Phosphorylated Amino Acid Residues Research Articles

Structural Alterations in the Nucleosome upon H3 Tail-Truncation Reveals a Crucial Role for the H2A C Terminal Docking Domain in Nucleosome Destabilization

The nucleosomal organization of eukaryotic chromatin offers a physical barrier to DNA access and also acts as a repository of epigenetic marks controlling chromosomal behavior during different periods of cell cycle. Post-translational modifications of histones play a key role in the regulation of gene access in eukaryotes. The majority of these modifications occur in the N-terminal extensions of the histone H3 in the form of methylation, acetylation or phosphorylation of amino acid residues. Here, we report on a total of 400 ns of all-atom molecular dynamics simulations of intact and tail-truncated nucleosomes and examine the effect of H3 tail truncation on nucleosome structure in atomic detail. During the intact nucleosome simulation one of the H3 tail domains showed propensity of alpha-helix formation. Upon truncation of the H3 tail containing the alpha-helical domain structural alterations occurred in the close by H2A(alpha)3 domain involving arginine residues and in the H2A C terminal docking domain suggesting a pathway for allosteric regulation of nucleosome stability. The relation between the present observations and corresponding experiments [1] is discussed.Reference:1. Ferreira et. al., Molecular and Cellular Biology 2007.

Nestin Protein Is Phosphorylated in Adult Neural Stem/Progenitor Cells and Not Endothelial Progenitor Cells

An intermediate filament protein, Nestin, is known as a neural stem/progenitor cell marker. It was shown to be required for the survival and self-renewal of neural stem cells according to the phenotypes of Nestin knockout mice. Nestin expression has also been reported in vascular endothelial cells, and we recently reported Nestin expression in proliferating endothelial progenitor cells, but not in mature endothelial cells. Using quantitative phosphoproteome analysis, we studied differences in phosphorylation levels between CNS Nestin in adult neural stem cells and vascular Nestin in adult bone-marrow-derived endothelial progenitor cells. We detected 495 phosphopeptides in the cell lysates of adult CNS stem/progenitor cells and identified 11 significant phosphorylated amino acid residues in the Nestin protein. In contrast, endothelial progenitor cells showed no significant phosphorylation of Nestin. We also measured neoplastic endothelial cells of the mouse brain and identified 13 phosphorylated amino acid residues in the Nestin protein. Among the 11 phosphorylated amino acids of adult CNS Nestin, five (S565, S570, S819, S883, and S886) were CNS Nestin-specific phosphorylation sites. Detection of the CNS-specific phosphorylation sites in Nestin, for example, by a phospho-specific Nestin antibody, may allow the expression of CNS Nestin to be distinguished from vascular Nestin.

Identification of new in vivo phosphosites on lamin Dm - the evidence of heterogeneity of phosphorylation sites in different Drosophila tissues

Changes in the nuclear structure and function during the cell cycle are thought to be correlated with lamins phosphorylation. Here, we report the identification of new in vivo phosphorylation sites on Drosophila melanogater lamin Dm using immunoisolation and mass spectrometry with collision-induced peptide fragmentation (Electrospray-Linear Trap Quadrupole- Fourier Transform Ion Cyclotron Resonance MS/MS). We identified S19 and confirmed previously suggested S595 as phosphorylated amino acid residues on embryonic lamin Dm. We also found that T597 is phosphorylated in vivo in cultured Kc cells while S595 in embryos, which suggests that different neighboring phosphoacceptors may be modified within the same region. We demonstrate also that Drosophila melanogaster lamin Dm in very early (syncytial) embryos is almost completely dispersed through the entire embryo. Only fraction of lamin Dm is associated with nuclei and nuclear envelopes. In later stages, due to the synchronization of mitosis, lamin Dm may be both nuclear and cytoplasmic in the same embryo. Our results provide a new and essential data for better understanding of the lamin phosporylation in development and cell cycle regulation in Drosophila.

Mass spectrometric analysis of protein phosphorylation

Phosphorylation is one of the most common posttranslational modifications of proteins in eukaryotic cells; it plays an important role in a wide spectrum of biological processes. This makes its study an important task for understanding cell functioning mechanisms. The aim of phosphoproteomics is a global mass spectral analysis of the phosphoprotein composition of cells, i.e., phosphoproteome. Nowadays, new effective methods are actively developed, which succeed not only in the detection of phosphorylated proteins but also in the determination of phosphorylated amino acid residues (phosphorylation sites) and in the quantitative comparison of phosphorylation among several specimens. Despite the analysis of protein phosphorylation remains a complicated problem, the available methods nowadays allow the detection of thousands of phosphorylation sites in the very same experiment. The present review covers the main methods utilized in contemporary phosphoproteomics: phosphoprotein and phosphopeptides enrichment as well as the mass spectrometric analysis of protein phosphorylation.

Identification of phosphorylation sites of equine β‐casein isoforms

AbstractEquine β‐casein is phosphorylated at variable degrees and isoforms carrying 3 to 7 phosphate groups (3P–7P) have been found in milk, but the phosphorylated amino acid residues of each isoform are not yet identified. In the present work, the different phosphorylation variants were first isolated by ion‐exchange chromatography and then hydrolysed by trypsin to generate caseinophosphopeptides (CPPs), each containing all the potential phosphorylation sites. The equine CPPs were prepared by metal oxide affinity chromatography, a method based on the affinity of phosphate groups towards titanium dioxide immobilized onto a micro‐column. This method turned out to be an efficient tool to separate the CPPs Arg1–Lys34 and Glu4–Lys34 from non‐phosphorylated peptides. Purification was achieved by reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and each CPP was hydrolyzed by endoproteinase Glu‐C. Finally, the digests were analyzed by RP‐HPLC/electrospray ionization mass spectrometry (RP‐HPLC/ESI‐MS) and identified by nano‐electrospray ionization tandem mass spectrometry (nESI‐MS/MS) to locate the phosphorylated sites of the β‐casein isoforms 4P–7P with accuracy. Thus, the isoform 4P was found to be phosphorylated on residues Ser9, Ser23, Ser24, and Ser25. Addition of phosphate groups on Ser18, Thr12, and Ser10 led to the formation of the isoforms 5P–7P, respectively. The results indicated that the in vivo phosphorylation of the equine β‐casein follows a sequential way and is not randomly performed. Copyright © 2010 John Wiley & Sons, Ltd.

The Cytotoxic Pathway Triggered by Palytoxin Involves a Change in the Cellular Pool of Stress Response Proteins

We have analyzed the proteome of MCF-7 cells exposed to palytoxin (PlTX), to characterize protein components involved in the death response induced by the toxin. The protein profiles of cell lysates were obtained by two-dimensional (2D) electrophoresis, and we found that four components were increased by PlTX treatment. By tryptic digestion of protein spots in the gels and LC-ESI-MS/MS analysis of resulting peptides, those four components were found to include three isoforms of the heat shock protein (hsp) 27 differing with regard to their phosphrylation state, as well as DJ-1/PARK7. The effects exerted by PlTX on hsp 27 and DJ-1 proteins were further quantified by immunoblotting analyses of proteins separated by monodimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and using antibodies recognizing total hsp 27, the hsp 27 forms phosphorylated in Ser(82), and DJ-1 protein. Dose-response and time-course experiments yielded results that only partially confirmed those found by protein staining after 2D electrophoresis. These findings were further checked by immunoblotting of proteins after fractionation by 2D electrophoresis, and we found that only some forms of those comigrating in a single band upon monodimensional SDS-PAGE were actually increased in extracts from PlTX-treated cells. We obtained evidence that the three hsp 27 isoforms whose relative abundance was increased in MCF-7 cells exposed to PlTX comprised two proteins phosphorylated in Ser(82), whereas the third form most likely contains a phosphorylated amino acid residue other than Ser(82). Moreover, we could show that PlTX treatment determined the accumulation of an oxidized isoform of DJ-1 in MCF-7 cells. We conclude that the toxicity pathway of PlTX in MCF-7 cells involves post-translational modifications of hsp 27 and DJ-1 stress response proteins, comprising a shift in the equilibria among hsp 27 isoforms toward those phosphorylated in Ser(82), as well as the oxidation of DJ-1.

Binding of phosphorylated peptides and inhibition of their interaction with disease‐relevant human proteins by synthetic metal‐chelate receptors

The modulation of biological signal transduction pathways by masking phosphorylated amino acid residues represents a viable route toward pharmacologic protein regulation. Binding of phosphorylated amino acid residues has been achieved with synthetic metal-chelate receptors. The affinity and selectivity of such receptors can be enhanced if combined with a second binding site. We demonstrate this principle with a series of synthetic ditopic metal-chelate receptors, which were synthesized and investigated for their binding affinity to phosphorylated short peptides under conditions of physiological pH. The compounds showing highest affinity were subsequently used to inhibit the interaction of the human STAT1 protein to a peptide derived from the interferon-gamma receptor, and between the checkpoint kinase Chk2 and its preferred binding motif. Two of the investigated ditopic synthetic receptors show a significant increase in inhibition activity. The results show that regulation of protein function by binding to phosphorylated amino acids is possible. The introduction of additional binding sites into the synthetic receptors increases their affinity, but the flexibility of the structures investigated so far prohibited stringent amino acid sequence selectivity in peptide binding.

Ala Scanning of the Inhibitory Region of Cardiac Troponin I

In skeletal and cardiac muscles, troponin (Tn), which resides on the thin filament, senses a change in intracellular Ca(2+) concentration. Tn is composed of TnC, TnI, and TnT. Ca(2+) binding to the regulatory domain of TnC removes the inhibitory effect by TnI on the contraction. The inhibitory region of cardiac TnI spans from residue 138 to 149. Upon Ca(2+) activation, the inhibitory region is believed to be released from actin, thus triggering actin-activation of myosin ATPase. In this study, we created a series of Ala-substitution mutants of cTnI to delineate the functional contribution of each amino acid in the inhibitory region to myofilament regulation. We found that most of the point mutations in the inhibitory region reduced the ATPase activity in the presence of Ca(2+), which suggests the same region also acts as an activator of the ATPase. The thin filaments can also be activated by strong myosin head (S1)-actin interactions. The binding of N-ethylmaleimide-treated myosin subfragment 1 (NEM-S1) to actin filaments mimics such strong interactions. Interestingly, in the absence of Ca(2+) NEM-S1-induced activation of S1 ATPase was significantly less with the thin filaments containing TnI(T144A) than that with the wild-type TnI. However, in the presence of Ca(2+), there was little difference in the activation of ATPase activity between these preparations.

Fluorescent BODIPY-Based Zn(II) Complex as a Molecular Probe for Selective Detection of Neurofibrillary Tangles in the Brains of Alzheimer’s Disease Patients

We have developed a new fluorescent binuclear Zn(II) complex for the detection of neurofibrillary tangles (NFTs) of hyperphosphorylated tau proteins, a representative hallmark of Alzheimer's disease (AD). The probe 1 incorporates a fluorescent BODIPY unit and two Zn(II)-2,2'-dipicolylamine (Dpa) complexes as a binding site for phosphorylated amino acid residues. Using fluorescence titration to evaluate the binding and sensing properties of 1 toward several phosphorylated peptide segments derived from hyperphosphorylated tau protein, we found that 1 binds preferentially to peptides presenting phosphorylated groups at the i and i+4 positions with dissociation constants (K(d)) in the micromolar range. Fluorescence titration with an artificially prepared aggregate of the phosphorylated tau protein (p-Tau) revealed that 1 binds strongly to p-Tau (EC(50) = 9 nM). In contrast, the interactions of 1 were weaker toward artificially prepared aggregates of the nonphosphorylated tau protein (n-Tau; EC(50) = 80 nM) and Abeta(1-42) fibrils (EC(50) = 650 nM). Histological imaging of a hippocampus tissue section obtained from an AD patient revealed that 1 fluorescently visualizes deposits of NFTs and clearly discriminates between NFTs and the amyloid plaques assembled from amyloid-beta peptides, confirming our strategy toward the rational design of a molecular probe for the selective fluorescence detection of NFTs in brain tissue sections.

Detection of Protein Phosphorylation on SDS-PAGE Using Probes with a Phosphate-Sensitive Emission Response

Fluorescent probes for the detection of protein phosphorylation on SDS-PAGE are presented. The probes were designed using a dinuclear metal-chelate phosphate recognition unit and an environmentally sensitive fluorophore. The specificity of the probes is determined by their binding site selectivity toward phosphate ions and the emission response induced by the change in the electrostatic environment of the fluorophore upon binding to a phosphorylated amino acid residue. The staining is fully reversible due to the noncovalent binding of the probe. Gel bands with less than 100 ng of phosphorylated alpha-casein are detectable with the new probes on a normal UV-table without specialized equipment like a laser-based gel scanner or a cooled camera detector.

Phosphorylation of the Consensus Sites of Protein Kinase A on α1D L-type Calcium Channel

The novel alpha(1D) L-type Ca(2+) channel is expressed in supraventricular tissue and has been implicated in the pacemaker activity of the heart and in atrial fibrillation. We recently demonstrated that PKA activation led to increased alpha(1D) Ca(2+) channel activity in tsA201 cells by phosphorylation of the channel protein. Here we sought to identify the phosphorylated PKA consensus sites on the alpha(1) subunit of the alpha(1D) Ca(2+) channel by generating GST fusion proteins of the intracellular loops, N terminus, proximal and distal C termini of the alpha(1) subunit of alpha(1D) Ca(2+) channel. An in vitro PKA kinase assay was performed for the GST fusion proteins, and their phosphorylation was assessed by Western blotting using either anti-PKA substrate or anti-phosphoserine antibodies. Western blotting showed that the N terminus and C terminus were phosphorylated. Serines 1743 and 1816, two PKA consensus sites, were phosphorylated by PKA and identified by mass spectrometry. Site directed mutagenesis and patch clamp studies revealed that serines 1743 and 1816 were major functional PKA consensus sites. Altogether, biochemical and functional data revealed that serines 1743 and 1816 are major functional PKA consensus sites on the alpha(1) subunit of alpha(1D) Ca(2+) channel. These novel findings provide new insights into the autonomic regulation of the alpha(1D) Ca(2+) channel in the heart.

Characteristic negative ion fragmentations of deprotonated peptides containing post‐translational modifications: mono‐phosphorylated Ser, Thr and Tyr. A joint experimental and theoretical study

Peptides and proteins may contain post-translationally modified phosphorylated amino acid residues, in particular phosphorylated serine (pSer), threonine (pThr) and tyrosine (pTyr). Following earlier work by Lehmann et al., the [M-H]- anions of peptides containing pSer and pThr functionality show loss of the elements of H3PO4. This process, illustrated for Ser (and using a model system), is CH3CONH-C(CH2OPO3H2)CONHCH(3) --> [CH3CONHC(==CH2)CONHCH3 (-OPO3H2)] (a) --> [CH3CONHC(==CH2)CONHCH3-H]- + H3PO4, a process endothermic by 83 kJ mol(-1) at the MP2/6-31++G(d,p)//HF/6-31++G(d,p) level of theory. In addition, intermediate (a) may decompose to yield CH3CONHC(==CH2)CONHCH3 + H2PO4 - in a process exothermic by 3 kJ mol(-1). The barrier to the transition state for these two processes is 49 kJ mol(-1). Characteristic cleavages of pSer and pThr are more energetically favourable than the negative ion backbone cleavages of peptides described previously. In contrast, loss of HPO3 from [M-H]- is characteristic of pTyr. The cleavage [NH2CH(CH2-C6H4-OPO3H-)CO2H] --> [NH2C(CH2-C6H4-O-)CO2H (HPO3)] (b) --> NH2CH(CH2-C6H4-O-)CO2H + HPO3 is endothermic by 318 kJ mol(-1) at the HF/6-31+G(d)//AM1 level of theory. In addition, intermediate (b) also yields NH2CH(CH2-C6H4-OH)CO2H + PO3 - (reaction endothermic by 137 kJ mol(-1)). The two negative ion cleavages of pTyr have a barrier to the transition state of 198 kJ mol(-1) (at the HF/6-31+G(d)//AM1 level of theory) comparable with those already reported for negative ion backbone cleavages.

The Role of Phosphorylated Residues in Peptide–Peptide Noncovalent Complexes Formation

Electrospray mass spectrometry (ESI-MS) has become the tool of choice for the study of noncovalent complexes. Our previous work has highlighted the role of phosphorylated amino acid residues in the formation of noncovalent complexes through electrostatic interaction with arginine residues' guanidinium groups. In this study, we employ tandem mass spectrometry to investigate the gas-phase stability and dissociation pathways of these noncovalent complexes. The only difference in the three phosphopeptides tested is the nature of the phosphorylated amino acid residue. In addition the absence of acidic residues and an amidated carboxyl terminus insured that the only negative charge came from the phosphate, which allowed for the comparison of the noncovalent bond between arginine residues and each of the different phosphorylated residues. Dissociation curves were generated by plotting noncovalent complex ion intensities as a function of the nominal energy given to the noncovalent complex ion before entering the collision cell. These results showed that noncovalent complexes formed with phosphorylated tyrosine were the most stable, followed by serine and threonine, which had similar stability.

Adrenergic beta receptors in anesthesia and intensive care medicine

beta-Adrenoceptors (beta-AR) are divided into a beta 1-, a beta 1-, and a recently cloned beta 3-subtype. beta-AR belong to the group of 7-transmembrane spanners which are coupled via GS-proteins to the enzyme adenylyl cyclase. Instead of being static entities, beta-AR undergo dynamic regulation upon repeated or prolonged exposure to agonists (down-regulation) or antagonists (up-regulation). After exposure to agonists, an early desensitisation caused by phosphorylation of amino acid residues can be distinguished from receptor sequestration. Finally, the internalised receptor is digested by proteolytic enzymes. The present article describes the basic patterns of molecular biology of beta-AR, signal transduction, and receptor desensitisation. In the human heart, beta-AR are the most important receptor system to provide positive inotropic and chronotropic effects. The alterations of the beta-adrenergic system in cardiac insufficiency, after heart transplantation, and after extracorporeal circulation, as well as changes induced by surgery and single anaesthetic agents, are reviewed.

Identification of in Vivo Phosphorylation Sites and Their Functional Significance in the Sodium Iodide Symporter

The Na+/I- symporter (NIS)-mediated iodide uptake activity is the basis for targeted radioiodide ablation of thyroid cancers. Although it has been shown that NIS protein is phosphorylated, neither the in vivo phosphorylation sites nor their functional significance has been reported. In this study, Ser-43, Thr-49, Ser-227, Thr-577, and Ser-581 were identified as in vivo NIS phosphorylation sites by mass spectrometry. Kinetic analysis of NIS mutants of the corresponding phosphorylated amino acid residue indicated that the velocity of iodide transport of NIS is modulated by the phosphorylation status of Ser-43 and Ser-581. We also found that the phosphorylation status of Thr-577 may be important for NIS protein stability and that the phosphorylation status of Ser-227 is functionally silent. Thr-49 appears to be critical for proper local structure/conformation of NIS because mutation of Thr-49 to alanine, aspartic acid, or serine results in reduced NIS activity without alterations in total or cell surface NIS protein levels. Taken together, we showed that NIS protein levels and functional activity could be modulated by phosphorylation through distinct mechanisms.

Determination of phosphorylated amino acid residues of Rab8 from Bombyx mori

The Rab family of small GTPases are key regulators of membrane trafficking. Partially purified Rab8 from Bombyx mori (BRab8) was phosphorylated by protein kinase C in mammalian cells in vitro. To determine which of the seven serines and four threonines are phosphorylated, we generated deletion and site-directed mutants of BRab8, inserted them in Escherichia coli, partially purified the encoded fusion proteins by affinity chromatography, and examined their phosphorylation by protein kinase C in vitro. We found that Ser-132 of BRab8 was specifically phosphorylated by protein kinase C. In addition, Western blotting using an antiserum against BRab8 and in-gel staining for phosphorylated proteins revealed that BRab8 is phosphorylated in vivo.

Yeast two-hybrid analysis of the origin recognition complex ofSaccharomyces cerevisiae: interaction between subunits and identification of binding proteins

Origin recognition complex (ORC), a six-protein complex (Orc1p-6p), is the most likely initiator of chromosomal DNA replication in eukaryotes. Although ORC of Saccharomyces cerevisiae has been studied extensively from biochemical and genetic perspectives, its quaternary structure remains unknown. Previous studies suggested that ORC has functions other than DNA replication, such as gene silencing, but the molecular mechanisms of these functions have not been determined. In this study, we used yeast two-hybrid analysis to examine the interaction between ORC subunits and to search for ORC-binding proteins. As well as the known Orc4p-Orc5p interaction, we revealed strong interactions between Orc2p and Ord3p (2p-3p), Orc2p and Ord5p (2p-5p), Orc2p and Ord6p (2p-6p) and Orc3p and Ord6p (3p-6p) and weaker interactions between Orc1p and Ord4p (1p-4p), Orc3p and Ord4p (3p-4p), Orc2p and Ord3p (3p-5p) and Orc5p and Ord3p (5p-6p). These results suggest that 2p-3p-6p may form a core complex. Orc2p and Orc6p are phosphorylated in vivo, regulating initiation of DNA replication. However, replacing the phosphorylated amino acid residues with others that cannot be phosphorylated, or that mimic phosphorylation, did not affect subunit interactions. We also identified several proteins that interact with ORC subunits; Sir4p and Mad1p interact with Orc2p; Cac1p and Ykr077wp with Orc3p; Rrm3p and Swi6p with Orc5p; and Mih1p with Orc6p. We discuss roles of these interactions in functions of ORC.

Liquid chromatographic analysis of phosphoamino acids at femtomole level using chemical derivatization with N-hydroxysuccinimidyl fluorescein- O-acetate

Phosphorylation of amino acid residues in proteins plays a major role in biological systems. In this paper, a reversed-phase high performance liquid chromatographic (HPLC) method based on chemical derivatization has been described for the separation and quantification of phosphoamino acids at femtomole level, using fluorimetric detection (FLD). The protocol involved pre-column derivatization of phosphoamino acids with N-hydroxysuccinimidyl fluorescein- O-acetate (SIFA) and subsequent separation on ZORBAX Eclipse XDB-C8 column. Several experimental factors that influenced derivatization and separation were carefully investigated. The derivatization was performed at 40 °C for 40 min in borate buffer (pH 8.5). Under the optimum conditions, phosphoserine (P-Ser), phosphothreonine (P-Thr) and phosphotyrosine (P-Tyr) were satisfactorily separated in 8 min. The detection limits (signal-to-noise ratio = 3) for the phosphoamino acids could reach 10–20 fmol, which was the lowest value reported for HPLC methods and comparable to those obtained by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection methods. The proposed method has been validated and used to characterize the phosphoamino acids in the hydrolyzed phosphorylated protein samples. The results clearly demonstrated the potential of this technique to study phosphoamino acids as well as provided a new analytical methodology that should be applicable to the study of phosphorylation of protein in biological system.

Phosphorylation of the 12 S globulin cruciferin in wild-type and abi1-1 mutant Arabidopsis thaliana (thale cress) seeds

Cruciferin (a 12 S globulin) is the most abundant storage protein in the seeds of Arabidopsis thaliana (thale cress) and other crucifers, sharing structural similarity with the cupin superfamily of proteins. Cruciferin is synthesized as a precursor in the rough endoplasmic reticulum. Subunit assembly is accompanied by structural rearrangements involving proteolysis and disulfide-bond formation prior to deposition in protein storage vacuoles. The A. thaliana cv. Columbia genome contains four cruciferin loci, two of which, on the basis of cDNA analysis, give rise to three alternatively spliced variants. Using MS, we confirmed the presence of four variants encoded by genes At4g28520.1, At5g44120.3, At1g03880.1 and At1g3890.1 in A. thaliana seeds. Two-dimensional gel electrophoresis, along with immunological detection using anti-cruciferin antiserum and antibodies against phosphorylated amino acid residues, revealed that cruciferin was the major phosphorylated protein in Arabidopsis seeds and that polymorphism far exceeded that predicted on the basis of known isoforms. The latter may be attributed, at least in part, to phosphorylation site heterogeneity. A total of 20 phosphorylation sites, comprising nine serine, eight threonine and three tyrosine residues, were identified by MS. Most of these are located on the IE (interchain disulfide-containing) face of the globulin trimer, which is involved in hexamer formation. The implications of these findings for cruciferin processing, assembly and mobilization are discussed. In addition, the protein phosphatase 2C-impaired mutant, abi1-1, was found to exhibit increased levels of cruciferin phosphorylation, suggesting either that cruciferin may be an in vivo target for this enzyme or that abi1-1 regulates the protein kinase/phosphatase system required for cruciferin phosphorylation.

Use of Intein-Mediated Phosphoprotein Arrays to Study Substrate Specificity of Protein Phosphatases

Synthetic peptides incorporating various chemical moieties, for example, phosphate groups, are convenient tools for investigating protein modification enzymes, such as protein phosphatases (PPs). However, short peptides are sometimes poor substrates, and their binding to commonly used matrices is unpredictable and variable. In general, protein substrates for PPs are superior for enzymatic assays, binding to various matrices, and Western blot analysis. The preparation and characterization of phosphoproteins, however can be difficult and technically demanding. In this study, the intein-mediated protein ligation (IPL) technique was used to readily generate phosphorylated protein substrates by ligating a synthetic phosphopeptide to an intein-generated carrier protein (CP) possessing a carboxyl-terminal thioester with a one-to-one stoichiometry. The ligated phosphoprotein (LPP) substrate was treated with a PP and subsequently subjected to array or Western blot analysis with a phospho-specific antibody. This approach is highly effective in producing arrays of protein substrates containing phosphorylated amino acid residues and has been applied for screening of PPs with specificity toward phosphorylated tyrosine, serine, or threonine residues, resulting in an approximately 240-fold increase in sensitivity in dot blot analysis compared with the use of synthetic peptides. The IPL technique overcomes the disadvantages of current methods and is a versatile system for the facile production of protein substrates containing well-defined structural motifs for the study of protein modification enzymes.