Protein kinase casein kinase 2 holoenzyme produced ectopically in human cells can be exported to the external side of the cellular membrane

Abstract

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␣ or CK2␣ ) and regulatory (CK2␤) subunits with hemaglutinin tags allowed us to study the export of ectopically synthesized enzyme. When the catalytic (CK2␣ or CK2␣ ) and the CK2␤ 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␣ that eliminate phosphorylating activity [CK2␣(Asp-156-Ala)] or that make it less sensitive to heparin inhibition [CK2␣(Lys-75-Glu,Lys-76-Glu)] demonstrated that these mutations do not prevent the holoenzyme to be exported from the cells.ectokinase ͉ protein phosphorylation T here are a number of reports that protein kinases are present on the external side of the cellular membrane and that these ectokinases are responsible for phosphorylating proteins of the extracellular matrix and extracellular domains of proteins that are attached to cells (1). Specifically, there are several reports that an ectokinase has the characteristics of protein kinase casein kinase 2 (CK2) (2-4). A CK2-like ectokinase activity has been reported to be responsible for the phosphorylation of vitronectin, an extracellular matrix protein (5, 6), and of the external domain of the ␤-amyloid precursor protein (7) and T lymphocyte surface proteins (8). However, there is no information about the structural features that are responsible for the export of CK2 or other ectokinases.Protein kinase CK2 is ubiquitous in eukaryotes and is responsible for the phosphorylation of hundreds of proteins (9-11). There is strong evidence that CK2 is involved in the control of cell proliferation, apoptosis, and circadian rhythms (12)(13)(14). In addition, CK2 may play a role in controlling the activity of a number of protein kinases through a positive feedback loop with the Cdc37 protein that acts as a chaperone that activates these kinases (15,16).The CK2 holoenzyme is a heterotetramer composed of catalytic subunits (␣ and ␣Ј) and regulatory ␤ subunits conforming ␣ 2 ␤ 2 , ␣␣Ј␤ 2 and ␣Ј 2 ␤ 2 combination...

Protein kinase CK2 as an ectokinase: The role of the regulatory CK2β subunit

“…Fig. 2D shows that transfection with (Myc) 6 -CK2␤ ⌬1-19 yields as much ectokinase activity as observed with the wild-type subunit. The possibility that the (Myc) 6 tag was affecting the results was discarded by experiments in which the (Myc) 6 tag was replaced for an HA tag attached to CK2␤ constructs with deletion mutants that yielded ectokinase activity and no ectokinase activity.…”

“…In Fig. 2E, lanes 2, 4, 6, 8, and 10 it is possible to see that the antibodies directed against the HA tag present in HA-CK2␣ were able to coimmunoprecipitate the (Myc) 6 -tagged CK2␤ constructs as revealed by the anti-Myc antibodies. These results mean that these mutant CK2␤ proteins coimmunoprecipitate with the catalytic subunit and therefore can form strong associations with CK2␣ and probably attain a holoenzyme structure (9).…”

“…2B shows ectokinase activity of the same cells whose lysates are analyzed in Fig. 2 A, and it can be seen that the (Myc) 6 -CK2␤ ⌬1-8 construct that lacks the first eight amino acids of the regulatory subunit was able to generate as much CK2 ectokinase activity as the wild-type subunit. Cells transfected with mutant (Myc) 6 -CK2␤ ⌬1-32 , on the other hand, did not have significant ectokinase activity, just as cells that were transfected only with Ha-CK2␣ WT .…”

“…The possibility that the (Myc) 6 tag was affecting the results was discarded by experiments in which the (Myc) 6 tag was replaced for an HA tag attached to CK2␤ constructs with deletion mutants that yielded ectokinase activity and no ectokinase activity. The results observed with the HA tags were similar to those found with the (Myc) 6 tags (data not shown).…”

“…Previous work in the laboratories of Pinna and Issinger (10,11) with CK2␤ fragments also demonstrated that amino-truncated CK2␤ could interact with and stimulate CK2␣. In separate experiments, cells were transfected with (Myc) 6 -CK2␤ ⌬1-49 , (Myc) 6 -CK2␤ ⌬1-79 , and (Myc) 6 -CK2␤ ⌬1-104 together with HA-CK2␣. These three other deletion mutants also failed to generate detectable CK2 ectokinase activity (data not shown).…”