Binding of Polyamines to an Autonomous Domain of the Regulatory Subunit of Protein Kinase CK2 Induces a Conformational Change in the Holoenzyme

doi:10.1074/jbc.272.33.20820

Binding of Polyamines to an Autonomous Domain of the Regulatory Subunit of Protein Kinase CK2 Induces a Conformational Change in the Holoenzyme

A PROPOSED ROLE FOR THE KINASE STIMULATION*

From the Laboratoire de Biochimie des Régulations Cellulaires Endocrines, Département de Biologie Moléculaire et Structurale, INSERM Unité 244, CEA Grenoble, 17 rue des Martyrs, F-38054 Grenoble Cedex 9, France

Abstract

The means by which the cell regulates protein kinase CK2 remain obscure. However, natural polyamines, cellular compounds required for cell proliferation, have been reported to strongly stimulate CK2-mediated phosphorylation of a number of substrates. Using spermine analogs, we have shown that polyamines directly interact with the CK2 β subunit, and the chemical features of the highly acidic binding site (Asp⁵¹-Tyr⁸⁰) have been determined. In the present study, we show that the isolated β subunit region extending from residue Asp⁵¹ to Pro¹¹⁰ exhibits a specific and efficient polyamine binding activity similar to that of the entire β subunit. Moreover, the replacement of Glu⁶⁰, Glu⁶¹, and Glu⁶³ of the β subunit by 3 alanine residues leads to a loss of the spermine-induced stimulation of CK2 activity which correlates with a decrease in spermine binding affinity. Thermal stability studies indicate that the binding of spermine induces a 4 °C decrease of the T _m value for the holoenzyme. This was confirmed by circular dichroism analyses, which show that the 6 °C negative shift of the CK2T _m value provoked by spermine binding, reflects a conformational change in the kinase. Together, these observations strongly suggest that this newly defined polyamine-binding domain is involved in the intrasteric regulation of CK2 activity.

Footnotes

↵* This work was supported by INSERM (Unité 244), CEA (DVS/DBMS/BRCE), Association pour la Recherche contre le Cancer, Fondation pour la Recherche Médicale, Ligue Nationale Frana̧ise contre le Cancer, and the Commission of the European Communities (BIOMED 2).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed. Tel.: 33-4-76-88-42-04; Fax: 33-4-76-88-50-58.
↵1 The abbreviations used are: CK2, protein kinase CK2; MBP, maltose-binding protein; MBPβ51–110, fusion protein of MBP and the CK2 β subunit domain Asp⁵¹-Pro¹¹⁰; MBPβ1–215, fusion protein of MBP and the entire CK2 β subunit; MBPβ3, fusion protein of MBP and the entire CK2 βAla60,Ala61,Ala63 subunit; βHA, fusion of the hemagglutinin tag at the C terminus of the CK2 regulatory subunit; β3HA, fusion of the hemagglutinin tag at the C terminus of the βAla60,Ala61,Ala63 subunit; CD, circular dichroism; PCR, polymerase chain reaction; BSA, bovine serum albumin.
↵2 D. Leroy. J.-K. Heriche, O. Filhol, E. M. Chambaz, and C. Cochet, submitted for publication.
↵3 D. Leroy and C. Cochet, unpublished results.
- Received October 22, 1996.
- Revision received June 11, 1997.