title: Submembranous recruitment of creatine kinase B supports formation of dynamic actin-based protrusions of macrophages and relies on its C-terminal flexible loop.
abstract: Subcellular partitioning of creatine kinase contributes to the formation of patterns in intracellular ATP distribution and the fuelling of cellular processes with a high and sudden energy demand. We have previously shown that brain-type creatine kinase (CK-B) accumulates at the phagocytic cup in macrophages where it is involved in the compartmentalized generation of ATP for actin remodeling. Here, we report that CK-B catalytic activity also helps in the formation of protrusive ruffle structures which are actin-dependent and abundant on the surface of both unstimulated and LPS-activated macrophages. Recruitment of CK-B to these structures occurred transiently and inhibition of the enzyme's catalytic activity with cyclocreatine led to a general smoothening of surface morphology as visualized by scanning electron microscopy. Comparison of the dynamics of distribution of YFP-tagged CK-mutants and isoforms by live imaging revealed that amino acid residues in the C-terminal segment (aa positions 323-330) that forms one of the protein's two mobile loops are involved in partitioning over inner regions of the cytosol and nearby sites where membrane protrusions occur during induction of phagocytic cup formation. Although wt CK-B, muscle-type CK (CK-M), and a catalytically dead CK-B-E232Q mutant with intact loop region were normally recruited from the cytosolic pool, no dynamic transition to the phagocytic cup area was seen for the CK-homologue arginine kinase and a CK-B-D326A mutant protein. Bioinformatics analysis helped us to predict that conformational flexibility of the C-terminal loop, independent of conformational changes induced by substrate binding or catalytic activity, is likely involved in exposing the enzyme for binding at or near the sites of membrane protrusion formation.
authors.affiliation: Department of Cell Biology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, PO Box 9101, 6500 HB, Nijmegen, The Netherlands.
authors.forename: Gerda
authors.initials: G
authors.lastname: Venter
authors.affiliation: Department of Cell Biology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, PO Box 9101, 6500 HB, Nijmegen, The Netherlands.
authors.forename: Saskia
authors.initials: S
authors.lastname: Polling
authors.affiliation: Department of Cell Biology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, PO Box 9101, 6500 HB, Nijmegen, The Netherlands.
authors.forename: Helma
authors.initials: H
authors.lastname: Pluk
authors.affiliation: Centre for Molecular and Biomolecular Informatics, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, PO Box 9101, 6500 HB, Nijmegen, The Netherlands.
authors.forename: Hanka
authors.initials: H
authors.lastname: Venselaar
authors.affiliation: Department of Cell Biology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, PO Box 9101, 6500 HB, Nijmegen, The Netherlands.
authors.forename: Mietske
authors.initials: M
authors.lastname: Wijers
authors.affiliation: Department of Cell Biology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, PO Box 9101, 6500 HB, Nijmegen, The Netherlands.
authors.forename: Marieke
authors.initials: M
authors.lastname: Willemse
authors.affiliation: Department of Cell Biology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, PO Box 9101, 6500 HB, Nijmegen, The Netherlands.
authors.forename: Jack A M
authors.initials: JA
authors.lastname: Fransen
authors.affiliation: Department of Cell Biology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, PO Box 9101, 6500 HB, Nijmegen, The Netherlands. Electronic address: b.wieringa@ncmls.ru.nl.
authors.forename: Bé
authors.initials: B
authors.lastname: Wieringa
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D022081:Cell Surface Extensions / Q000187:drug effects / Q000378:metabolism*; D019295:Computational Biology; D052276:Creatine Kinase, BB Form / Q000378:metabolism*; D003404:Creatinine / Q000031:analogs & derivatives / Q000494:pharmacology; D004331:Drosophila melanogaster; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008264:Macrophages / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D017434:Protein Structure, Tertiary
chemical_list: D000199:Actins; D004791:Enzyme Inhibitors; C012260:cyclocreatine; D003404:Creatinine; D052276:Creatine Kinase, BB Form
keywords: Actin cytoskeleton.; Creatine kinase; Macrophages; Phagocytosis; Ruffle formation