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Biochem. J. (2002) 362 (375–382) (Printed in Great Britain)

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Phosphorylation of Xenopus transcription factor IIIA by an oocyte protein kinase CK2¹

Cara J. WESTMARK, Romi GHOSE and Paul W. HUBER²

Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, Indiana 46556, U.S.A.

Transcription factor IIIA (TFIIIA), isolated from the cytoplasmic 7S ribonucleoprotein complex of Xenopus oocytes, is phosphorylated when incubated with [g-³²P]ATP. This modification is due to a trace kinase activity that remains associated with the factor through several steps of purification. The kinase can use either ATP or GTP, and will phosphorylate casein and phosvitin to the exclusion of TFIIIA. The kinase is reactive with a ten-amino-acid peptide that is a specific substrate for protein kinase CK2 (CK2; formerly casein kinase II). In addition, inhibition of phosphorylation by heparin and stimulation by spermidine indicate that the activity can be ascribed to CK2. Phospho amino acid analysis established that serine is the sole phosphoryl acceptor in TFIIIA. There are four consensus sites for CK2 in TFIIIA; all contain serine residues at the putative site of phosphorylation. TFIIIA immunoprecipitated from oocytes, which were incubated with [³²P]orthophosphate, is also phosphorylated exclusively on serine residues. Only the cyanogen bromide fragment, which was derived from the N-terminal end of TFIIIA, is labelled in vivo. A recognition sequence for CK2, located at Ser¹⁶ in the b-turn of the first zinc-finger domain, is the only protein kinase consensus sequence present in this peptide. Assays in vitro with site-specific mutants of TFIIIA established that Ser¹⁶ is the preferred site of phosphorylation, with some secondary modification at Ser³¹⁴.

Key words: 5S rRNA, RNA polymerase III, zinc-finger protein.

¹This paper is dedicated to the memory of Dr. Veronica C. Blasquez

Abbreviations used: CK2, protein kinase CK2 (formerly casein kinase II); CNBr, cyanogen bromide; DTT, dithiothreitol; GV, germinal vesicle; pol III, RNA polymerase III; RNP, ribonucleoprotein; TFIIIA, transcription factor IIIA; S314E, Ser³¹⁴ Glu substitution.

²To whom correspondence should be addressed (e-mail Huber.1@nd.edu).

Received 8 August 2001/31 October 2001; accepted 6 November 2001

The Biochemical Society, London © 2002