Protein kinase casein kinase 2 holoenzyme produced ectopically in human cells can be exported to the external side of the cellular membrane.
Ectokinases can phosphorylate extracellular proteins and external domains of membrane proteins influencing cell adhesion, movement, and cellular interactions. An ectokinase with the properties of casein kinase 2 (CK2) has been previously described, but little is known about the structural characteristics that allow this enzyme to be exported from the cell. Transfection of human embryonic kidney-293 cells with cDNAs coding for the catalytic (CK2alpha or CK2alpha') and regulatory (CK2beta) subunits with hemaglutinin tags allowed us to study the export of ectopically synthesized enzyme. When the catalytic (CK2alpha or CK2alpha') and the CK2beta regulatory subunits are cotransfected, the tetrameric enzyme composed of both subunits (holoenzyme) is detected outside the cell. This observation has been confirmed by assaying protein kinase activity in immunoprecipitates obtained with antihemaglutinin antibody by using a CK2-specific peptide substrate and by Western blots as well as by immunofluorescence of nonpermeabilized cells. Transfection with cDNA of catalytic or regulatory subunit alone does not result in export of these subunits. A study of the kinetics of appearance of the ectopically synthesized protein at different times after transfection indicates that a 5- to 7-h delay after the synthesis of the protein before it appears in the extracellular compartment. Using mutations of CK2alpha that eliminate phosphorylating activity [CK2alpha(Asp-156-Ala)] or that make it less sensitive to heparin inhibition [CK2alpha(Lys-75-Glu,Lys-76-Glu)] demonstrated that these mutations do not prevent the holoenzyme to be exported from the cells.
Rodríguez
Fernando
F
Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Independencia 1027, 8380453 Santiago, Chile.
Allende
Catherine C
CC
Allende
Jorge E
JE
D015153:Blotting, Western; D047390:Casein Kinase II / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000502:physiology*; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D047468:Immunoprecipitation; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D021381:Protein Transport / Q000502:physiology; D014162:Transfection
D018076:DNA, Complementary; D016326:Extracellular Matrix Proteins; D047390:Casein Kinase II