Expression and biochemical properties of a protein serine/threonine phosphatase encoded by bacteriophage lambda.

Abstract

The predicted amino acid sequence encoded by the open reading frame 221 (orf221) of bacteriophage A exhibited a high degree of smibaity to the catalytic subunits of a variety of protein serine/threonine phosphatases belonging to PP1, PP2A, and PP2B groups. Cloning and expression of the orf221 gene in Escherichia coli provided direct evidence that the gene codes for a protein serine/threonine phosphatase. The single-subunit recombinant enzyme was purified in soluble form and shown to possess a unique repertoire of biochemical properties-e.g., an absolute requirement for Mn2+, resistance to okadaic acid, inhibitors 1 and 2, and ability to dephosphorylate casein, adenovirus ElA proteins, and the a subunit of phosphorylase kinase. No phosphotyrosine phosphatase activity was observed. Mutational and biochemical analyses identified the conserved residues 73-77 and Cys138 to be important for activity. The name PP-A is proposed for this unusual prokaryotic enzyme.Reversible phosphorylation and dephosphorylation of proteins have been recognized as the primary means of posttranslational regulation of protein function (1). However, while studies of protein kinases experienced a nearly exponential growth, those of protein phosphatases remained generally neglected. The complex structure and enzymology of the phosphatases and the paucity of functionally active recombinant enzymes seem to be the major reasons for the relative lack of progress. A potential breakthrough in phosphatase research has recently been achieved through the suggestion that the genomes ofbacteriophages A and 080 may encode a protein serine/threonine phosphatase (2, 3). The 663-bp open reading frame (orf) of phage A could potentially code for a 221-amino acid (orf221) polypeptide with significant sequence similarity to the catalytic subunits of a variety of eukaryotic protein serine/threonine phosphatases (3).Subsequently, a phosphatase with activity toward phosphorylated casein was detected in Escherichia coli infected with AgtlO (2); the activity was absent in uninfected E. coli cells or in those infected with Agtll mutant containing the large ninS deletion that removed ==6% (2805 bp) of the A genome including the N-terminal portion of orf221 (4). However, these in vivo studies could not be definitive as to whether the activity was an intrinsic property of orf221 or represented a bacterial phosphatase induced or activated by the expression of orf221 or any other orf in the Anin region.In this communication, I report the cloning and expression of the A orf221 gene in E. coli. The purified recombinant protein was shown to be a potent serine/threonine phosphatase and exhibited a unique combination of biochemical properties. These studies confirm preliminary results of Cohen and coworkers (2, 3) and establish the orf221 protein as the only documented serine/threonine phosphatase encoded by a bacteriophage. The (-D-thiogalactopyranoside (IPTG), and lysis of the cells were carried out as described (5). Unless otherwise stated, all chromatographic an...

Genome of Bacteriophage P1

“…An ORF221 mutation, known as byp, which abolishes the phosphatase activity of lambdaPP, caused a defect in the establishment of lysogeny, unlike a nin deletion (62,65). The defect was proposed to depend either on the inability of altered lambdaPP to dephosphorylate a host protein that normally participates in termination of transcription of early genes or on constitutive transcription, from a promoter created by the mutation, of the Q gene, encoding the antiterminator of late gene transcription (24,53). Whether the product of P1 ppp serves P1 by dephosphorylating P1-encoded proteins involved in virion morphogenesis or a host protein or both remains to be determined.…”

Regulation of protein phosphatase type 1 and cell cycle progression by PfLRR1, a novel leucine‐rich repeat protein of the human malaria parasite Plasmodium falciparum

“…The activity of PfPP1 with p-nitrophenylphosphate (pNPP) as substrate was assayed essentially as previously described (Barik, 1993). Initial experiments were performed to determine the optimal conditions for PfPP1 activity.…”

Interactions Between a Minimal Protein Serine/Threonine Phosphatase and Its Phosphopeptide Substrate Sequence

“…The unique biochemical properties of PP included resistance to okadaic acid and heat-stable inhibitors 1 and 2, sensitivity to orthovanadate, an absolute requirement of Mn 2ϩ or Ni 2ϩ that could not be substituted by Ca 2ϩ or Mg 2ϩ , and an apparent absence of a regulatory subunit. Thus, despite the sequence homology with eukaryotic phosphatases, a biochemical classification of PP has remained elusive (12). With regard to mutagenesis, deletion of one of the invariant peptides, viz.…”

“…Mutational evidence to support these analyses, however, remains fragmentory, primarily due to the difficulties in obtaining the catalytic subunits in large quantities and in soluble form. Recent cloning and expression of the bacteriophage lambda () phosphatase (PP) have helped to expedite biochemical and mutational analysis of Ser/Thr phosphatases (12,13). Recombinant PP, expressed in Escherichia coli, was shown to possess a phosphatase activity that was active against Ser(P), His(P), and Tyr(P) residues in a number of proteins, although dephosphorylation of Tyr(P) residues was pronouncedly slower than that of the other two (12,13).…”

“…Recent cloning and expression of the bacteriophage lambda () phosphatase (PP) have helped to expedite biochemical and mutational analysis of Ser/Thr phosphatases (12,13). Recombinant PP, expressed in Escherichia coli, was shown to possess a phosphatase activity that was active against Ser(P), His(P), and Tyr(P) residues in a number of proteins, although dephosphorylation of Tyr(P) residues was pronouncedly slower than that of the other two (12,13). The unique biochemical properties of PP included resistance to okadaic acid and heat-stable inhibitors 1 and 2, sensitivity to orthovanadate, an absolute requirement of Mn 2ϩ or Ni 2ϩ that could not be substituted by Ca 2ϩ or Mg 2ϩ , and an apparent absence of a regulatory subunit.…”