of insulin-stimulated glucose uptake in insulin resistance and Type 2 diabetes. Numerous studies have analysed the intracellular membrane compartments occupied by GLUT4 and the mechanisms by which insulin regulates GLUT4 exocytosis. However, until recently, GLUT4 internalization was less well understood. In the present paper, we review: (i) evidence supporting the co-existence of clathrin-dependent and independent GLUT4 internalization in adipocytes and muscle cells; (ii) the contrasting regulation of GLUT4 internalization by insulin in these cells; and (iii) evidence suggesting regulation of GLUT4 endocytosis in muscle cells by signals associated with muscle contraction