Type of Document	Dissertation
Author	Thurston, Barbara
Author's Email Address	none
URN	etd-58821132974710
Title	Protein Phosphorylation in Archaea
Degree	PhD
Department	Biochemistry & Nutrition
Advisory Committee	Advisor Name Title Peter J. Kennelly chair Robert H. White chair Bruce M. Anderson none David R. Bevan none Dennis R. Dean none James M. Tanko none
Keywords	succinyl-CoA synthetase Sulfolobus solfataricus protein-serine/threonine phosphatase hexose phosphate mutase Methanosarcina thermophila
Date of Defense	1997-10-03
Availability	unrestricted
Abstract Protein phosphorylation constitutes an important mechanism for cellular regulation in both Eucarya and Bacteria. All living organisms evolved from a common progenitor; this implies that protein phosphorylation as a means of regulation also exists in Archaea. Previously, in the sulfur-dependent archaeon Sulfolobus solfataricus a gene was cloned encoding a protein-serine/threonine phosphatase that was similar to eucaryal protein-serine/threonine phosphatases type 1, 2A, and 2B. To identify protein phosphatases in other archaeons, oligonucleotides encoding conserved regions of eucaryal protein-serine/threonine phosphatases were used in the polymerase chain reaction to amplify genomic DNA from the methanogenic archaeon Methanosarcina thermophila. From the PCR reaction a fragment of DNA was isolated that encoded a portion of a protein phosphatase. Using this DNA fragment as a probe, the entire phosphatase gene was isolated. The amino acid sequence of the phosphatase encoded by this gene displayed greater than 30% identity with eucaryal protein-serine/threonine phosphatase type 1. The gene encoding the Methanosarcina phosphatase was expressed in Escherichia coli. The expressed protein exhibited protein serine phosphatase activity that was sensitive to inhibitors of eucaryal phosphatases such as okadaic acid, microcystin, calyculin, and tautomycin. In order to identify potential endogenous substrates of archaeal protein-serine/threonine phosphatases and kinases, a study was initiated to characterize the most prominent phosphoproteins in S. solfataricus. Cell extracts were incubated with [g-32P] ATP, MgCl2, and MnCl2, and the proteins in the extracts were separated by SDS-PAGE. Autoradiography of the gels revealed four prominent phosphoproteins with apparent molecular masses of 35, 46, and 50 kDa. N-terminal sequence analysis and enzymatic assays of the 35 kDa phosphoprotein identified this phosphoprotein as the a-subunit of succinyl-CoA synthetase. N-terminal sequence analysis and enzymatic assays revealed that the 50 kDa phosphoprotein was a hexosephosphate mutase. Neither the 50 kDa nor the 35 kDa phosphoprotein appeared to be the target of protein kinases or phosphatases. Therefore, while protein-serine phosphatases exist in Archaea, the targets of these phosphatases have yet to be determined.
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