title: Conserved BK channel-protein interactions reveal signals relevant to cell death and survival.
abstract: The large-conductance Ca(2+)-activated K(+) (BK) channel and its β-subunit underlie tuning in non-mammalian sensory or hair cells, whereas in mammals its function is less clear. To gain insights into species differences and to reveal putative BK functions, we undertook a systems analysis of BK and BK-Associated Proteins (BKAPS) in the chicken cochlea and compared these results to other species. We identified 110 putative partners from cytoplasmic and membrane/cytoskeletal fractions, using a combination of coimmunoprecipitation, 2-D gel, and LC-MS/MS. Partners included 14-3-3γ, valosin-containing protein (VCP), stathmin (STMN), cortactin (CTTN), and prohibitin (PHB), of which 16 partners were verified by reciprocal coimmunoprecipitation. Bioinformatics revealed binary partners, the resultant interactome, subcellular localization, and cellular processes. The interactome contained 193 proteins involved in 190 binary interactions in subcellular compartments such as the ER, mitochondria, and nucleus. Comparisons with mice showed shared hub proteins that included N-methyl-D-aspartate receptor (NMDAR) and ATP-synthase. Ortholog analyses across six species revealed conserved interactions involving apoptosis, Ca(2+) binding, and trafficking, in chicks, mice, and humans. Functional studies using recombinant BK and RNAi in a heterologous expression system revealed that proteins important to cell death/survival, such as annexinA5, γ-actin, lamin, superoxide dismutase, and VCP, caused a decrease in BK expression. This revelation led to an examination of specific kinases and their effectors relevant to cell viability. Sequence analyses of the BK C-terminus across 10 species showed putative binding sites for 14-3-3, RAC-α serine/threonine-protein kinase 1 (Akt), glycogen synthase kinase-3β (GSK3β) and phosphoinositide-dependent kinase-1 (PDK1). Knockdown of 14-3-3 and Akt caused an increase in BK expression, whereas silencing of GSK3β and PDK1 had the opposite effect. This comparative systems approach suggests conservation in BK function across different species in addition to novel functions that may include the initiation of signals relevant to cell death/survival.
authors.affiliation: Otology Laboratory, Department of Otolaryngology - Head and Neck Surgery, University of South Florida, College of Medicine, Tampa, Florida, United States of America.
authors.forename: Bernd
authors.initials: B
authors.lastname: Sokolowski
authors.forename: Sandra
authors.initials: S
authors.lastname: Orchard
authors.forename: Margaret
authors.initials: M
authors.lastname: Harvey
authors.forename: Settu
authors.initials: S
authors.lastname: Sridhar
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Sakai
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016923:Cell Death; D002470:Cell Survival; D002645:Chickens; D003051:Cochlea / Q000166:cytology*; D017124:Conserved Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D047468:Immunoprecipitation; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010802:Phylogeny; D000091402:Prohibitins; D011485:Protein Binding; D060066:Protein Interaction Maps; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D013045:Species Specificity; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D051036:Large-Conductance Calcium-Activated Potassium Channels; C000717328:PHB protein, human; D000091402:Prohibitins; D034741:RNA, Small Interfering