Prediction of functional phosphorylation sites by incorporating evolutionary information

Protein phosphorylation is a ubiquitous protein post-translational modification, which plays an important role in cellular signaling systems underlying various physiological and pathological processes. Current in silico methods mainly focused on the prediction of phosphorylation sites, but rare methods considered whether a phosphorylation site is functional or not. Since functional phosphorylation sites are more valuable for further experimental research and a proportion of phosphorylation sites have no direct functional effects, the prediction of functional phosphorylation sites is quite necessary for this research area. Previous studies have shown that functional phosphorylation sites are more conserved than non-functional phosphorylation sites in evolution. Thus, in our method, we developed a web server by integrating existing phosphorylation site prediction methods, as well as both absolute and relative evolutionary conservation scores to predict the most likely functional phosphorylation sites. Using our method, we predicted the most likely functional sites of the human, rat and mouse proteomes and built a database for the predicted sites. By the analysis of overall prediction results, we demonstrated that protein phosphorylation plays an important role in all the enriched KEGG pathways. By the analysis of protein-specific prediction results, we demonstrated the usefulness of our method for individual protein studies. Our method would help to characterize the most likely functional phosphorylation sites for further studies in this research area.     

A large-scale method to measure absolute protein phosphorylation stoichiometries

The functional role of protein phosphorylation is impacted by its fractional stoichiometry. Thus, a comprehensive strategy to study phosphorylation dynamics should include an assessment of site stoichiometry. Here we report an integrated method that relies on phosphatase treatment and stable-isotope labeling to determine absolute stoichiometries of protein phosphorylation on a large scale. This approach requires the measurement of only a single ratio relating phosphatase-treated and mock-treated samples. Using this strategy we determined stoichiometries for 5,033 phosphorylation sites in triplicate analyses from Saccharomyces cerevisiae growing through mid-log phase. We validated stoichiometries at ten sites that represented the full range of values obtained using synthetic phosphopeptides and found excellent agreement. Using bioinformatics, we characterized the biological properties associated with phosphorylation sites with vastly differing absolute stoichiometries.     

Differential hormone-dependent phosphorylation of progesterone receptor A and B forms revealed by a phosphoserine site-specific monoclonal antibody

Human progesterone receptor (PR) is phosphorylated on multiple serine residues (at least seven sites) in a manner that involves distinct groups of sites coordinately regulated by hormone and different kinases. Progress on defining a functional role for PR phosphorylation has been hampered both by the complexity of phosphorylation and the lack of simple, nonradioactive methods to detect the influence of ligands and other signaling pathways on specific PR phosphorylation sites in vivo. Toward this end, we have produced monoclonal antibodies (MAbs) that recognize specific phosphorylation sites within human PR including a basal site at Ser 190 (MAb P190) and a hormone-induced site at Ser 294 (MAb P294). Biochemical experiments showed the differential reactivity of the P190 and P294 MAbs for phosphorylated and unphosphorylated forms of PR. Both MAbs recognize specific phosphorylated forms of PR under different experimental conditions including denatured PR protein by Western blots and PR in its native conformation in solution or complexed to specific target DNA. As detected by Western blot of T47D cells treated with hormone for different times, hormone-dependent down-regulation of total PR and the Ser 190 phosphorylation site occurred in parallel, whereas the Ser 294 phosphorylation site was down-regulated more rapidly. This difference in kinetics suggests that the Ser 294 site is more labile than basal sites and is acted upon by distinct phosphatases. A strong preferential hormone-dependent phosphorylation of Ser 294 was observed on PR-B as compared with the amino-terminal truncated A form of PR. This was unexpected because Ser 294 and flanking sequences are identical on both proteins, suggesting that a distinct conformation of the N-terminal domain of PR-A inhibits phosphorylation of this site. That Ser 294 lies within an inhibitory domain that mediates the unique repressive functions of PR-A raises the possibility that differential phosphorylation of Ser 294 is involved in the distinct functional properties of PR-A and PR-B.     

Protein abundance is key to distinguish promiscuous from functional phosphorylation based on evolutionary information

In eukaryotic cells, protein phosphorylation is an important and widespread mechanism used to regulate protein function. Yet, of the thousands of phosphosites identified to date, only a few hundred at best have a characterized function. It was recently shown that these functional sites are significantly more conserved than phosphosites of unknown function, stressing the importance of considering evolutionary conservation in assessing the global functional landscape of phosphosites. This leads us to review studies that examined the impact of phosphorylation on evolutionary conservation. While all these studies have shown that conservation is greater among phosphorylated sites compared with non-phosphorylated ones, the magnitude of this difference varies greatly. Further, not all studies have considered key factors that may influence the rate of phosphosite evolution. Such key factors are their localization in ordered or disordered regions, their stoichiometry or the abundance of their corresponding protein. Here we take into account all of these factors simultaneously, which reveals remarkable evolutionary patterns. First, while it is well established that protein conservation increases with abundance, we show that phosphosites partly follow an opposite trend. More precisely, Saccharomyces cerevisiae phosphosites present among abundant proteins are 1.5 times more likely to diverge in the closely related species Saccharomyces bayanus when compared with phosphosites present in the 5 per cent least abundant proteins. Second, we show that conservation is coupled to stoichiometry, whereby sites frequently phosphorylated are more conserved than those rarely phosphorylated. Finally, we provide a model of functional and noisy or 'accidental' phosphorylation that explains these observations.     

Kinetics of the two-sited enzyme. II. A method of distinguishing between anticooperative and independent active sites based on competitive inhibition

The presence of two active sites on an enzyme leads to downwardly curving Lineweaver-Burk plots if (A) the sites are independent, but have different Michaelis constants, or (B) if the sites interact anticooperatively to impair binding, but not catalysis, at the second site filled. Cases A and B are kinetically indistinguishable when only enzyme and substrate are present. However, equations derived by the rapid-equilibrium treatment show that the two cases have different patterns of competitive inhibition and become distinguishable in the presence of a suitable inhibitor. The inhibitor may decrease or increase the curvature of Lineweaver-Burk plots, but certain patterns have diagnostic value because they can occur only in case (B).In one type of diagnostic pattern, high concentrations of inhibitor cause the Lineweaver-Burk plots to curve upward, and cause the corresponding saturation curves to become sigmoid. The effect of the inhibitor is thus to make sites which are anticooperative appear to be cooperative. This suggests that the mere occurrence of sigmoid saturation curves is not necessarily evidence of cooperative binding effects, and may have uncertain significance in considerations of enzyme regulation.     

Identification of a CK2 phosphorylation site in mdm2.

Mdm2 is a cellular oncoprotein the most obvious function of which is the down-regulation of the growth suppressor protein p53. It represents a highly phosphorylated protein but only little is yet known about the sites phosphorylated in vivo, the kinases that are responsible for the phosphorylation or the functional relevance of the phosphorylation status. Recently, we have shown that mdm2 is a good substrate for protein kinase CK2 at least in vitro. Computer analysis of the primary amino acid sequence of mdm2 revealed 19 putative CK2 phosphorylation sites. By using deletion mutants of mdm2 and a peptide library we identified the serine residue at position 269 which lies within a canonical CK2 consensus sequence (EGQELSDEDDE) as the most important CK2 phosphorylation site. Moreover, by using the mdm2 S269A mutant for in vitro phosphorylation assays this site was shown to be phosphorylated by CK2. Binding studies revealed that phosphorylation of mdm2 at S269 does not have any influence on the binding of p53 to mdm2.     

Computational Analysis of the Predicted Evolutionary Conservation of Human Phosphorylation Sites

Protein kinase-mediated phosphorylation is among the most important post-translational modifications. However, few phosphorylation sites have been experimentally identified for most species, making it difficult to determine the degree to which phosphorylation sites are conserved. The goal of this study was to use computational methods to characterize the conservation of human phosphorylation sites in a wide variety of eukaryotes. Using experimentally-determined human sites as input, homologous phosphorylation sites were predicted in all 432 eukaryotes for which complete proteomes were available. For each pair of species, we calculated phosphorylation site conservation as the number of phosphorylation sites found in both species divided by the number found in at least one of the two species. A clustering of the species based on this conservation measure was concordant with phylogenies based on traditional genomic measures. For a subset of the 432 species, phosphorylation site conservation was compared to conservation of both protein kinases and proteins in general. Protein kinases exhibited the highest degree of conservation, while general proteins were less conserved and phosphorylation sites were least conserved. Although preliminary, these data tentatively suggest that variation in phosphorylation sites may play a larger role in explaining phenotypic differences among organisms than differences in the complements of protein kinases or general proteins.     

Positional oxygen isotope exchange as a probe for the mechanism of catalysis by Escherichia coli succinyl coenzyme A synthetase

Succinyl-CoA synthetase of Escherichia coli has an alpha 2 beta 2 subunit structure. The enzyme shows strict half-sites reactivity with respect to the phosphorylation of a histidine residue in the alpha subunit that represents a step in catalysis. Several lines of evidence indicate that this behavior may result from cooperative interactions between alternatingly functional active sites, so that subsequent steps in catalysis at one site may be promoted by phosphoryl transfer to the site on the neighboring half of the molecule. This study is directed toward learning more about the nature of these cooperative interactions. Here we have used positional isotope exchange (i.e., exchange of 18O between the beta, gamma bridge and the beta nonbridge position of ATP) as a test for transient bisphosphorylation. Succinyl-CoA synthetase was ATP) as a test for transient bisphosphorylation. Succinyl-CoA synthetase was prepared in which one of the two active sites was thiophosphorylated; this species thus has one of its two active-site histidine residues occupied and unavailable for further reaction with ATP. Treatment of this monothiophosphorylated enzyme with [beta, gamma-18O]ATP resulted in no significant scrambling of isotope into the nonbridge position, clearly indicating that the enzyme does not undergo even transient bisphosphorylation. We interpret the results in terms of a model of catalysis in which phosphoryl transfer to the second site occurs in concerted fashion with transfer from the first.     

Effect of diethyl pyrocarbonate modification on the calcium binding mechanism of the sarcoplasmic reticulum ATPase

Diethyl pyrocarbonate was used to modify histidyl residues on the sarcoplasmic reticulum ATPase. Difference spectra of the N-carbethoxyhistidyl derivative indicated that most all the histidyl residues on the enzyme had been modified. These residues could be divided into two populations on the basis of their reaction rate with the reagent. It could then be shown that enzyme inhibition followed modification of the slower reacting population. Reversal with hydroxylamine verified that the loss of activity was due specifically to histidyl modification. Using [32P]ATP as a substrate it was further determined that the modified ATPase could form a phosphoenzyme intermediate, but that the hydrolysis of this intermediate was inhibited. Size exclusion chromatography was used to obtain equilibrium binding curves for high affinity Ca2+ sites on the enzyme. With the normal ATPase a cooperative binding curve for two Ca2+ with a Hill coefficient of 1.8 was observed. With the modified ATPase binding to two independent sites was observed; however, the dissociation constants remained the same as in the cooperative mechanism (K1 = 14 microM; K2 = 0.5 microM). That is, modification had eliminated cooperativity without changing the site specific binding affinities. E-P formation was then shown to follow binding to the higher affinity of the two sites. This would be the second site to bind Ca2+ in a sequential, cooperative mechanism. A model is suggested in which the binding of Ca2+ to an initial site allows for the binding of a second Ca2+ to an occluded site, this second site being responsible for enzyme activation. Modification apparently allows the binding properties of both sites to be observed independently.     

Mixed mode of ligand-DNA binding results in S-shaped binding curves

S-shaped binding curves often characterize interactions of ligands with nucleic acid molecules as analyzed by different physico-chemical and biophysical techniques. S-shaped experimental binding curves are usually interpreted as indicative of the positive cooperative interactions between the bound ligand molecules. This paper demonstrates that S-shaped binding curves may occur as a result of the "mixed mode" of DNA binding by the same ligand molecule. Mixed mode of the ligand-DNA binding can occur, for example, due to 1) isomerization or dimerization of the ligands in solution or on the DNA lattice, 2) their ability to intercalate the DNA and to bind it within the minor groove in different orientations. DNA-ligand complexes are characterized by the length of the ligand binding site on the DNA lattice (so-called "multiple-contact" model). We show here that if two or more complexes with different lengths of the ligand binding sites could be produced by the same ligand, the dependence of the concentration of the complex with the shorter length of binding site on the total concentration of ligand should be S-shaped. Our theoretical model is confirmed by comparison of the calculated and experimental CD binding curves for bis-netropsin binding to poly(dA-dT) poly(dA-dT). Bis-netropsin forms two types of DNA complexes due to its ability to interact with the DNA as monomers and trimers. Experimental S-shaped bis-netropsin-DNA binding curve is shown to be in good correlation with those calculated on the basis of our theoretical model. The present work provides new insight into the analysis of ligand-DNA binding curves.     

Mixed Mode of Ligand-DNA Binding Results in S-Shaped Binding Curves

Abstract

S-shaped binding curves often characterize interactions of ligands with nucleic acid molecules as analyzed by different physicochemical and biophysical techniques. S-shaped experimental binding curves are usually interpreted as indicative of the positive cooperative interactions between the bound ligand molecules. This paper demonstrates that S-shaped binding curves may occur as a result of the “mixed mode” of DNA binding by the same ligand molecule. Mixed mode of the ligand-DNA binding can occur, for example, due to 1) isomerization or dimerization of the ligands in solution or on the DNA lattice, 2) their ability to intercalate the DNA and to bind it within the minor groove in different orientations. DNA- ligand complexes are characterized by the length of the ligand binding site on the DNA lattice (so-called “multiple-contact” model). We show here that if two or more complexes with different lengths of the ligand binding sites could be produced by the same ligand, the dependence of the concentration of the complex with the shorter length of binding site on the total concentration of ligand should be S-shaped. Our theoretical model is confirmed by comparison of the calculated and experimental CD binding curves for bis-netropsin binding to poly(dA-dT) poly(dA-dT). Bis-netropsin forms two types of DNA complexes due to its ability to interact with the DNA as monomers and trimers. Experimental S-shaped bis-netropsin-DNA binding curve is shown to be in good correlation with those calculated on the basis of our theoretical model. The present work provides new insight into the analysis of ligand-DNA binding curves.     

Identification of a phosphorylation site in the hinge region of the human progesterone receptor and additional amino-terminal phosphorylation sites

We have previously reported the identification of seven in vivo phosphorylation sites in the amino-terminal region of the human progesterone receptor (PR). From our previous in vivo studies, it was evident that several phosphopeptides remained unidentified. In particular, we wished to determine whether human PR contains a phosphorylation site in the hinge region, as do other steroid receptors including chicken PR, human androgen receptor, and mouse estrogen receptor. Previously, problematic trypsin cleavage sites hampered our ability to detect phosphorylation sites in large incomplete tryptic peptides. Using a combination of mass spectrometry and in vitro phosphorylation, we have identified six previously unidentified phosphorylation sites in human PR. Using nanoelectrospray ionization mass spectrometry, we have identified two new in vivo phosphorylation sites, Ser(20) and Ser(676), in baculovirus-expressed human PR. Ser(676) is analogous to the hinge site identified in other steroid receptors. Additionally, precursor ion scans identified another phosphopeptide that contains Ser(130)-Pro(131), a likely candidate for phosphorylation. In vitro phosphorylation of PR with Cdk2 has revealed five additional in vitro Cdk2 phosphorylation sites: Ser(25), Ser(213), Thr(430), Ser(554), and Ser(676). At least two of these, Ser(213) and Ser(676), are authentic in vivo sites. We confirmed the presence of the Cdk2-phosphorylated peptide containing Ser(213) in PR from in vivo labeled T47D cells, indicating that this is an in vivo site. Our combined studies indicate that most, if not all, of the Ser-Pro motifs in human PR are sites for phosphorylation. Taken together, these data indicate that the phosphorylation of PR is highly complex, with at least 14 phosphorylation sites.     

New model for activation of yeast pyruvate decarboxylase by substrate consistent with the alternating sites mechanism: demonstration of the existence of two active forms of the enzyme

Pyruvate decarboxylase from yeast (YPDC, EC 4.1.1.1) exhibits a marked lag phase in the progress curves of product (acetaldehyde) formation. The currently accepted kinetic model for YPDC predicts that, only upon binding of substrate in a regulatory site, a slow activation step converts inactive enzyme into the active form. This allosteric behavior gives rise to sigmoidal steady-state kinetics. The E477Q active site variant of YPDC exhibited hyperbolic initial rate curves at low pH, not consistent with the model. Progress curves of product formation by this variant were S-shaped, consistent with the presence of three interconverting conformations with distinct steady-state rates. Surprisingly, wild-type YPDC at pH < or =5.0 also possessed S-shaped progress curves, with the conformation corresponding to the middle steady state being the most active one. Reexamination of the activation by substrate of wild-type YPDC in the pH range of 4.5-6.5 revealed two characteristic transitions at all pH values. The values of steady-state rates are functions of both pH and substrate concentration, affecting whether the progress curve appears "normal" or S-shaped with an inflection point. The substrate dependence of the apparent rate constants suggested that the first transition corresponded to substrate binding in an active site and a subsequent step responsible for conversion to an asymmetric conformation. Consequently, the second enzyme state may report on "unregulated" enzyme, since the regulatory site does not participate in its generation. This enzyme state utilizes the alternating sites mechanism, resulting in the hyperbolic substrate dependence of initial rate. The second transition corresponds to binding a substrate molecule in the regulatory site and subsequent minor conformational adjustments. The third enzyme state corresponds to the allosterically regulated conformation, previously referred to as activated enzyme. The pH dependence of the Hill coefficient suggests a random binding of pyruvate in a regulatory and an active site of wild-type YPDC. Addition of pyruvamide or acetaldehyde to YPDC results in the appearance of additional conformations of the enzyme.     

A novel view of pH titration in biomolecules

When individual titratable sites in a molecule interact with each other, their pH titration can be considerably more complex than that of an independent site described by the classical Henderson-Hasselbalch equation. We propose a novel framework that decomposes any complex titration behavior into simple standard components. The approach maps the set of N interacting sites in the molecule onto a set of N independent, noninteracting quasi-sites, each characterized by a pK'(a) value. The titration curve of an individual site in the molecule is a weighted sum of Henderson-Hasselbalch curves corresponding to the quasi-sites. The total protonation curve is the unweighted sum of these Henderson-Hasselbalch curves. We show that pK'(a) values correspond to deprotonation constants available from methods that can be used to assess total proton uptake or release, and establish their connection to protonation curves of individual residues obtained by NMR or infrared spectroscopy. The new framework is tested on a small molecule diethylenetriaminepentaacetate (DTPA) exhibiting nonmonotonic titration curves, where it gives an excellent fit to experimental data. We demonstrate that the titration curve of a site in a group of interacting sites can be accurately reconstructed, if titration curves of the other sites are known. The application of the new framework to the protein rubredoxin demonstrates its usefulness in calculating and interpreting complicated titration curves.