Volume 263, number 2, 265-268 FEBS 08346 April 1990 Sequence homology between purple acid phosphatases and phosphoprotein phosphatases Are phosphoprotein phosphatases metalloproteins containing oxide-bridged dinuclear metal centers? John B. Vincent and Bruce A. Averill University of Virginia, Department of Chemistry, Charlottesville, VA 22901, USA Received 12 January 1990; revised version received 26 February 1990 The amino acid sequences of mammalian purple acid phosphatases and phosphoprotein phosphatases are shown to possess regions of significant homology. The conserved residues contain a high percentage of possible metal-binding residues. The phosphoprotein phosphatases I, 2A and 2B are proposed to be iron-zinc metalloenzymeswith active sites isostructural (or nearly so) with those of the purple phosphatases, Protein phosphatase; Purple acid phosphatase; Sequence homology 1. INTRODUCTION tain fungi and Drosophila have been shown to be highly homologous (50-90°70) to those of mammalian PP1 Phosphoprotein phosphatases (PPs) are a class of and PP2A [13,16-20], suggesting that the phosphopro- mammalian regulatory enzymes that catalyze the tein phosphatases may be widely distributed. dephosphorylation of phosphoserine and phospho- Mammalian purple acid phosphatases (PAPs) are threonine proteins [1,2]. Phosphoprotein phosphatase novel enzymes of molecular mass -37 kDa that contain 1 (PP1), which is inhibited by inhibitor-1 and -2, an oxide-bridged dinuclear iron active site. The amino generally occurs in a glycogen- or myosin-bound form. acid sequences of the enzymes from bovine spleen, por- The type 2 enzymes (insensitive to the above inhibitors) cine uterine fluid, and human placenta are highly are further subdivided into three classes. PP2A is a homologous (-90o70), again indicating a close relation- cytosolic enzyme that possesses broad reactivity, while ship [21,22]. A possibly related purple iron-zinc enzyme PP2B, better known as calcineurin, is a Ca 2+- and has been isolated from red kidney beans [23]; it differs calmodulin-binding protein that dephosphorylates significantly from the mammalian enzymes in both several brain-specific phosphoproteins. PP1 and PP2A molecular weight and amino acid composition. Yet contain a catalytic, -35 kDa subunit and a variety of another purple phosphatase has been isolated from regulatory subunits. However, PP2B is comprised of a fungi [24,25], but the metal composition of this form catalytic 60 kDa subunit and a smaller 19 kDa subunit; is unknown. PP2C is composed of a single 46 kDa subunit. Herein, w.e describe regions of the amino acid se- The nucleotide sequences of the cDNA coding (or quences of the catalytic subunits of PPs and mam- partially coding) for the catalytic subunits of PP1, malian PAPs that display surprising homology and PP2A, and PP2B have been determined from a variety may contain conserved metal-binding ligands. of sources [3-14]; the deduced amino acid sequences have very high degrees of homology (-50o70), indicating 2. RESULTS AND DISCUSSION that the catalytic subunits are closely related (and probably derived from a common ancestor). The amino Given the similarities in size and function between acid sequence of PP2C appears to be unrelated to those PAPs and the catalytic subunits of PP1 and PP2A of the other PPs [15], and the in vivo function of the (both mammalian PAPs and PPs possess phospho- enzyme is presently in question. Very recently, the serine/phosphothreonine protein phosphatase activity nucleotide sequence encoding some proteins from cer- [26,27], and all the enzymes are able to hydrolyze the artificial substrate p-nitrophenylphosphate [28-31]), Correspondence address: B.A. Averill, Department of Chemistry, the amino acid compositions of the human placental University of Virginia, Charlottesville, VA 22901, USA PAP and rabbit skeletal muscle PP2A proteins were Published by Elsevier Science Publishers B.V. (Biomedical Division) 00145793/90/$3.50 © 1990 Federation of European Biochemical Societies 265 Volume 263, number 2 FEBS LETTERS April 1990 compared (Table I). Using a statistical technique for Region I 4 5. 6' 7 8 testing the significance of amino acid composition in- 3 0 0 0 0 Human PAPa EdyVLvaghyPV-wsiaeHGptHcLVkQLrpLIATygvTaYLcghDhn dexes [32,33], the two proteins were found to have Porcine PAP,b EdyVLvaghyPV-wsiaeHGptHcLVkQLlpLITThkvTaYLqghDhn about a 90°7o probability of possessing significantly Beef PAP c EdyVXvaghyPV-wsxaeHGvvHcXVkcXxpXxnAhkvTaYXcghDhn Rabbit PP2ad EsnVqevrc-PVtvcgdvHGqfHdLM-ELfrlgGkspdTnYLfmgDyn related sequences (DI = 10.4, D = 0.0635, SAQ = 40.4). Beef PP2ae EsnVqevrc-PVtvrgdvHGqfHdLM-ELfrIgGkspdTnYLfmgDyv Porcine PP2af EsnVqevrc-PVtvrgdvHGqfHdLM-ELfrlgGkspdTnYLfmgDyv The major differences in the composition of the two en- Porcine PP2af EsnVqevrc-PVtvcgdvHGqfHdLM-ELfrlgGkspdTnYLfmgDyv zymes are in the mole fractions of alanine, glutamate Human PP2ag EsnVqevrc-PVtvcgdvHGqfHdLM-ELfrlgGkspaTnYLfmgDyv Human P@2ah EsnVqevrc-PVtvcgdvHGqfHdLM-ELfrlgGlspdTnYLfmgByv and aspartate, and cysteine. The higher mole fraction Rat 2ba! EknLLdida-PVtvcgdiHGqffdLM-kLdwcfGSpanTrYLflgDyv Rat 2b~ ~ . EktMlevea-PltvcgdiHGqffdLM-kLfeVgGSpanTrYLflgDyv of carboxylates is reflected in the overall charge of the Ruman 2b j EktMlevea-PltvcgdiHGqffdLM-kLfeVgGSpanTrYLflgDyv Rabbit PPI k QpiLLelea-PLkicgdiHGqyydLLr-LfeygGFppeSnYLflgDyv enzymes. PP2A, which possesses the greater number of Yeast HIS41 Esnlqpvqt-PVtvcgdiHGqfHd~LelfrtagGfpddinYlflgDyv carboxylates, binds to DEAE-cellulose, while PAPs are strongly cationic. The amino terminus of PAP is large- 9 1 1 ly responsible for the relatively high content of alanine. 0 0 3 This hydrophobic region of the protein has been found 0 Region I[ 0 to be highly homologous to the noncatalytic Fo subunit Human PAP l-qYlqdEn-GVgyvL SgAgn~mDpsKR Porcine PAP l-qYlqdEn-GLgfvL SgAgnFmDpsKK of maize mitochondrial ATPase [34]. Beef PAP x-qYxqqEn-GXgfvX SgAgnFmDpsKK Rabbit PP2a drgYysvEtvTLlvaL SqvygFyDeclR A search of Genbank revealed no significant Beef PP2a drgYysvEtvTLlvaL SqvygFyDeclR similarities of the human PAP with the rabbit PP2Ace, Porcine PP2a drgYysvEtvTLlvaL SqvygFyDeclR Porcine PP2a drgYysvEtvTLlvaL SqvygFyDeclR confirming an earlier report [22]. However, careful in- Human PP2a drgYysvEtvTLlvaL SqvygFyDeclR Human PP2a drgYysvEtvTLlvaL SqvygFyDeclR spection of the sequences of a representative PAP and Rat 2ba drgYfsiEcvlylwaL SeyftFkQecKi PP did reveal a region of appreciable sequence Rat 2b~ drgYfsiEcvlylwaL SeyftFkQecKi Human 2b drgYfsiEcvlylwaL SeyftFkQecKi homology between the two proteins. Between residues Rabbit PPI drgkqslEtieLl]ay nrTygFyDecKR Rabbit PPXm TqvygFyDeclR 186-237 of the human PAP [22] and residues 51-103 Yeast HIS4 drgYyslEtfTLlmcL TqvygFyEecln of the rabbit PP2Ac~ enzyme [7], 16 residues were con- 2 2 2 served (-30% absolute homology). Extension of the 4 5 6 Region III 0 0 0 comparison to other available PAP and PP sequences Human PAP HQrkVpNGYIcFH-yGT...... edSL-GGfaY and partial sequences (Fig. 1) showed that the region of Porcine PAP HQrkVpNGYIrFH-fGA...... enSL-GGfaY sequence homology was significantly larger, but that Beef PAP H..qVpDGYxrFH-yGA...... enSX-GGfaY Rabbit PP2a HQI-VmEGYnwcH-drn...... vvTlfSApnY the sequences of the phosphoprotein phosphatases had Beef PP2a HQI-VmEGYnwcH-drn...... vvTlfSApnY Porcine PP2a HQl-VmEGYnccH-drn ...... vvTlfSApnY two sizeable insertions compared to the sequences of Porcine PP2a HQI-VmEGYnccH-drn...... vvTlfSApnY Human PP2a HQI-VmEGYnwcH-drn...... vvTlfSApnY the purple acid phosphatases (Fig. 2). Of note also is Human PP2a HQI-VmEGYnwcH-drn...... vvTlfSApnY the fact that the amino terminus of the PAP sequences, Rat 2ba HEaq-daGYrmYr-kSqttgfpsliTlfSApnY Rat 2b~ HEaq-daGYrmYr-kSqttgfpsliTlfSApnY Human 2b HEaq-daGYrmYr-kSqttgfpsliTlfSApnY Rabbit PPI HQv-VeDGYefFa-krq...... IvTLfSApnY Rabbit PPX HQI-VmEGYkwhf-neT...... vlTVwSApnY Table I Yeast HIS4 HQI-VmEGFkyhfpekd...... vvTVwgApn~ Comparison of amino acid content of rabbit PP2Aoe and human pAp c Fig. 1. Sequence comparison of purple acid phosphatases and selected protein phosphatases. Numbers correspond to positions in the rabbit PP2A sequence. PPN, protein phosphatase N; X (in Amino acid Mole fraction PP2A ~ Mole fraction PAP b sequences), isoleucine or leucine; -, not determined; *, possible metal Met 0.0194 0.0132 ligands, a [22]; b [21,22]; c [21]; a [7]; e [3,4]; ~ [4]; g [8]; h [9]; i [44]; Asp 0.0777 0.0559 J [45]; k [6]; f [16]; m [13]. Conserved residues in all or all but one Ash 0.0388 0.0428 sequence are notated with a bar at the top and bottom of the column Trp 0.0162 0.0164 and capitalized; conservative substitutions are capitalized. For Thr 0.0583 0.0559 convenience, the list of PP sequences is not inclusive; however, the Ala 0.0388 0.0789 conserved metal binding ligands are unaffected by incorporation of Leu 0.100 0.0921 the additional sequences [5,10,11,17-19]. Ile 0.0421 0.0362 Gin 0.0453 0.0427 Glu 0.0647 0.0395 Pro 0.0421 0.0559 which contains the region homologous to the Fo Ser 0.0485 0.0625 subunit of ATPase and the glycosylation site, has no Gly 0.0744 0.0789 counterpart in the PP sequences. As can be readily Arg 0.0647 0.0625 observed in Fig. 1, possible metal-binding ligands (in- Phe 0.0453 0.0789 dicated by asterisks) constitute a large portion of the Val 0.0680 0.0625 Tyr 0.0518 0.0559 conserved residues between the PAP and PP sequences, Cys 0.0324 0.0724 i.e. two tyrosines (80 and 264), two histidines (59 and His 0.0324 0.0362 240), two aspartic acids (86 and 131),