title: Calcium Stimulates Self-Assembly of Protein Kinase C α In Vitro.
abstract: Protein kinase C α (PKCα) is a nodal regulator in several intracellular signaling networks. PKCα is composed of modular domains that interact with each other to dynamically regulate spatial-temporal function. We find that PKCα specifically, rapidly and reversibly self-assembles in the presence of calcium in vitro. This phenomenon is dependent on, and can be modulated by an intramolecular interaction between the C1a and C2 protein domains of PKCα. Next, we monitor self-assembly of PKC-mCitrine fusion proteins using time-resolved and steady-state homoFRET. HomoFRET between full-length PKCα molecules is observed when in solution with both calcium and liposomes containing either diacylglycerol (DAG) or phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Surprisingly, the C2 domain is sufficient to cluster on liposomes containing PI(4,5)P2, indicating the C1a domain is not required for self-assembly in this context. We conclude that three distinct clustered states of PKCα can be formed depending on what combination of cofactors are bound, but Ca2+ is minimally required and sufficient for clustering.
authors.affiliation: Biophysics Program, University of Michigan, Ann Arbor, 48109, United States of America.
authors.forename: Carter J
authors.identifier: http://orcid.org/0000-0001-6697-9475
authors.initials: CJ
authors.lastname: Swanson
authors.affiliation: Dept. of Genetics, Cell Biology and Development, University of Minnesota, Twin Cities, 55455, United States of America.
authors.forename: Ruth F
authors.initials: RF
authors.lastname: Sommese
authors.affiliation: Dept. of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Twin Cities, 55455, United States of America.
authors.forename: Karl J
authors.initials: KJ
authors.lastname: Petersen
authors.affiliation: Dept. of Genetics, Cell Biology and Development, University of Minnesota, Twin Cities, 55455, United States of America.
authors.forename: Michael
authors.initials: M
authors.lastname: Ritt
authors.affiliation: Biophysics Program, University of Michigan, Ann Arbor, 48109, United States of America.
authors.forename: Joshua
authors.initials: J
authors.lastname: Karslake
authors.affiliation: Dept. of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Twin Cities, 55455, United States of America.
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
authors.affiliation: Dept. of Genetics, Cell Biology and Development, University of Minnesota, Twin Cities, 55455, United States of America.
authors.forename: Sivaraj
authors.initials: S
authors.lastname: Sivaramakrishnan
mesh_terms: D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002850:Chromatography, Gel; D006224:Cricetinae; D003412:Cricetulus; D004075:Diglycerides / Q000737:chemistry / Q000378:metabolism; D000067493:Dynamic Light Scattering; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry / Q000378:metabolism; D000072417:Protein Domains; D017510:Protein Folding; D051571:Protein Kinase C-alpha / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D061987:Sf9 Cells
chemical_list: C011439:1,2-diacylglycerol; D004075:Diglycerides; D008081:Liposomes; D019269:Phosphatidylinositol 4,5-Diphosphate; D011994:Recombinant Proteins; D051571:Protein Kinase C-alpha; D002118:Calcium