Archaeal phosphoproteins. Identification of a hexosephosphate mutase and the α‐subunit of succinyl‐CoA synthetase in the extreme acidothermophile <i>Sulfolobus solfataricus</i>

Solow, Barbara T.; Bischoff, Kenneth M.; Zylka, Mark J.; Kennelly, Peter J.

doi:10.1002/pro.5560070111

Cited by 20 publications

(13 citation statements)

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“…Phosphorylated proteins have been detected in several halophilic, methanogenic, and thermophilic archaeons (11,26,48,62,63,65,66,68,69), and in several cases the observed patterns of protein phosphorylation exhibited the type of environmentally sensitive changes suggestive of regulatory control (48,62,68,69). A CheA-like two-component cascade responsible for modulating chemo-and phototaxis has been characterized for the halophilic archaeon Halobacterium halobium (52,53), while protein-serine/threonine phosphatases belonging to the PPP family of enzymes first discovered in the Eucarya have been described for the archaeons Sulfolobus solfataricus (32,37), Methanosarcina thermophila TM-1 (49,67), and Pyrodictium abyssi TAG11 (41).…”

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

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Kennelly

2003

J Bacteriol

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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, Ser 548 , with alanine blocked autophosphorylation but not phosphorylation of an exogenous protein, casein.The reversible alteration of the functional properties of strategically selected proteins by phosphorylation-dephosphorylation represents one of nature's most widely employed means for controlling cellular processes (reviewed in reference 30). The scientific literature provides numerous examples of the prominent role played by this versatile regulatory mechanism in members of the Eucarya (reviewed in references 17, 18, and 24) and the Bacteria (reviewed in references 2, 7, and 22). By contrast, we know very little concerning the chemical nature, enzymatic catalysts, or physiological roles of the protein phosphorylation-dephosphorylation events that take place in members of the so-called third domain of life, the Archaea. Such knowledge is important not only for understanding how these biologically diverse organisms adapt to the extreme environments in which they typically reside but also for tracing the origins and evolution of a fundamentally important regulatory mechanism.The available evidence suggests that protein phosphorylation is a fairly general phenomenon among the Archaea. Phosphorylated proteins have been detected in several halophilic, methanogenic, and thermophilic archaeons (11,26,48,62,63,65,66,68,69), and in several cases the observed patterns of protein phosphorylation exhibited the type of environmentally sensitive changes suggestive of regulatory control (48,62,68,69). A CheA-like two-compo...

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

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Kennelly

2003

J Bacteriol

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“…It is clear that archaeal proteins also become phosphorylated on serine, threonine, and tyrosine residues (34,35,37,38,40,41). However, little hard data concerning the enzymes responsible exist (34,35).…”

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The Archaeon Sulfolobus solfataricus Contains a Membrane-Associated Protein Kinase Activity That Preferentially Phosphorylates Threonine Residues In Vitro

2000

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The extreme acidothermophilic archaeon Sulfolobus solfataricus harbors a membrane-associated protein kinase activity. Its solubilization and stabilization required detergents, suggesting that this activity resides within an integral membrane protein. The archaeal protein kinase utilized purine nucleotides as phosphoryl donors in vitro. A noticeable preference for nucleotide triphosphates over nucleotide diphosphates and for adenyl nucleotides over the corresponding guanyl ones was observed. The molecular mass of the solubilized, partially purified enzyme was estimated to be Ϸ125 kDa by gel filtration chromatography. Catalytic activity resided in a polypeptide with an apparent molecular mass of Ϸ67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Challenges with several exogenous substrates revealed the protein kinase to be relatively selective. Only casein, histone H4, reduced carboxyamidomethylated and maleylated lysozyme, and a peptide modeled after myosin light chains (KKRAARATSNVFA) were phosphorylated to appreciable levels in vitro. All of the aforementioned substrates were phosphorylated on threonine residues, while histone H4 was phosphorylated on serine as well. Substitution of serine for the phosphoacceptor threonine in the myosin light chain peptide produced a noticeably inferior substrate. The protein kinase underwent autophosphorylation on threonine and was relatively insensitive to a set of known inhibitors of "eukaryotic" protein kinases.For over 3 decades following the discovery of protein phosphorylation-dephosphorylation events, a clear dichotomy between those that occur in eukaryotes and those that occur in prokaryotes was perceived. Initially, it was believed that only eukaryotes had evolved the ability to transduce signals and regulate protein function via this covalent modification process. In the late 1970s the universality of this protein phosphorylation-dephosphorylation became apparent. However, the disparity in sequence between the characteristic protein kinases from eukaryotes, i.e., the homologs of the cyclic AMP (cAMP)-dependent protein kinase, and those from prokaryotes, i.e., the histidine kinases and the isocitrate dehydrogenase kinase/phosphatase, suggested that the members of each phylogenetic domain evolved unique molecular archetypes to carry out this process.The past several years have witnessed the demolition of this dichotomy. The "bacterial" two-component regulatory system, for example, has been discovered in several eukaryotes, while "eukaryotic" protein kinases in bacterial organisms have been identified and characterized and a number of eukaryotic protein phosphatases from members of both the Archaea and the Bacteria have been identified and characterized (reviewed in reference 19). Adding to the foment has been the recent discovery of several distinct new protein kinases, e.g., the HPr protein kinase from Bacillus subtilis (10, 28), the Rsb/Spo family protein kinases of bacteria (8,25,32,43), and the myosin heavy chain kinase/eIF-2 protein kinase famil...

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“…Among the more visually prominent of these was a polypeptide with an apparent mass of ϳ46-kDa that contained [ 32 P]phosphoserine (41). The large number of polypeptides present in the extract rendered the unambiguous identification of the Coomassie stained band corresponding to the ϳ46-kDa phosphoprotein problematic.…”

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“…In a previous study, we observed that when extracts of the hyperthermophilic archaeon Sulfolobus solfataricus were incubated with [␥- 32 P]ATP, numerous polypeptides incorporated radiolabeled phosphate (41). One of the more visually prominent of these was a phosphoserine-containing polypeptide with a mass of ϳ46 kDa.…”

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Phosphoprotein with Phosphoglycerate Mutase Activity from the Archaeon Sulfolobus solfataricus

et al. 2003

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When soluble extracts of the extreme acidothermophilic archaeon Sulfolobus solfataricus were incubated with [␥-32 P]ATP, several proteins were radiolabeled. One of the more prominent of these, which migrated with a mass of ϳ46 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was purified by column chromatography and SDS-PAGE and subjected to amino acid sequence analysis via both the Edman technique and mass spectroscopy. The best match to the partial sequence obtained was the potential polypeptide product of open reading frame sso0417, whose DNA-derived amino acid sequence displayed many features reminiscent of the 2,3-diphosphoglycerate-independent phosphoglycerate (PGA) mutases [iPGMs]. Open reading frame sso0417 was therefore cloned, and its protein product was expressed in Escherichia coli. Assays of its catalytic capabilities revealed that the protein was a moderately effective PGA mutase that also exhibited low levels of phosphohydrolase activity. PGA mutase activity was dependent upon the presence of divalent metal ions such as Co 2؉ Interest in the members of the Archaea has grown steadily over the last two decades, stimulated to a large degree by their extremophilic lifestyles and distinct phylogenetic status. Within archaeal proteomes resides a wealth of information concerning unique metabolic pathways for extracting energy from sulfur, producing methane from CO 2 , etc., as well as biochemical and biophysical mechanisms for sustaining life processes under conditions of temperature, pH, and salinity hostile to "conventional" organisms (reviewed in references 1, 12, 23, and 37). Archaeal genomes combine unique, bacterial, and eucaryal features in a complex puzzle whose deconvolution will provide important insights into the nature and order of the evolutionary process (reviewed in references 13, 14, and 17). However, our current library of information concerning fundamental metabolic, sensory, regulatory, and other processes in the Archaea remains lacking in both breadth and depth.In a previous study, we observed that when extracts of the hyperthermophilic archaeon Sulfolobus solfataricus were incubated with [␥-32 P]ATP, numerous polypeptides incorporated radiolabeled phosphate (41). One of the more visually prominent of these was a phosphoserine-containing polypeptide with a mass of ϳ46 kDa. Using the recently released genome sequence of S. solfataricus P2 (38), we have been able to link this phosphoprotein to the open reading frame (ORF) that encodes it. We describe here the identification of the ϳ46-kDa phosphoprotein as an enzyme with phosphoglycerate (PGA) mutase activity that is encoded by ORF sso0417. MATERIALS AND METHODS Materials. [␥-32 P]ATP, [␣-32 P]ATP, and [␥-32 P]GTP were purchased from NEN Research Products (Boston, Mass.). Matrix Gel Blue A and Matrix Gel Blue B were purchased from Amicon Corp. (Danvers, Mass.). Hydroxyapatite was from Bio-Rad (Richmond, Calif.). Enolase was obtained from Sigma (St. Louis, Mo.). Oligonucleotide primers were from Life Technol...

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Archaeal phosphoproteins. Identification of a hexosephosphate mutase and the α‐subunit of succinyl‐CoA synthetase in the extreme acidothermophile Sulfolobus solfataricus

Cited by 20 publications

References 35 publications

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

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

The Archaeon Sulfolobus solfataricus Contains a Membrane-Associated Protein Kinase Activity That Preferentially Phosphorylates Threonine Residues In Vitro

Phosphoprotein with Phosphoglycerate Mutase Activity from the Archaeon Sulfolobus solfataricus

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