期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2005
卷号：102
期号：13
页码：4718-4723
DOI：10.1073/pnas.0501074102
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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 (CK2{alpha} or CK2{alpha}') and regulatory (CK2{beta}) subunits with hemaglutinin tags allowed us to study the export of ectopically synthesized enzyme. When the catalytic (CK2{alpha} or CK2{alpha}') and the CK2{beta} 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 CK2{alpha} that eliminate phosphorylating activity [CK2{alpha}(Asp-156-Ala)] or that make it less sensitive to heparin inhibition [CK2{alpha}(Lys-75-Glu,Lys-76-Glu)] demonstrated that these mutations do not prevent the holoenzyme to be exported from the cells.
