title: Interaction of c-Cbl with myosin IIA regulates Bleb associated macropinocytosis of Kaposi's sarcoma-associated herpesvirus.
abstract: KSHV is etiologically associated with Kaposi's sarcoma (KS), an angioproliferative endothelial cell malignancy. Macropinocytosis is the predominant mode of in vitro entry of KSHV into its natural target cells, human dermal microvascular endothelial (HMVEC-d) cells. Although macropinocytosis is known to be a major route of entry for many viruses, the molecule(s) involved in the recruitment and integration of signaling early during macropinosome formation is less well studied. Here we demonstrate that tyrosine phosphorylation of the adaptor protein c-Cbl is required for KSHV induced membrane blebbing and macropinocytosis. KSHV induced the tyrosine phosphorylation of c-Cbl as early as 1 min post-infection and was recruited to the sites of bleb formation. Infection also led to an increase in the interaction of c-Cbl with PI3-K p85 in a time dependent manner. c-Cbl shRNA decreased the formation of KSHV induced membrane blebs and macropinocytosis as well as virus entry. Immunoprecipitation of c-Cbl followed by mass spectrometry identified the interaction of c-Cbl with a novel molecular partner, non-muscle myosin heavy chain IIA (myosin IIA), in bleb associated macropinocytosis. Phosphorylated c-Cbl colocalized with phospho-myosin light chain II in the interior of blebs of infected cells and this interaction was abolished by c-Cbl shRNA. Studies with the myosin II inhibitor blebbistatin demonstrated that myosin IIA is a biologically significant component of the c-Cbl signaling pathway and c-Cbl plays a new role in the recruitment of myosin IIA to the blebs during KSHV infection. Myosin II associates with actin in KSHV induced blebs and the absence of actin and myosin ubiquitination in c-Cbl ShRNA cells suggested that c-Cbl is also responsible for the ubiquitination of these proteins in the infected cells. This is the first study demonstrating the role of c-Cbl in viral entry as well as macropinocytosis, and provides the evidence that a signaling complex containing c-Cbl and myosin IIA plays a crucial role in blebbing and macropinocytosis during viral infection and suggests that targeting c-Cbl could lead to a block in KSHV infection.
authors.affiliation: H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, United States of America.
authors.forename: Mohanan
authors.initials: M
authors.lastname: Valiya Veettil
authors.forename: Sathish
authors.initials: S
authors.lastname: Sadagopan
authors.forename: Nagaraj
authors.initials: N
authors.lastname: Kerur
authors.forename: Sayan
authors.initials: S
authors.lastname: Chakraborty
authors.forename: Bala
authors.initials: B
authors.lastname: Chandran
mesh_terms: D021961:Cell Membrane Structures / Q000821:virology*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000821:virology*; D004730:Endothelium, Vascular / Q000166:cytology; D019288:Herpesvirus 8, Human / Q000502:physiology*; D006801:Humans; D024507:Nonmuscle Myosin Type IIA / Q000378:metabolism*; D010873:Pinocytosis; D011485:Protein Binding; D050721:Proto-Oncogene Proteins c-cbl / Q000378:metabolism*; D015398:Signal Transduction; D053586:Virus Internalization
chemical_list: D050721:Proto-Oncogene Proteins c-cbl; D024507:Nonmuscle Myosin Type IIA; C067419:CBL protein, human