The Ubiquitin/Proteasome System Mediates Entry and Endosomal Trafficking of Kaposi's Sarcoma-Associated Herpesvirus in Endothelial Cells

Ubiquitination, a post-translational modification, mediates diverse cellular functions including endocytic transport of molecules. Kaposi's sarcoma-associated herpesvirus (KSHV), an enveloped herpesvirus, enters endothelial cells primarily through clathrin-mediated endocytosis. Whether ubiquitination and proteasome activity regulates KSHV entry and endocytosis remains unknown. We showed that inhibition of proteasome activity reduced KSHV entry into endothelial cells and intracellular trafficking to nuclei, thus preventing KSHV infection of the cells. Three-dimensional (3-D) analyses revealed accumulation of KSHV particles in a cytoplasmic compartment identified as EEA1+ endosomal vesicles upon proteasome inhibition. KSHV particles are colocalized with ubiquitin-binding proteins epsin and eps15. Furthermore, ubiquitination mediates internalization of both KSHV and one of its receptors integrin β1. KSHV particles are colocalized with activated forms of the E3 ligase c-Cbl. Knock-down of c-Cbl or inhibition of its phosphorylation reduced viral entry and intracellular trafficking, resulting in decreased KSHV infectivity. These results demonstrate that ubiquitination mediates internalization of both KSHV and one of its cognate receptors integrin β1, and identify c-Cbl as a potential E3 ligase that facilitates this process

Degradation of Internalized αvβ5 Integrin Is Controlled by uPAR Bound uPA: Effect on β1 Integrin Activity and α-SMA Stress Fiber Assembly

Myofibroblasts (Mfs) that persist in a healing wound promote extracellular matrix (ECM) accumulation and excessive tissue contraction. Increased levels of integrin αvβ5 promote the Mf phenotype and other fibrotic markers. Previously we reported that maintaining uPA (urokinase plasminogen activator) bound to its cell-surface receptor, uPAR prevented TGFβ-induced Mf differentiation. We now demonstrate that uPA/uPAR controls integrin β5 protein levels and in turn, the Mf phenotype. When cell-surface uPA was increased, integrin β5 levels were reduced (61%). In contrast, when uPA/uPAR was silenced, integrin β5 total and cell-surface levels were increased (2–4 fold). Integrin β5 accumulation resulted from a significant decrease in β5 ubiquitination leading to a decrease in the degradation rate of internalized β5. uPA-silencing also induced α-SMA stress fiber organization in cells that were seeded on collagen, increased cell area (1.7 fold), and increased integrin β1 binding to the collagen matrix, with reduced activation of β1. Elevated cell-surface integrin β5 was necessary for these changes after uPA-silencing since blocking αvβ5 function reversed these effects. Our data support a novel mechanism by which downregulation of uPA/uPAR results in increased integrin αvβ5 cell-surface protein levels that regulate the activity of β1 integrins, promoting characteristics of the persistent Mf

Cdc28-Cln3 phosphorylation of Sla1 regulates actin patch dynamics in different modes of fungal growth

10.1091/mbc.E12-03-0231Molecular Biology of the Cell23173485-3497MBCE

Integrin endosomal signalling suppresses anoikis

Integrin containing focal adhesions (FAs) transmit extracellular signals across the plasma membrane to modulate cell adhesion, signalling and survival. Although integrins are known to undergo continuous endo/exocytic traffic, potential impact of endocytic traffic on integrin-induced signals is unknown. Here, we demonstrate that integrin signalling is not restricted to cell-ECM adhesions and identify an endosomal signalling platform that supports integrin signalling away from the plasma membrane. We show that active focal adhesion kinase (FAK), an established marker of integrin-ECM downstream signalling, localises with active integrins on endosomes. Integrin endocytosis positively regulates adhesion-induced FAK activation, which is early endosome antigen-1 (EEA1) and small GTPase Rab21 dependent. FAK binds directly to purified endosomes and becomes activated on them, suggesting a role for endocytosis in enhancing distinct integrin downstream signalling events. Finally, endosomal integrin signalling contributes to cancer-related processes such as anoikis resistance, anchorage-independence and metastasis. Integrins are heterodimeric cell surface adhesion receptors functioning as integrators of the extra-cellular matrix (ECM) driven cues, the cellular cytoskeleton and the cellular signalling apparatus 1.Upon adhesion, integrins trigger the formation of plasma-membrane proximal large mechanosensing and signal-transmitting protein clusters depicted as “adhesomes” 2, 3. In addition, integrins undergo constant endocytic traffic to facilitate focal adhesion turnover, cell migration, invasion and cytokinesis 4. For other receptor systems it is well established that endocytic membrane traffic regulates bioavailability of cell-surface molecules and therefore the intensity and/or specificity of receptor-initiated signals 5, 6. Although active integrins and their ligands have been detected in endosomes 7–9 and increased integrin recycling to the plasma membrane contributes to enhanced signalling of co-trafficked receptor tyrosine kinases10, 11 it has remained unclear whether endocytosed active integrins signal in endosomes. Here, we demonstrate that integrin signalling is not restricted to focal adhesions as previously described and that endocytosis is necessary for full ECM-induced, integrin mediated ERK, AKT and FAK signalling. We find that FAK binds directly to and can become activated on purified endosomes. Moreover, the FERM-domain of FAK is able to bind purified integrin containing endosomes, suggesting the potential for integrin signalling complexes to assemble on endosomes after internalization of active integrins. Importantly, FAK is required for anchorage-independent growth and suppression of anoikis 12. Integrin endosomal signalling correlates with reduced anoikis sensitivity in normal cells and anchorage-independent growth and metastasis in breast cancer cells