Isolation and cloning of a protein-serine/threonine phosphatase from an archaeon

Abstract

A divalent metal ion-stimulated protein-serine/threonine phosphatase, PP1-arch, was purified approximately 1,000-fold from the extreme acidothermophilic archaeon Sulfolobus solfataricus (ATCC 35091). Purified preparations contained 40 to 70% of total protein as PP1-arch, as determined by assay-ing sodium dodecyl sulfatepolyacrylamide gels for protein phosphatase activity. The first 25 amino acids of the protein's sequence were identified, as well as an internal sequence spanning some 20 amino acids. Using this information, we cloned the gene for PP1-arch via the application of PCR and conventional cloning techniques. The gene for PP1-arch predicted a protein of 293 amino acids that bore striking resemblance to the members of the major family of protein-serine/threonine phosphatases from members of the domain Eucarya, the PP1/2A/2B superfamily. The core of the protein, spanning residues 4 to 275, possessed 29 to 31% identity with these eucaryal protein phosphatases. Of the 42 residues found to be absolutely conserved among the known eucaryal members of the PP1/2A/2B superfamily, 33 were present in PP1-arch. If highly conservative substitutions are included, this total reached 37. The great degree of sequence conservation between molecules from two distinct phylogenetic domains implies that the members of this enzyme superfamily had evolved as specialized, dedicated protein phosphatases prior to the divergence of members of the Archaea and Eucarya from one another.Protein phosphorylation-dephosphorylation represents a ubiquitous and prominent mechanism for regulating cellular processes. Protein kinases, protein phosphatases, and the phosphoproteins that they modify have been found in virtually every living organism. A priori, such universality might suggest that protein phosphorylation-dephosphorylation constitutes a very ancient means for exerting regulatory control. However, for many years, comparisons between the components of bacterial and eucaryal protein phosphorylation networks showed them to be structurally distinct, suggesting that each arose independently, at different times and places (reviewed in reference 18). For example, while all eucaryal protein kinases could be grouped into a single giant superfamily (14), the protein kinases from bacterial organisms showed no significant resemblance to this group (9). While members of the domain Eucarya phosphorylated some proteins on tyrosine residues, members of the domain Bacteria apparently did not (13). Therefore, it was and still is widely believed that protein phosphorylationdephosphorylation constitutes a relatively recent addition to the cell's regulatory repertoire, one that arose separately in each phylogenetic domain after it had diverged from its predecessors.Recent evidence has eroded the foundation upon which this consensus is based. Tyrosine-phosphorylated proteins have been detected in several members of the Bacteria (1,10,33,34,43). Genes encoding bacterial protein-histidine kinases have been discovered in eucaryal species ranging from rats (31...

Posttranslational Protein Modification inArchaea

“…The genes encoding PP1-arch1, PP1-arch2, and Py-PP1 were cloned from Sulfolobus solfataricus (216,252), Methanosarcina thermophila TM-1 (321,403), and Pyrodictium abyssi TAG11 (268), respectively. In addition, other archaeal PPP family phosphorylases have been predicted following analysis of genome sequences, relying on the presence of conserved sequence motifs (24,215).…”

Cloning of a potential cytochrome P450 from the Archaeon Sulfolobus solfataricus

“…'Touchdown' PCR was performed by the procedure of Roux [7] as modified by Leng et al [4]. The synthesis of the 920 bp primer was performed according to the standard PCR protocol of Leng et al [4], with the exception that the quantity of DNA used was reduced from 1000 to 500 ng and the quantity of each primer was increased from 100 to 500 pmol.…”

Eukaryote-Like Serine/Threonine Kinases and Phosphatases in Bacteria

“…For example, PrpE, a B. subtilis PPP family member, exclusively removes phosphate groups from phosphotyrosines in vitro (70). Other PPP family members have been annotated for bacteria and archaea, and in vitro, most are dual-specificity phosphatases that dephosphorylate tyrosine residues in addition to serine and threonine residues (85,88,108,164,170).…”