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Journal of Bacteriology, June 2003, p. 3436-3445, Vol. 185, No. 11
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.11.3436-3445.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Open Reading Frame sso2387 from the Archaeon Sulfolobus solfataricus Encodes a Polypeptide with Protein-Serine Kinase Activity

Brian H. Lower and Peter J. Kennelly*

Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061

Received 30 September 2002/ Accepted 21 March 2003

The predicted polypeptide product of open reading frame sso2387 from the archaeon Sulfolobus solfataricus, SsoPK2, displayed several of the sequence features conserved among the members of the "eukaryotic" protein kinase superfamily. sso2387 was cloned, and its polypeptide product was expressed in Escherichia coli. The recombinant protein, rSsoPK2, was recovered in insoluble aggregates that could be dispersed by using high concentrations (5 M) of urea. The solubilized polypeptide displayed the ability to phosphorylate itself as well as several exogenous proteins, including mixed histones, casein, bovine serum albumin, and reduced carboxyamidomethylated and maleylated lysozyme, on serine residues. The source of this activity resided in that portion of the protein displaying homology to the catalytic domain of eukaryotic protein kinases. By use of mass spectrometry, the sites of autophosphorylation were found to be located in two areas, one immediately N terminal to the region corresponding to subdomain I of eukaryotic protein kinases, and the second N terminal to the presumed activation loop located between subdomains VII and VIII. Autophosphorylation of rSsoPK2 could be uncoupled from the phosphorylation of exogenous proteins by manipulation of the temperature or mutagenic alteration of the enzyme. Autophosphorylation was detected only at temperatures >=60°C, whereas phosphorylation of exogenous proteins was detectable at 37°C. Similarly, replacement of one of the potential sites of autophosphorylation, Ser548, with alanine blocked autophosphorylation but not phosphorylation of an exogenous protein, casein.

* Corresponding author. Mailing address: Department of Biochemistry, 111 Engel Hall (0308), Virginia Polytechnic Institute and State University, Blacksburg, VA 24061. Phone: (540) 231-4317. Fax: (540) 231-9070. E-mail: pjkennel{at}vt.edu.

Journal of Bacteriology, June 2003, p. 3436-3445, Vol. 185, No. 11
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.11.3436-3445.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Eichler, J., Adams, M. W. W. (2005). Posttranslational Protein Modification in Archaea. Microbiol. Mol. Biol. Rev. 69: 393-425 [Abstract] [Full Text]  
  • Ray, W. K., Keith, S. M., DeSantis, A. M., Hunt, J. P., Larson, T. J., Helm, R. F., Kennelly, P. J. (2005). A Phosphohexomutase from the Archaeon Sulfolobus solfataricus Is Covalently Modified by Phosphorylation on Serine. J. Bacteriol. 187: 4270-4275 [Abstract] [Full Text]  
  • Albers, S.-V., Driessen, A. J. M. (2005). Analysis of ATPases of putative secretion operons in the thermoacidophilic archaeon Sulfolobus solfataricus. Microbiology 151: 763-773 [Abstract] [Full Text]  
  • Lower, B. H., Potters, M. B., Kennelly, P. J. (2004). A Phosphoprotein from the Archaeon Sulfolobus solfataricus with Protein-Serine/Threonine Kinase Activity. J. Bacteriol. 186: 463-472 [Abstract] [Full Text]  


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