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

Abstract

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...

Posttranslational Protein Modification inArchaea

“…In Haloferax volcanii, growth at elevated salt concentrations may lead to the appearance of several serine-phosphorylated proteins not detected during growth under optimal salt conditions (32). A threonine-phoshorylated 67-kDa membrane protein displaying serine kinase activity has been found in Sulfolobus solfataricus, although the pathway in which this protein participates remains to be defined (261,264).…”

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

“…tokodaii, are the best-studied Archaea regarding protein phosphorylation. Four protein phosphatases (427)(428)(429) and five protein kinases (430)(431)(432)(433)(434) were identified and biochemically investigated. Furthermore, the first signal transduction pathway involved in archaella expression mediated by reversible protein phosphorylation was discovered in Sul.…”

The Membrane-Associated Protein-Serine/Threonine Kinase fromSulfolobus solfataricusIs a Glycoprotein

“…32 P-labeled phosphoproteins were visualized by electronic autoradiography with a Packard (Meriden, Conn.) Instantimager. Protein kinase activity was assayed at 65°C with [␥-32 P]ATP, using myosin light-chain (MLC) peptide as the phosphoacceptor substrate, as described previously (15).…”

“…While the catalytic polypeptide appeared to behave as a single species during SDS-PAGE, on two-dimensional gels it migrated in a noticeably diffuse manner, even under conditions where it presumably was in a dephosphorylated state (15). Electrophoretic behavior of this type oftentimes is indicative of covalent modification by glycosylation (22), as complex carbohydrate moieties constitute a frequent source of structural microheterogeneity.…”

“…Recently, protein-serine/threonine kinase activity was detected in the membrane fraction of the extreme acidothermophilic archaeon Sulfolobus solfataricus (15). Solubilization of this protein kinase activity required detergents such as Triton X-100 or octyl glucoside, indicating that it was associated with an integral membrane protein.…”