Sulfate acts as phosphate analog on the monomeric catalytic fragment of the CPx-ATPase CopB from Sulfolobus solfataricus

The crystal structure of the catalytic fragment of a Sulfolobus solfataricus P-type ATPase, CopB-B, was determined with a 2.6 A resolution. CopB-B is the major soluble fragment of the archaeal CPx-ATPase CopB and is comprized of a nucleotide and a phosphorylation domain. In the crystalline state two molecules of CopB-B are in close contact to each other, although the presence of dimers in free solution could be ruled out by analytical ultracentrifugation. The overall architecture of CopB-B is similar to that of other P-type ATPases such as Ca-ATPase. Short peptide segments are linking the nucleotide binding to the phosphorylation domain. CopB-B exhibits 33% sequence identity (of 216 aligned residues) with the respective fragment of the Archaeoglobus fulgidus ATPase CopA. The CopB-B nucleotide-binding domain has the most primitive fold yet identified for this enzyme class. It is 24% identical to the nucleotide-binding domain of the disease-related Wilson ATPase ATP7B (80 structurally aligned residues). Structural superposition with Ca-ATPase suggests a putative nucleotide-binding site in CopB-B. The phosphorylation domain of CopB-B is structurally related to the corresponding part of Ca-ATPase in the anion-bound E2 state. In CopB-B crystals, a bound sulfate anion was identified at the phosphate-binding location. In solution state, the potential binding of CopB-B to phosphate was probed with (32)P(i). Bound phosphate could be readily displaced by orthovanadate at submillimolar concentration as well as by sulfate at millimolar concentration. It is possible therefore to assign the structure of the sulfate-bound phosphorylation domain of CopB-B to a state related to the E2.P(i) intermediate state of the catalytic cycle.     

Archaeoglobus fulgidus CopB is a thermophilic Cu2+-ATPase: functional role of its histidine-rich-N-terminal metal binding domain

P1B-type ATPases transport heavy metal ions across cellular membranes. Archaeoglobus fulgidus CopB is a member of this subfamily. We have cloned, expressed in Escherichia coli, and functionally characterized this enzyme. CopB and its homologs are distinguished by a metal binding sequence Cys-Pro-His in their sixth transmembrane segment (H6) and a His-rich N-terminal metal binding domain (His-N-MBD). CopB is a thermophilic protein active at 75 degrees C and high ionic strength. It is activated by Cu2+ with high apparent affinity (K1/2 = 0.28 microm) and partially by Cu+ and Ag+ (22 and 55%, respectively). The higher turnover was associated with a faster phosphorylation rate in the presence of Cu2+. A truncated CopB lacking the first 54 amino acids was constructed to characterize the His-N-MBD. This enzyme showed reduced ATPase activity (50% of wild type) but no changes in metal selectivity, ATP dependence, or phosphorylation levels. However, a slower rate of dephosphorylation of the E2P(Cu2+) form was observed for truncated CopB. The data suggest that the presence of the His residue in the putative transmembrane metal binding site of CopB determines a selectivity for this enzyme that is different for that observed in Cu+/Ag+-ATPases carrying a Cys-Pro-Cys sequence. The His-NMBD appears to have a regulatory role affecting the metal transport rate by controlling the metal release/dephosphorylation rates.     

Expression, purification and refolding of the phosphatase domain of protein phosphatase 1 (Ppt1) from Saccharomyces cerevisiae

Here we report the recombinant expression of the catalytically active phosphatase domain of the Saccharomyces cerevisiae protein phosphatase 1 (Ppt1) in E. coli. Ppt1 consists of two domains: a 20 kDa TPR (tetratricopeptide repeat) domain, which mediates protein-protein interactions and directs Ppt1 to potential substrate proteins, e.g. the molecular chaperone Hsp90. The second, a 40 kDa phosphatase domain, exhibits catalytic activity and dephosphorylates phosphorylated serine/threonine residues of respective substrate proteins. The Ppt1 phosphatase domain was cloned and expressed in E. coli in unsoluble inclusion bodies. After isolating these, the aggregates were denatured with guanidinium hydrochloride and soluble protein was purified using affinity chromatography. Optimal renaturation conditions led to large amounts of the refolded phosphatase domain in high purity. Interestingly, further enzymatic studies revealed that the domain is not only correctly folded, but also shows higher catalytic activity compared to the full length protein.     

Conformations of the apo-, substrate-bound and phosphate-bound ATP-binding domain of the Cu(II) ATPase CopB illustrate coupling of domain movement to the catalytic cycle

Heavy metal P1B-type ATPases play a critical role in cell survival by maintaining appropriate intracellular metal concentrations. Archaeoglobus fulgidus CopB is a member of this family that transports Cu(II) from the cytoplasm to the exterior of the cell using ATP as energy source. CopB has a 264 amino acid ATPBD (ATP-binding domain) that is essential for ATP binding and hydrolysis as well as ultimately transducing the energy to the transmembrane metal-binding site for metal occlusion and export. The relevant conformations of this domain during the different steps of the catalytic cycle are still under discussion. Through crystal structures of the apo- and phosphate-bound ATPBDs, with limited proteolysis and fluorescence studies of the apo- and substrate-bound states, we show that the isolated ATPBD of CopB cycles from an open conformation in the apo-state to a closed conformation in the substrate-bound state, then returns to an open conformation suitable for product release. The present work is the first structural report of an ATPBD with its physiologically relevant product (phosphate) bound. The solution studies we have performed help resolve questions on the potential influence of crystal packing on domain conformation. These results explain how phosphate is co-ordinated in ATPase transporters and give an insight into the physiologically relevant conformation of the ATPBD at different steps of the catalytic cycle.     

Kinetic comparison of the catalytic domains of SHP-1 and SHP-2

The phosphatase activity of SH2-containing protein tyrosine phosphatase (SHP) is inhibited by its SH2 domains and C-terminal tail. In order to determine the inhibitory effects of the SH2 domains and C-terminal tail, we have expressed and purified the catalytic domains of SHP-1 and SHP-2, and the SH2 domain truncated SHP-1 and SHP-2. We have then measured their kinetic parameters using p-nitrophenyl phosphate (p-NPP) and phosphotyrosine (pY) as substrates under the same experimental conditions. The results indicate that the pH-dependent profiles of SHP-1 and SHP-2 are mainly determined by their catalytic domains. Both enzymes have maximum activity at pH 5.0. In addition, the phosphatase activity of different forms of SHP-1 and SHP-2 decreases as the salt concentration increases. Without SH2 domains, both SHP-1 and SHP-2 are no longer inhibited by their C-terminal tails. However, the C-terminal tail of SHP-1 can further prevent the salt inhibition of the phosphatase activity. Under the same experimental conditions, the catalytic domain of SHP-1 is two times more active than the catalytic domain of SHP-2.     

Characterization of the catalytic activities of the PhoQ histidine protein kinase of Salmonella enterica serovar Typhimurium

Studies of Escherichia coli membranes that were highly enriched in the Salmonella enterica serovar Typhimurium PhoQ protein showed that the presence of ATP and divalent cations such as Mg2+, Mn2+, Ca2+, or Ba2+ resulted in PhoQ autophosphorylation. However, when Mg2) or Mn2+ was present at concentrations higher than 0.1 mM, the kinetics of PhoQ autophosphorylation were strongly biphasic, with a rapid autophosphorylation phase followed by a slower dephosphorylation phase. A fusion protein lacking the sensory and transmembrane domains retained the autokinase activity but could not be dephosphosphorylated when Mg2+ or Mn2+ was present at high concentrations. The instability of purified [32P]phospho-PhoP in the presence of PhoQ-containing membranes indicated that PhoQ also possesses a phosphatase activity. The PhoQ phosphatase activity was stimulated by increasing the Mg2+ concentration. These data are consistent with a model in which Mg2+ binding to the sensory domain of PhoQ coordinately regulates autokinase and phosphatase activities.     

Structural and enzymatic characterization of DR1281: A calcineurin-like phosphoesterase from Deinococcus radiodurans

We have determined the crystal structure of DR1281 from Deinococcus radiodurans. DR1281 is a protein of unknown function with over 170 homologs found in prokaryotes and eukaryotes. To elucidate the molecular function of DR1281, its crystal structure at 2.3 A resolution was determined and a series of biochemical screens for catalytic activity was performed. The crystal structure shows that DR1281 has two domains, a small alpha domain and a putative catalytic domain formed by a four-layered structure of two beta-sheets flanked by five alpha-helices on both sides. The small alpha domain interacts with other molecules in the asymmetric unit and contributes to the formation of oligomers. The structural comparison of the putative catalytic domain with known structures suggested its biochemical function to be a phosphatase, phosphodiesterase, nuclease, or nucleotidase. Structural analyses with its homologues also indicated that there is a dinuclear center at the interface of two domains formed by Asp8, Glu37, Asn38, Asn65, His148, His173, and His175. An absolute requirement of metal ions for activity has been proved by enzymatic assay with various divalent metal ions. A panel of general enzymatic assays of DR1281 revealed metal-dependent catalytic activity toward model substrates for phosphatases (p-nitrophenyl phosphate) and phosphodiesterases (bis-p-nitrophenyl phosphate). Subsequent secondary enzymatic screens with natural substrates demonstrated significant phosphatase activity toward phosphoenolpyruvate and phosphodiesterase activity toward 2',3'-cAMP. Thus, our structural and enzymatic studies have identified the biochemical function of DR1281 as a novel phosphatase/phosphodiesterase and disclosed key conserved residues involved in metal binding and catalytic activity.     

Molecular dissection of the mechanisms of substrate recognition and F-actin-mediated activation of cofilin-phosphatase Slingshot-1

Slingshot-1 (SSH1), a member of a dual-specificity protein phosphatase family, regulates actin dynamics by dephosphorylating and reactivating cofilin, an actin-depolymerizing factor. SSH1 has the SSH family-specific, N-terminal, noncatalytic (SSH-N) domain, consisting of the A and B subdomains. SSH1 is activated by binding to actin filaments. In this study, we examined the mechanisms of SSH1 substrate recognition of phospho-cofilin (P-cofilin) and SSH1 activation by F-actin. We found that P-cofilin binds to a phosphatase-inactive mutant, SSH1(CS), in which the catalytic Cys-393 is replaced by Ser. Using a series of deletion mutants, we provided evidence that both the phosphatase (P) domain and the adjacent B domain are indispensable for P-cofilin binding of SSH1(CS) and cofilin-phosphatase activity of SSH1. In contrast, the A domain is required for the F-actin-mediated activation of SSH1, but not for P-cofilin binding or basal cofilin-phosphatase activity. The P domain alone is sufficient for the phosphatase activity toward p-nitrophenyl phosphate (pNPP), indicating that the SSH-N domain is not essential for the basal phosphatase activity of SSH1. Addition of F-actin increased the cofilin-phosphatase activity of SSH1 more than 1200-fold, but the pNPP-phosphatase activity only 2.2-fold, which suggests that F-actin principally affects the cofilin-specific phosphatase activity of SSH1. When expressed in cultured cells, SSH1, but not its mutant deleted of SSH-N, accumulated in the rear of the lamellipodium. Together, these findings suggest that the conserved SSH-N domain plays critical roles in P-cofilin recognition, F-actin-mediated activation, and subcellular localization of SSH1.     

Non-catalytic domains of subunit A negatively regulate the activity of calcineurin

Calcineurin is composed of a catalytic subunit A (CNA) and a regulatory subunit B (CNB). In addition to the catalytic core, CNA further contains three non-catalytic domains--CNB binding domain (BBH), calmodulin binding domain (CBD), and autoinhibitory domain (AI). To investigate the effect of these three domains on the activity of CNA, we have constructed domain deletion mutants CNAa (catalytic domain only), CNAac (CNAa and CBD), and CNAaci (CNAa, CBD and AI). By using p-nitrophenylphosphate and (32)P-labeled R(II) peptide as substrates, we have systematically examined the phosphatase activities, kinetics, and regulatory effects of Mn(2+)/Ni(2+) and Mg(2+). The results show that the catalytic core has the highest activity and the order of activity of the remaining constructs is CNAac>CNAaci>CNA. Sequential removal of the non-catalytic domains corresponds to concurrent increases of the phosphatase activity assayed under several conditions. This observation clearly demonstrates that non-catalytic domains negatively regulate the enzyme activity and act as intra-molecular inhibitors, possibly through restraining the conformation elasticity of the catalytic core required for optimal catalysis or interfering with substrate access. The sequential domain deletion favors activation of the enzyme by Mn(2+)/Ni(2+) but not by Mg(2+) (except for CNAa), suggesting that enzyme activation by Mn(2+)/Ni(2+) is mainly mediated via the catalytic domain, whereas activation by Mg(2+) is via both the catalytic core and non-catalytic domains.     

Function and structure of N-terminal and C-terminal domains of calcineurin B subunit

Calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, plays a critical role in T-cell activation by regulating the activity of NF-AT. CN is a heterodimer consisting of a catalytic subunit (CNA) and a Ca2+-binding regulatory subunit (CNB). CNB is composed of two global domains: the C-terminal domain (DC) and the N-terminal domain (DN), each containing two Ca2+ binding sites. In this study, using purified DN and DC derived from constructed expression systems, we revealed that intact CNB and DC can stimulate the phosphatase activity of CNA, about 2.2 and 1.6 times the phosphatase activity of CNA alone, respectively; DN itself has little effect on the phosphatase activity of CNA. Fluorescence spectroscopy of an ANS-hydrophobic fluorescence probe shows that binding of Ca2+ to CNB, DC or DN leads to exposure of the hydrophobic surface of the proteins and that the hydrophobicity of CNB is the greatest, that of DC is less, and that of DN is the least. The hydrophobic surface of CNB may be an important structural basis for stimulating CN phosphatase activity.     

Lack of constitutive activity of the free kinase domain of protein kinase C zeta. Dependence on transphosphorylation of the activation loop

Following the induction of apoptosis in mammalian cells, protein kinase C zeta (PKC zeta) is processed between the regulatory and catalytic domains by caspases, which increases its kinase activity. The catalytic domain fragments of PKC isoforms are considered to be constitutively active, because they lack the autoinhibitory amino-terminal regulatory domain, which includes a pseudosubstrate segment that plugs the active site. Phosphorylation of the activation loop at Thr(410) is known to be sufficient to activate the kinase function of full-length PKC zeta, apparently by inducing a conformational change, which displaces the amino-terminal pseudosubstrate segment from the active site. Amino acid substitutions for Thr(410) of the catalytic domain of PKC zeta (CAT zeta) essentially abolished the kinase function of ectopically expressed CAT zeta in mammalian cells. Similarly, substitution of Ala for a Phe of the docking motif for phosphoinositide-dependent kinase-1 prevented activation loop phosphorylation and abolished the kinase activity of CAT zeta. Treatment of purified CAT zeta with the catalytic subunit of protein phosphatase 1 decreased activation loop phosphorylation and kinase activity. Recombinant CAT zeta from bacteria lacked detectable kinase activity. Phosphoinositide-dependent kinase-1 phosphorylated the activation loop and activated recombinant CAT zeta from bacteria. Treatment of HeLa cells with fetal bovine serum markedly increased the phosphothreonine 410 content of CAT zeta and stimulated its kinase activity. These findings indicate that the catalytic domain of PKC zeta is intrinsically inactive and dependent on the transphosphorylation of the activation loop.     

Purification and characterization of the CopB replication control protein, and precise mapping of its target site in the R1 plasmid

The CopB regulatory loop from plasmid R1 has been analyzed. The CopB protein was partially purified, but proteolytic activity in vitro resulted in the recovery of two molecular forms of the polypeptide. Both of these acted as repressors of the repA promoter and had identical activities. The smaller of the proteins was found to be the result of a specific cleavage in the normal in vivo translation product. The active form of the CopB protein is most likely a tetramer, which binds to a DNA region overlapping the repA promoter that also contains a stretch of dyad symmetry. Footprinting analysis and mutant analysis (including nucleotide sequence determination) identified this binding site within 20-25 base pairs. In agreement with in vivo results the binding between CopB and its target site is moderate compared with other operons like lac and trp.     

Role of the diacylglycerol kinase alpha-conserved domains in membrane targeting in intact T cells

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to phosphatidic acid, modifying the cellular levels of these two lipid mediators. Ten DGK isoforms, grouped into five subtypes, are found in higher organisms. All contain a conserved C-terminal domain and at least two cysteine-rich motifs of unknown function. DGKalpha is a type I enzyme that acts as a negative modulator of diacylglycerol-based signals during T cell activation. Here we studied the functional role of the DGKalpha domains using mutational analysis to investigate membrane binding in intact cells. We show that the two atypical C1 domains are essential for plasma membrane targeting of the protein in intact cells but unnecessary for catalytic activity. We also identify the C-terminal sequence of the protein as essential for membrane binding in a phosphatidic acid-dependent manner. Finally we demonstrate that, in the absence of the calcium binding domain, receptor-dependent translocation of the truncated protein is regulated by phosphorylation of Tyr(335). This functional study provides new insight into the role of the so-called conserved domains of this lipid kinase family and demonstrates the existence of additional domains that confer specific plasma membrane localization to this particular isoform.     

Molecular characterization of laforin, a dual-specificity protein phosphatase implicated in Lafora disease

Lafora disease is a progressive myoclonus epilepsy with an early fatal issue. Two genes were identified thus far, the mutations of which cause the disease. The first one, EPM2A, encodes the consensus sequence of a protein tyrosine phosphatase. Its product, laforin, is the object of the present work. We analysed in detail the amino acid sequence of this protein. This suggested, as also observed by others, that it could present two domains, a carbohydrate-binding domain (CBM20, known as a starch-binding domain) and the catalytic domain of a dual-specificity protein phosphatase. We produced the enzyme as two different GST-fused proteins and as an N-terminally His-tagged protein. Differences in solubility were observed between the constructs. Moreover, the N-terminal carbohydrate-binding domain contains a thrombin cleavage site, which is hidden in the simplest GST-fusion protein we produced, but was accessible after introducing a five-residue linker between the engineered cleavage site and the enzyme N-terminus. The two types of constructs hydrolyse pNPP and OMFP with kinetic parameters consistent with those of a dual-specificity phosphatase. We show in addition that the protein not only binds glycogen, but also starch, amylose and cyclodextrin. Neither binding of glycogen nor of beta-cyclodextrin appreciably affects the phosphatase activity. These results suggest that the role of the N-terminal domain is rather that of targeting the protein in the cell, probably to glycogen and the protein complexes attached to it, rather than that of directly modulating the catalytic activity.     

Substrate conformational restriction and CD45-catalyzed dephosphorylation of tail tyrosine-phosphorylated Src protein

Hydrolysis of the tail phosphotyrosine in Src family members is catalyzed by the protein-tyrosine phosphatase CD45, activating Src family-related signaling pathways. Using purified recombinant phospho-Src (P-Src) (amino acid residues 83-533) and purified recombinant CD45 catalytic (cytoplasmic) domain (amino acid residues 565-1268), we have analyzed the kinetic behavior of dephosphorylation. A time course of phosphatase activity showed the presence of a burst phase. By varying the concentration of P-Src, it was shown that the amplitude of this burst phase increased linearly with respect to P-Src concentration. Approximately 2% of P-Src was shown to be rapidly dephosphorylated followed by a slower linear phase. A P-Src protein substrate containing a functional point mutation in the Src homology domain 2 (SH2) led to more rapid dephosphorylation catalyzed by CD45, and this reaction showed only a single linear kinetic phase. These results were interpreted in terms of a model in which P-Src exists in a relatively slow dynamic equilibrium between "closed" and "open" conformational forms. Combined mutations in the SH2 and SH3 domain or the addition of an SH3 domain ligand peptide enhanced the accessibility of P-Src to CD45 by biasing P-Src to a more open form. Consistent with this model, a phosphotyrosine peptide that behaved as an SH2 domain binding ligand showed approximately 100-fold greater affinity for unphosphorylated Src versus P-Src. Surprisingly, P-Src possessing combined SH3 and SH2 functional inactivating point mutations was dephosphorylated by CD45 more slowly compared with P-Src completely lacking SH3 and SH2 domains. Additional data suggest that the SH3 and SH2 domains can inhibit accessibility of the P-Src tail to CD45 by interactions other than direct phosphotyrosine binding by the SH2 domain. Taken together, these results suggest how activation of Src family member signaling pathways by CD45 may be influenced by the presence or absence of ligand interactions remote from the tail.     

Function analysis of conserved amino acid residues in a Mn2+-dependent protein phosphatase, Pph3, from Myxococcus xanthus

The Myxococcus xanthus protein phosphatase Pph3 belongs to the Mg(2+)- or Mn(2+)-dependent protein phosphatase (PPM) family. Bacterial PPMs contain three divalent metal ions and a flap subdomain. Putative metal- or phosphate-ion binding site-specific mutations drastically reduced enzymatic activity. Pph3 contains a cyclic nucleotide monophosphate (cNMP)-binding domain in the C-terminal region, and it requires 2-mercaptoethanol for phosphatase activity; however, the C-terminal deletion mutant showed high activity in the absence of 2-mercaptoethanol. The phosphatase activity of the wild-type enzyme was higher in the presence of cAMP than in the absence of cAMP, whereas a triple mutant of the cNMP-binding domain showed slightly lower activities than those of wild-type, without addition of cAMP. In addition, mutational disruption of a disulphide bond in the wild-type enzyme increased the phosphatase activity in the absence of 2-mercaptoethanol, but not in the C-terminal deletion mutant. These results suggested that the presence of the C-terminal region may lead to the formation of the disulphide bond in the catalytic domain, and that disulphide bond cleavage of Pph3 by 2-mercaptoethanol may occur more easily with cAMP bound than with no cAMP bound.     

Regulation of protein kinases by lipids

Membranes are sites of intense signaling activity within the cell, serving as dynamic scaffolds for the recruitment of signaling molecules and their substrates. The specific and reversible localization of these signaling molecules to membranes is critical for the appropriate activation of downstream signaling pathways. Phospholipid-binding domains, including C1, C2, PH, and PX domains, play critical roles in the membrane targeting of protein kinases. Recent structural studies have identified a new membrane association domain, the Kinase Associated 1 (KA1) domain, which targets a number of yeast and mammalian protein kinases to membranes containing acidic phospholipids. Despite an abundance of localization studies on lipid-binding proteins and structural studies of the isolated lipid-binding domains, the question of how membrane binding is coupled to the activation of the kinase catalytic domain has been virtually untouched. Recently, structural studies on protein kinase C (PKC) have provided some of the first structural insights into the allosteric regulation of protein kinases by lipid second messengers.     

Structural basis for domain-domain communication in a protein tyrosine kinase, the C-terminal Src kinase

The catalytic activity of protein tyrosine kinases is commonly regulated by domain-domain interactions. The C-terminal Src kinase (Csk) contains a catalytic domain and the regulatory SH3 and SH2 domains. Both the presence of the regulatory domains and binding of specific phosphotyrosine-containing proteins to the SH2 domain activate Csk. The structural basis for both modes of activation is investigated here. First, the SH3-SH2 linker is crucial for Csk activation. Mutagenic and kinetic studies demonstrate that this activation is mediated by a cation-pi interaction between Arg68 and Trp188. Second, Ala scanning and kinetic analyses on residues in the SH2-catalytic domain interface identify three functionally distinct types of residues in mediating the communication between the SH2 and the catalytic domains. Type I residues are important in mediating a ligand-triggered activation of Csk because their mutation severely reduces Csk activation by the SH2 domain ligand. Type II residues are involved in suppressing Csk activity, and their mutation activates Csk, but makes Csk less sensitive to activation by the SH2 ligand. Both type I and type II residues are likely involved in mediating SH2 ligand-triggered activation of Csk. Type III residues are those located in the SH2 domain whose mutation severely decreases Csk catalytic activity without affecting the SH2 ligand-triggered activation. These residues likely mediate SH2 activation of Csk regardless of SH2-ligand interaction. These studies lead us to propose a domain-domain communication model that provides functional insights into the topology of Csk family of protein tyrosine kinases.     

Evolution of the multifunctional protein tyrosine phosphatase family

The protein tyrosine phosphatase (PTP) family plays a central role in signal transduction pathways by controlling the phosphorylation state of serine, threonine, and tyrosine residues. PTPs can be divided into dual specificity phosphatases and the classical PTPs, which can comprise of one or two phosphatase domains. We studied amino acid substitutions at functional sites in the phosphatase domain and identified putative noncatalytic phosphatase domains in all subclasses of the PTP family. The presence of inactive phosphatase domains in all subclasses indicates that they were invented multiple times in evolution. Depending on the domain composition, loss of catalytic activity can result in different consequences for the function of the protein. Inactive single-domain phosphatases can still specifically bind substrate and protect it from dephosphorylation by other phosphatases. The inactive domains of tandem phosphatases can be further subdivided. The first class is more conserved, still able to bind phosphorylated tyrosine residues and might recruit multiphosphorylated substrates for the adjacent active domain. The second has accumulated several variable amino acid substitutions in the catalytic center, indicating a complete loss of tyrosine-binding capabilities. To study the impact of substitutions in the catalytic center to the evolution of the whole domain, we examined the evolutionary rates for each individual site and compared them between the classes. This analysis revealed a release of evolutionary constraint for multiple sites surrounding the catalytic center only in the second class, emphasizing its difference in function compared with the first class. Furthermore, we found a region of higher conservation common to both domain classes, suggesting a new regulatory center. We discuss the influence of evolutionary forces on the development of the phosphatase domain, which has led to additional functions, such as the specific protection of phosphorylated tyrosine residues, substrate recruitment, and regulation of the catalytic activity of adjacent domains.