The Physical Association of Casein Kinase 2 with Nucleolin*
- ‡ To whom correspondence should be addressed: Dept. of Pharmacology, University of Washington, Seattle, WA 98195. Tel.: 206-543-8500; Fax: 206-543-0858.
Abstract
CK2 (formerly called casein kinase 2) is a ubiquitous messenger-independent serine/threonine protein kinase implicated in cell growth and proliferation. To investigate the regulation and functions of this enzyme, experiments were carried out to search for CK2-interacting proteins. The methods employed included an overlay technique, co-purification, co-immunoprecipitation, and the use of glutathione S-transferase (GST) CK2 fusion proteins. By the CK2 overlay technique, one protein of 110 kDa was found to bind to CK2 with very high affinity. The binding was inhibited by CK2 effectors such as heparin, polyarginine, and histone H1, but was not affected by the CK2 substrate, casein. Protein p110 was also detected by co-immunoprecipitation using anti-CK2 antiserum, suggesting an in vivo association of this protein with CK2. Co-purification of p110 with CK2 from Sf-9 cells that overexpressed CK2 was also observed through sequential chromatographic steps. Using GST fusion proteins of CK2, the CK2-p110 interaction was investigated further and was found to occur primarily through CK2 α or α′ subunits, but not the β subunit. Protein p110 was purified from 3T3 L1 mouse fibroblast cell lines using a GST-CK2 affinity resin. Amino acid sequence analysis of peptides obtained from the protein indicated that it is the nuclear protein, nucleolin. Furthermore, p110 was recognized by anti-nucleolin antiserum. At present, the physiological significance of the strong interaction between CK2 and nucleolin, an excellent substrate for the enzyme, is not clear. However, this association may be important for regulating rDNA transcription.
Footnotes
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↵* This work was supported by Grant DK42528 from National Institutes of Health and by a grant from International Human Frontier Science Program Organization. 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.
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↵1 The abbreviations used are:
- CK2
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casein kinase 2
- PAGE
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polyacrylamide gel electrophoresis
- DTT
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dithiothreitol
- HPLC
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high performance liquid chromatography
- PCR
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polymerase chain reaction
- PVDF
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polyvinylidene difluoride
- PMSF
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phenylmethylsulfonyl fluoride
- BSA
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bovine serum albumin
- GST
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glutathione S-transferase.
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↵2 G. Dobrowolska, F. J. Lozeman, D. Li, and E. G. Krebs, manuscript in preparation.
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- Received February 7, 1996.
- Revision received April 9, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
