Computational Molecular Biology, Max-Planck-Institute for Molecular Genetics, Berlin, Germany
Correspondence: E-mail: joerg.schultz{at}biozentrum.uni-wuerzburg.de.
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Abstract |
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Key Words: protein tyrosine phosphatase antiphosphatase signaling enzymes functional divergence evolutionary site rates
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Introduction |
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Functionally, two types of PTPs, conserved in sequence and structure can be distinguished: The classical PTPs, which are specific for tyrosine residues, and the dual-specificity phosphatases (DSPs), which can additionally dephosphorylate serine and threonine residues.
Obviously, the catalytic residues of an enzyme are key to its molecular function. Therefore, it came as a surprise when a member of the DSP family, Sfb1, with a replacement of the catalytically essential cysteine was described (Cui et al. 1998). As expected, this protein had lost its enzymatic activity, raising the question about its molecular function. Experimentally, it could be shown that Sbf1 has maintained its ability to stably bind phosphorylated substrate, protecting the substrate from other phosphatases at this specific site. Because of its antagonistic mechanism, this phosphatase has been termed "antiphosphatase" (De Vivo et al. 1998; Hunter 1998). Sbf1 function differs also on the cellular level. In contrast to active phosphatases exposing growth inhibitory behavior, it shows transforming abilities (Cui et al. 1998). A similar substitution of the catalytic cysteine was found in another subgroup of the DSPs, called STYX (Wishart et al. 1995). Within the classical PTPs, substitutions of functional residues have been described in the receptor protein tyrosine phosphatase (RPTP) family. Most RPTPs contain two phosphatase domains, of which the second phosphatase domain is inactive or remains with very low activity because of substitutions at functional sites. Although the detailed function of this catalytically inactive domain is not yet totally understood, it is of absolute importance for the function of the receptors. Partial or entire deletion of the second domain completely abolishes or severely reduces activity of the first domain, so that a role in regulating the catalytic activity or substrate specificity of the first domain has been hypothesized (Streuli et al. 1990; Johnson et al. 1992). Furthermore, specific interaction of domain II with domain I leading to active site blocking of domain I (Bilwes et al. 1996; Majeti et al. 1998; Blanchetot and den Hertog 2000) has been shown experimentally. Inactive phosphatase domains have also been studied experimentally in the single-phosphatase domain RPTPs (Kambayashi et al. 1995; Cui et al. 1996). Here, the PTP typical signature with the catalytic cysteine is present, but two other catalytically important residues are substituted, leading to loss of phosphatase activity.
On the basis of these more anecdotal reports, we analyze here on a genomic scale how frequent substitutions of functional residues in the PTP family are and whether they were invented multiple times in evolution. Furthermore, we use site-specific evolutionary rates to unravel the evolutionary implications of these substitutions for the whole domain, leading to the prediction of distinct functional classes and the delineation of an additional functional site.
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Methods |
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Scan for Inactive Phosphatases
For each PTP subclass, a multiple sequence alignment of all found members was created according to the family alignment in SMART, using HMMalign (HMMER). The alignments were manually curated to remove unnecessary gaps and to connect interrupted secondary structure elements, partially with the help of secondary structure information, using a representative structure from PDB (1LAR for the classical PTPs, 1VHR for the DSPs). Knowing the positions of functional residues from literature, we were able to scan the sequences for substitutions at these sites and to extract the substituted amino acid from the alignment. If these sites were occupied by gaps, absence of the functional residue was not considered in the further analysis.
Evolutionary Rate Analysis
After definition of the subclasses (D2A, D2B, membrane proximal, and cytosolic), we compared the evolutionary rates of all sites between the subclasses. Therefore, we selected the following genes from human and mouse for further analysis: Homo sapiens: ENSP00000175756, ENSP00000246887, ENSP00000248594, ENSP00000256635, ENSP00000262539, ENSP00000263708, and ENSP00000311857; Mus musculus: ENSMUSP00000022508, ENSMUSP00000025420, ENSMUSP00000027633, ENSMUSP00000029053, ENSMUSP00000029433, ENSMUSP00000030556, and ENSMUSP00000048119. The evolutionary site rates were estimated with Tree-Puzzle version 5.1 (Strimmer and von Haeseler 1996) using the quartet puzzling algorithm under the substitution model of Jones-Taylor-Thornton with an eight site-rate category discretized gamma model (Yang 1994). This model sets the average rate of a site to 1 and assigns each site to one of eight rate classes. As the rate of one class can differ between analyses of different subfamilies, we considered a site as differentially evolving if its rate was below 0.8 in the conserved subfamily and above 1.6 in the fast-evolving one. These values were chosen because within all analyses, they covered classes 1 to 4 for the conserved domain and classes 7 and 8 in the fast-evolving domain (total rates range approximately from 0 to 3 within all analyses). We performed the analyses separately for mouse and human and accepted only sites that were classified as differentially evolving in at least three of the four possible comparisons.
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Results and Discussion |
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The analysis revealed that all subclasses of the DSPs and classical PTPs, including the membrane-proximal, membrane-distal, and cytosolic domains, carry substitutions in functional positions.
Table 2 shows the distribution of phosphatase domains among subclasses for each investigated species and the number of domains with substitutions at functional sites within each subclass. The number of putative inactive domains varies strongly among the different species; however, it accounts for a significant part of all subclasses, even in the single-domain phosphatase subfamily. One might argue that the catalytically inactive proteins are nonfunctional relics in the genome, but their conservation between species and also within subfamilies, strongly indicates their functional importance. In many of the observed cases, the protein contains either a single nonactive phosphatase domain or if it contains two, both are inactive. Depending on the type of substitution, these proteins are good candidates as potential antiphosphatases.
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In the second subclass (D2B) substitutions in functional sites are more frequent and more heterogeneous than in subclass D2A. The high number of substitutions and the high variety in amino acids makes it seem unlikely that these domains maintained their catalytic activity. Indeed, Streuli et al. (1990) experimentally showed that the D2B domain of CD45 completely lost its phosphatase activity. The fact that the inactive phosphatase domain has not been lost during evolution and that orthologs are found in all species investigated in our study excludes the mutation to a nonfunctional "pseudodomain." On the contrary, because the PTP structure is still conserved, a specialization on other functions is likely to have occurred during evolution.
In summary, substitutions in functional sites reside in all subclasses of the phosphatase family. This raises the question of whether the inactive phosphatase was invented once or multiple times. If invented once, its widespread presence would indicate an origin before the split into the subclasses. Assuming this monophyletic origin would imply that inactive phophatases of different subclasses are more related to each other than to other members of the subclass, which is not the case. Furthermore, it was shown that the major subclasses evolved monophyletically (Andersen et al. 2001). Therefore, we conclude that the invention of inactive phophatases happened multiple times independently during evolution. The percentage of nonfunctional phosphatases in metazoan genomes is surprisingly high. The regulation of signaling pathways by protection of phosphorylated serine/threonine or tyrosine residues might, therefore, turn out to be an important mechanism to modulate signaling pathways. It has to be further investigated whether the phenomenon of nonfunctional enzymes is restricted to the phosphatase family or whether other signaling enzymes show a similar behavior.
Analysis of Altered Evolutionary Rates Between Phosphatase Subclasses
The strikingly high number of amino acid substitutions in functional residues in the membrane-distal phosphatase domain of receptor PTPs, which is associated with loss of activity, raises the question how on the one hand these substitutions evolved and how on the other hand they influenced the evolution of the whole domain. As a change in function should be mirrored in a change of evolutionary constraints at the involved sites, we compared the site-specific evolutionary rates between the active (cytosolic and membrane-proximal) and the inactive (D2A and D2B) PTP subclasses, a method that has been used to identify functionally important sites (Gu and Vander Velden 2002; Blouin, Boucher, and Roger 2003). Because of the lack of a representative quantity of PTP sequences in the single subclasses of most species, evolutionary rates could only be estimated reliably for human and mouse. Indeed, we found a substantial number of sites with changing evolutionary constraints (table 3). To further understand how these work together, we mapped these sites onto the structure of the PTP domain (pdb 1LAR) (fig. 1). This revealed that these sites cluster within two regions. One group is located around the catalytic center, and the other group is located on the backside of the protein. In the following section we discuss these regions separately.
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The evolutionary events in D2B after domain duplication might have been triggered by a single mutation of a functional residue, which led to a noncatalytic domain. Subsequently, the evolutionary constraint of the catalytic center was relaxed, leading to the accumulation of mutations in the surrounding.
In contrast to domain D2B, there are only five sites that are fast evolving in D2A but conserved in the active domains. One of them (position 185) is located next to the functional site 184, occupied by aspartate in the active domain but replaced by glutamate in domain D2A. This mutation might have freed the immediate surrounding from selective constraint and allowed a faster evolutionary rate at site 185. The functional residues in the catalytic center of D2A are affected by only two amino acid substitutions. The catalytic aspartate is replaced by glutamate acid and the substrate binding tyrosine either by valine or leucine. These substitutions maintain the biochemical properties, and although the catalytic activity of this domain is barely detectable, it is still able to stably bind its substrate (Bliska et al. 1992). The analysis demonstrates that the catalytic center is still under selective pressure because residues that are fast evolving in D2B and predicted to function in substrate binding are conserved in D2A. This is confirmed experimentally by regaining a catalytically fully active domain if the two substituted functional residues are converted to their original amino acids (Lim et al. 1998). We conclude that the catalytic center of D2A, in contrast to D2B, plays a pivotal role in the function of tandem domain phosphatases, leading to the question of what this function is. As the domain has lost its catalytic activity but still can bind to phosphotyrosine, one could assume two complementary molecular functions. First, the domain could function as "antiphosphatase" as described for DSPs (Cui et al. 1998). Second, it might work as an adaptor domain for phosphotyrosine substrates, similar to SH2 and PTB domains, revealing an additional function of the PTP domain family.
Slow Evolving Sites on the Backside of the Domain
The complete loss of evolutionary pressure on the catalytic center of the D2B family leads to the question of what the function of this domain is and whether there is a similar role of the D2A domains. If a new function was acquired, this should be reflected in novel conserved sites. Therefore, we searched for sites that evolve at a higher rate in the active domains while they are conserved in D2A or D2B. The comparison found 11 sites conserved in D2B and 12 sites conserved in D2A, of which five sites are found in both analyses (table 3). Almost all sites are located on the surface of the "backside" of the domain (fig. 1). This could indicate, that a new functional center has evolved in this region. The solvent exposure as well as the nature of the conserved amino acids might hint that this region is involved in protein-protein interactions. Indeed, interactions of the membrane-distal and membrane-proximal domains have been described recently. The direct interaction of the membrane-distal domain with the membrane-proximal domain stabilizes the enzyme and enhances catalytic activity (Felberg and Johnson 2000). This effect can be abrogated by deletion of the two carboxy-terminal -helices of the membrane-distal domain (244 to 278) (Johnson et al. 1992). These two helices host two residues (245 and 256), which are significantly more conserved in D2A and D2B than in the active domains, and one residue (250) that is more conserved if D2B is compared against the active domains. Another highly conserved site (240) found in both comparisons is preceding the two helices. These sites might play an important role in the interaction between the phosphatase domains. The additional sites with altered selective constraint might contribute to the stable binding but are not sufficient for stable interaction without presence of the two carboxy-terminal helices. Experimental mutation of these sites might give further insight into the molecular mechanism of regulation of RPTPs.
Together with the variation of the catalytic site, our results indicate, that domain D2A and D2B have distinct influences on the activity of membrane-proximal domains. We suggest that both domains can control activity of the first domain by interaction of residues from the "backside" of the membrane-distal domain and residues from the membrane-proximal domain. In addition, D2A can regulate substrate specificity of the membrane-proximal domain and remain associated with the substrate protein, which is accomplished by the inactive catalytic center of D2A.
The results of our analyses allow delineating a possible scenario for the evolution of the membrane-distal domain of RPTPs. The overlap of conserved residues on the "backside" of both D2A and D2B indicates that their common ancestor already had evolved this novel functional site. Whether the membrane-distal domain of the first RPTP was still active remains unclear, but the complete absence of a domain without substitutions of functional residues within the catalytic center hints that it indeed was inactive. This ancestral RPTP gave rise to one lineage with a conserved catalytic center that is still able to bind substrate (D2A) and one lineage that accumulated substitutions around the catalytic center, completely loosing the substrate binding function (D2B).
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Conclusions |
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Acknowledgements |
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Footnotes |
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