Catalytic Activities of the PPP Family Members

PPPs dephosphorylate phosphoserine and phosphothreo-nine residues in proteins in vivo and in vitro. Mammalian PP1, PP2A, and PP2C have also been shown to dephosphorylate histidine residues in proteins in vitro [14]. Mammalian PPPs expressed in Escherichia coli display properties that are slightly different from the native enzymes, including dependence or partial dependence on divalent metal ions such as Mn2+ or Mg2+ [15]. Bacterially expressed mammalian Ppplc also exhibits protein-phosphotyrosine activity, in addition to serine/threonine phosphatase activities, but this disappears when the enzyme is refolded in the presence

Figure 1 Phylogenetic tree, depicting the relationships between Homo sapiens, Drosophila melanogaster, budding yeast Saccharomyces cerevisiae, and prokaryotic protein serine/threonine phosphatases in the PPP family. Prokaryotes include archaebacteria Methanosarcina thermophila and Sulfolobus solfataricus, eubacteria Escherichia coli and Microcystis aeruginosa (cyanobacterium), and bacteriophages Lambda and Phi80. The chromosomal location is included for the Drosophila PPPs. The unrooted tree is derived from multiple alignment in CLUSTALW (http://www.clustalw.genome.ad.jp/) of the phosphatase catalytic domain (starting = 30 amino acids prior to the invariant GDXHG motif and terminating = 30 amino acids following the conserved SAXNY motif [13]). Note that the eubacterial and bacteriophage catalytic domains are only homologous to the N-terminal half of the eukaryotic and archeabacterial catalytic domains. Ppplc and Ppp2c show approximately 40% sequence identity, isoforms of Ppplc show >85% sequence identity, and novel Drosophila phosphatases in the Pppl subfamily show <65% sequence identity. Similar values occur in the PPP2A subfamily. Genbank accession numbers for human PPP sequences are Ppplca (J04759/M63960), Ppplcß (X80910), PPpplcy (X74008), Ppp2ca (J03804), Ppp2cß (X12656), Ppp4c(X702l8), Ppp6c (X92972), Ppp3ca (Ll4778), Ppp3cß (M2955l), Ppp3cy (S46622), Ppp5c (X894l6), Ppp7ca (X97867), and Ppp7cß (AF023456). Drosophila PPP sequences are Pppl-l3C (X69974), Pppl-87B (Xl5583), Pppl-96A (X56438), Pppl-9C (X56439), PpplY2 (AF427494), PppY-55A (Y075l0), Ppp-23D (AE00358l), Ppp-58B (AE003456), PpplYl (AF427493), PppN-58A (Yl7355), Ppp2-28D (X78577), Ppp4-l9C (Yl42l3), PppV-6A (X75980), Ppp-68D (AY058490), Ppp3- l00B(M970l2), Ppp3-l4F (AE003502), Ppp3-l4D (X77768), Ppp5-85E (AJ27l78l), and rdgC-77B(M89628). S. cerevisiae PPP sequences are Glc7 (M27070), Ppzl (X74l35), Ppz2 (X74l36), Ppql (X75485), Pph2l (X5626l), Pph22 (X56262), Pph3 (X58858), Sit4 (M24395), Ppgl (M94269), Can/Cmpl (M64839), Can/Cmp2 (M64840), and Pptl (X894l7); Trypanosoma brucei Pppl(X52746), T. brucei Ppp2 (M74l68), T. brucei Ppp5 (AF305085); M. thermophila Ppp (U96772); S. solfataricus Ppp(U35278); M. aeruginosa Ppp (U80886); Escherichia coli PrpA(U5l99l), E. coli PrpB (U29579); lambda Ppp (J02459); Phi80 Ppp [7]. (A color version of this figure can be viewed in the CD edition of Handbook of Cell Signaling, v. 1. )

Figure 2 Domain organization of protein phosphatases in the PPP family. Bacteriophage and eubacteria Ppps show homology to the eukaryotic and archeabacterial protein phosphatases in the amino-terminal half of the catalytic domain, but the carboxy-terminal halves show little similarity. Autoinhibitory regions (AIs), Ca2+-binding EF hands, tetratricopeptide repeats (TPRs), and calmodulin (CaM) binding sites are indicated. The numbers of amino acids in different phosphatases with each type of structure are given on the right. The three invariant amino acid motifs found in all PPP family members are shown at the bottom of the figure (numbers refer to the amino acid position in PP1a1/PP1y1).

Figure 2 Domain organization of protein phosphatases in the PPP family. Bacteriophage and eubacteria Ppps show homology to the eukaryotic and archeabacterial protein phosphatases in the amino-terminal half of the catalytic domain, but the carboxy-terminal halves show little similarity. Autoinhibitory regions (AIs), Ca2+-binding EF hands, tetratricopeptide repeats (TPRs), and calmodulin (CaM) binding sites are indicated. The numbers of amino acids in different phosphatases with each type of structure are given on the right. The three invariant amino acid motifs found in all PPP family members are shown at the bottom of the figure (numbers refer to the amino acid position in PP1a1/PP1y1).

of inhibitor-2, indicating that it is unlikely to be a property of the native enzyme in vivo [16]. Ppp2 (PP2A) exhibits some protein tyrosine phosphatase activity in the presence of the protein PTPA [17]. Bacterially expressed phage lambda Ppp exhibits protein-serine, -threonine, -histidine, and -tyrosine phosphatase activities [18], while native cyanobacterial PPP exhibits protein-serine, -tyrosine, -histidine, and -lysine phos-phatase activities in vitro [11]. Thus, the activities of some Ppps may be wider than dephosphorylation of protein-bound phosphoserine and phosphothreonine residues. The discovery that the diadenosine tetraphosphatases have sequence similarities to the PPP family provides evidence that the essential PPP signature motifs for phosphomonoesterase activity have been utilized for the hydrolysis of biomolecules other than protein-bound phosphoesters [13].

PPPs dephosphorylate serine and threonine residues that lie in unrelated amino acid sequences, suggesting that, in contrast to many protein kinases, specificity is not determined primarily by the linear sequence of amino acids of either side of the phosphorylated residue. Rather, these observations suggest that higher order structures and/or interaction with regulatory subunits are involved [19].

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