Phosphorylated Amino Acid Research Articles

Forcefield_PTM: Ab Initio Charge and AMBER Forcefield Parameters for Frequently Occurring Post-Translational Modifications.

In this work, we introduce Forcefield_PTM, a set of AMBER forcefield parameters consistent with ff03 for 32 common post-translational modifications. Partial charges were calculated through ab initio calculations and a two-stage RESP-fitting procedure in an ether-like implicit solvent environment. The charges were found to be generally consistent with others previously reported for phosphorylated amino acids, and trimethyllysine, using different parameterization methods. Pairs of modified and their corresponding unmodified structures were curated from the PDB for both single and multiple modifications. Background structural similarity was assessed in the context of secondary and tertiary structures from the global dataset. Next, the charges derived for Forcefield_PTM were tested on a macroscopic scale using unrestrained all-atom Langevin molecular dynamics simulations in AMBER for 34 (17 pairs of modified/unmodified) systems in implicit solvent. Assessment was performed in the context of secondary structure preservation, stability in energies, and correlations between the modified and unmodified structure trajectories on the aggregate. As an illustration of their utility, the parameters were used to compare the structural stability of the phosphorylated and dephosphorylated forms of OdhI. Microscopic comparisons between quantum and AMBER single point energies along key χ torsions on several PTMs were performed and corrections to improve their agreement in terms of mean squared errors and squared correlation coefficients were parameterized. This forcefield for post-translational modifications in condensed-phase simulations can be applied to a number of biologically relevant and timely applications including protein structure prediction, protein and peptide design, docking, and to study the effect of PTMs on folding and dynamics. We make the derived parameters and an associated interactive webtool capable of performing post-translational modifications on proteins using Forcefield_PTM available at http://selene.princeton.edu/FFPTM.

A 2D gel electrophoresis-based snapshot of the phosphoproteome in the cyanobacterium Synechocystis sp. strain PCC 6803

Cyanobacteria are photoautotrophic prokaryotes that occur in highly variable environments. Protein phosphorylation is one of the most widespread means to adjust cell metabolism and gene expression to the demands of changing growth conditions. Using a 2D gel electrophoresis-based approach and a phosphoprotein-specific dye, we investigated the protein phosphorylation pattern in cells of the model cyanobacterium Synechocystis sp. strain PCC 6803. The comparison of gels stained for total and phosphorylated proteins revealed that approximately 5 % of the protein spots seemed to be phosphoproteins, from which 32 were identified using MALDI-TOF MS. For eight of them the phosphorylated amino acid residues were mapped by subsequent mass spectrometric investigations of isolated phosphopeptides. Among the phosphoproteins, we found regulatory proteins, mostly putative anti-sigma factor antagonists, and proteins involved in translation. Moreover, a number of enzymes catalysing steps in glycolysis or the Calvin-Benson cycle were found to be phosphorylated, implying that protein phosphorylation might represent an important mechanism for the regulation of the primary carbon metabolism in cyanobacterial cells.

Phosphorylation Influences The Binding Of Bim To Anti-Apoptotic Proteins In Multiple Myeloma

Multiple myeloma cells are strongly dependent on the anti-apoptotic protein Mcl-1 for survival. However, several studies have shown that some myeloma cells are sensitive to treatment with ABT-737 and related compounds, which target Bcl-2 and Bcl-xL, but not Mcl-1. These studies suggest that sensitive cells have more of the direct-activating, pro-apoptotic protein Bim bound to Bcl-2 and Bcl-xL than to Mcl-1. We have previously shown that the expression levels of pro- and anti-apoptotic proteins have little influence on which anti-apoptotic protein binds Bim, leading us to further explore what factors influence the preferential binding of Bim. We have found that IL-6 stimulation of myeloma cells results in both the phosphorylation of Bim at serine 69, and a shift in Bim from Bcl-2 and Bcl-xL to Mcl-1. These observations suggest a role for phosphorylation in determining the anti-apoptotic protein that Bim binds to. To determine if constitutive phosphorylation of Bim regulates the steady state binding of Bim to anti-apoptotic Bcl-2 family members, we utilized phospho-affinity gel electrophoresis (Phos-Tag). We determined that Bim is constitutively phosphorylated in unstimulated myeloma cells. However, unlike what is observed following IL-6 stimulation, phosphorylation does not occur at serine 69. Through use of co-immunopreciptation studies, we found that phosphorylation status of Bim affects its binding to anti-apoptotic proteins. The phosphorylated species are preferentially bound to Bcl-2 and Bcl-xL, and not to Mcl-1. Additionally, we observed that phosphorylated Bim species bound to Bcl-2 and Bcl-xL were present at a significantly lower level in myeloma cells that were more dependent on Mcl-1, such as OPM2, than in KMS18 cells, which exhibited greater Bcl-2/Bcl-xL dependency. This pattern also held true for myeloma cells that had been selected for resistance to ABT-737. ABT-737 resistant KMS18 cells had no detectable phosphorylated species of Bim, and all of the Bim was bound to Mcl-1. We have begun to identify the phosphorylated amino acids in Bim through the use of phospho-Bim-specific antibodies. In addition to serine 69, we have determined that the constitutive phosphorylation does not occur at serine 59 or serine 87. However, preliminary data suggests that Bim phosphorylated at threonine 116 preferentially binds to Bcl-xL in KMS18 cells. We have also taken initial steps to identify signaling pathways and kinases that are phosphorylating Bim, and altering its affinity. We treated KMS18 myeloma cells for 12 hours with 10 μM of the MEK inhibitor U0126, the JNK inhibitor SP600125, or the p38 inhibitor SB203580, and ran lysates from each of these conditions on PhosTag gels. While U0126 and SB203580 treatment did not result in the loss of any phosphorylated Bim species, treatment with SP600125 resulted in a loss of phosphorylated Bim species comparable to treatment of myeloma cell lysates with lambda protein phosphatase. These results suggest that JNK mediates phosphorylation of Bim in myeloma cells. Interestingly, threonine 116 is contained within a JNK consensus sequence. Therefore, JNK could directly influence the preferential binding of Bim. Together, these results suggest that combining Bim kinase inhibitors with agents that result in decreased Mcl-1 (e.g. CDK9 inhibitors) or inducers of the Mcl-1 inhibitor Noxa (proteasome inhibitors) represents a novel approach in cancer cells that demonstrate Mcl-1 dependence. Disclosures: Boise: Onyx Pharmaceuticals: Consultancy.

Phosphorylation of the N‐ and C‐terminal UPF1 domains plays a critical role in plant nonsense‐mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is an essential quality control system that degrades aberrant transcripts containing premature termination codons and regulates the expression of several normal transcripts. Targets for NMD are selected during translational termination. If termination is slow, the UPF1 NMD factor binds the eRF3 protein of the termination complex and then recruits UPF2 and UPF3. Consequently, the UPF1-2-3 NMD complex induces SMG7-mediated degradation of the target mRNA. It is unknown how formation of the NMD complex and transcript degradation are linked in plants. Previously we have shown that the N- and C-terminal domains of UPF1 act redundantly and that the N-terminal domain is phosphorylated. To clarify the role of UPF1 phosphorylation in plant NMD, we generated UPF1 mutants and analyzed their phosphorylation status and the NMD competency of the mutants. We show that although several residues in the N-terminal domain of UPF1 are phosphorylated, only three phosphorylated amino acids, S3, S13 and T29, play a role in NMD. Moreover, we found that the C-terminal domain consists of redundant S/TQ-rich segments and that S1076 is involved in NMD. All NMD-relevant phosphorylation sites were in the S/TQ context. Co-localization and fluorescence resonance energy transfer-fluorescence lifetime imaging assays suggest that N-terminal and probably also C-terminal phosphorylated S/TQ residues are the binding platform for SMG7. Our data support the hypothesis that phosphorylation of UPF1 connects NMD complex formation and the SMG7-mediated target transcript degradation steps of NMD. SMG7 binds the phosphorylated S/TQ sites of the UPF1 component of the NMD complex, and then it induces the degradation of the NMD target.

Identification and functional analysis of novel phosphorylation sites in the RNA surveillance protein Upf1

One third of inherited genetic diseases are caused by mRNAs harboring premature termination codons as a result of nonsense mutations. These aberrant mRNAs are degraded by the Nonsense-Mediated mRNA Decay (NMD) pathway. A central component of the NMD pathway is Upf1, an RNA-dependent ATPase and helicase. Upf1 is a known phosphorylated protein, but only portions of this large protein have been examined for phosphorylation sites and the functional relevance of its phosphorylation has not been elucidated in Saccharomyces cerevisiae. Using tandem mass spectrometry analyses, we report the identification of 11 putative phosphorylated sites in S. cerevisiae Upf1. Five of these phosphorylated residues are located within the ATPase and helicase domains and are conserved in higher eukaryotes, suggesting a biological significance for their phosphorylation. Indeed, functional analysis demonstrated that a small carboxy-terminal motif harboring at least three phosphorylated amino acids is important for three Upf1 functions: ATPase activity, NMD activity and the ability to promote translation termination efficiency. We provide evidence that two tyrosines within this phospho-motif (Y-738 and Y-742) act redundantly to promote ATP hydrolysis, NMD efficiency and translation termination fidelity.

Incorporating substrate sequence motifs and spatial amino acid composition to identify kinase-specific phosphorylation sites on protein three-dimensional structures

BackgroundProtein phosphorylation catalyzed by kinases plays crucial regulatory roles in cellular processes. Given the high-throughput mass spectrometry-based experiments, the desire to annotate the catalytic kinases for in vivo phosphorylation sites has motivated. Thus, a variety of computational methods have been developed for performing a large-scale prediction of kinase-specific phosphorylation sites. However, most of the proposed methods solely rely on the local amino acid sequences surrounding the phosphorylation sites. An increasing number of three-dimensional structures make it possible to physically investigate the structural environment of phosphorylation sites.ResultsIn this work, all of the experimental phosphorylation sites are mapped to the protein entries of Protein Data Bank by sequence identity. It resulted in a total of 4508 phosphorylation sites containing the protein three-dimensional (3D) structures. To identify phosphorylation sites on protein 3D structures, this work incorporates support vector machines (SVMs) with the information of linear motifs and spatial amino acid composition, which is determined for each kinase group by calculating the relative frequencies of 20 amino acid types within a specific radial distance from central phosphorylated amino acid residue. After the cross-validation evaluation, most of the kinase-specific models trained with the consideration of structural information outperform the models considering only the sequence information. Furthermore, the independent testing set which is not included in training set has demonstrated that the proposed method could provide a comparable performance to other popular tools.ConclusionThe proposed method is shown to be capable of predicting kinase-specific phosphorylation sites on 3D structures and has been implemented as a web server which is freely accessible at http://csb.cse.yzu.edu.tw/PhosK3D/. Due to the difficulty of identifying the kinase-specific phosphorylation sites with similar sequenced motifs, this work also integrates the 3D structural information to improve the cross classifying specificity.

Why Has It Taken So Long to Learn What We Still Don’t Know?

Ca2+ is an essential ion and second messenger that plays a central role in determining membrane excitability and contractility in heart, while also participating in diverse and fundamental activities, such as gene transcription, cell metabolism and survival, vesicle secretion, learning, and memory. Voltage-gated Ca2+ channels (CaVx) are the primary entry point for extracellular Ca2+ to cross cell membranes and gain access to the intracellular space. In ventricular myocytes, CaV1.2 is the most important pathway for Ca2+ entry and provides a trigger for release of intracellular Ca2+ from the sarcoplasmic reticulum that activates myofilament crossbridge formation to grade the strength of each heart beat. Thus, improved understanding of Ca2+ homeostatic mechanisms, generally, and CaV1.2 regulation, specifically, are important goals for cardiovascular science. Article, see p 871 The increase in CaV1.2 current (ICa) following adrenergic receptor agonist stimulation is a fundamental aspect of fight or flight physiology, but the molecular mechanisms underlying this pathway are incompletely understood. Although Ca2+ was identified as a necessary ingredient for myocardial excitability by experiments by Sydney Ringer, over 100 years ago,1 knowledge of the signaling pathways that enhance cardiac contraction, in part, by increasing ICa has accrued more recently. Highlights in this discovery time line include the concept of receptors for extracellular chemical signals by Alquist,2 the identity of cAMP as a second messenger by Hardman et al,3 and enzymatic catalysis of amino acid phosphorylation by the cAMP kinase (protein kinase A [PKA]) to modify protein function by Kemp et al.4 Pioneering work by many investigators, including Mikami et al5 who first cloned the pore forming α-subunit of CaV1.2 and De Jongh et al6 who identified key biochemical features of several …

Epigenetic Basis of Molecular Changes in Animal Cells with Particular Regard to Embryonic Development – A Review / Epigenetyczne Podstawy Przemian Molekularnych Zachodzących W Komórkach Zwierzęcych, Ze Szczególnym Uwzględnieniem Rozwoju Embrionalnego – Artykuł Przeglądowy

Abstract Regulation of gene expression is a complex process. Epigenetics is the study of heritable changes in gene expression independently of DNA sequence. Epigenetic control of gene transcription is based on two main processes. The first is reversible DNA methylation, primarily of cytosine at position C5, rarely in position N3, or of adenine at position C6 (Xu et al., 2010). The second process is the change in chromatin structure and function by chemical modification of histones, including mainly methylation, acetylation, and phosphorylation of histone amino acids (Zamudio et al., 2008). During development and differentiation of cells, changes occur in DNA methylation of genes. After fertilization there are dynamic histone modifications and changes in DNA methylation in zygotes. Use of methylation sensitive restriction enzymes causes a global demethylation in the early embryonic stage (Sulewska et al., 2007 b). De novo methylation of CpG sites is followed by embryo implantation. Next, during gastrulation most genes are methylated except the tissue-specific genes. The last wave of de novo methylation takes place during the gametogenesis and is dependent on sex (Sulewska et al., 2007 b). The aim of this work is to review the current knowledge about epigenetic mechanism of molecular changes in animal cells with particular regard to embryonic development.

Internalisation and multiple phosphorylation of γ-Conglutin, the lupin seed glycaemia-lowering protein, in HepG2 cells

Lupin seed γ-Conglutin is a protein capable of reducing glycaemia in mammalians and increasing glucose uptake by model cells. This work investigated whether γ-Conglutin is internalised into the target cells and undergoes any covalent change during the process, as a first step to understanding its mechanism of action.To this purpose, γ-Conglutin-treated and untreated HepG2 cells were submitted to confocal and transmission electron microscopy. Immune-revelation of γ-Conglutin at various intervals revealed its accumulation inside the cytosol.In parallel, 2D-electrophoresis of the cell lysates and antibody reaction of the blotted maps showed the presence of the protein intact subunits inside the treated cells, whilest no trace of the protein was found in the control cells. However, γ-Conglutin-related spots with an unexpectedly low pI were also observed in the maps. These spots were excised, trypsin-treated and submitted to MS/MS spectrometric analysis. The presence of phosphorylated amino acids was detected.These findings, by showing that γ-Conglutin is internalised by HepG2 cells in an intact form and is modified by multiple phosphorylation, open the way to the understanding of the lupin γ-Conglutin insulin-mimetic activity.

Mutation of Light-dependent Phosphorylation Sites of the Drosophila Transient Receptor Potential-like (TRPL) Ion Channel Affects Its Subcellular Localization and Stability

The Drosophila phototransduction cascade terminates in the opening of the ion channel transient receptor potential (TRP) and TRP-like (TRPL). Contrary to TRP, TRPL undergoes light-dependent subcellular trafficking between rhabdomeric photoreceptor membranes and an intracellular storage compartment, resulting in long term light adaptation. Here, we identified in vivo phosphorylation sites of TRPL that affect TRPL stability and localization. Quantitative mass spectrometry revealed a light-dependent change in the TRPL phosphorylation pattern. Mutation of eight C-terminal phosphorylation sites neither affected multimerization of the channels nor the electrophysiological response of flies expressing the mutated channels. However, these mutations resulted in mislocalization and enhanced degradation of TRPL after prolonged dark-adaptation. Mutation of subsets of the eight C-terminal phosphorylation sites also led to a reduction of TRPL content and partial mislocalization in the dark. This suggests that a light-dependent switch in the phosphorylation pattern of the TRPL channel mediates stable expression of TRPL in the rhabdomeres upon prolonged dark-adaptation.

A Phos-tag-based magnetic-bead method for rapid and selective separation of phosphorylated biomolecules

A simple and efficient method based on magnetic-bead technology has been developed for the separation of phosphorylated and nonphosphorylated low-molecular-weight biomolecules, such as nucleotides, phosphorylated amino acids, or phosphopeptides. The phosphate-binding site on the bead is an alkoxide-bridged dinuclear zinc(II) complex with 1,3-bis(pyridin-2-ylmethylamino)propan-2-olate (Phos-tag), which is linked to a hydrophilic cross-linked agarose coating on a magnetic core particle. All steps for the phosphate-affinity separation are conducted in buffers of neutral pH with 50 μL of the magnetic beads in a 1.5-mL microtube. The entire separation protocol for phosphomonoester-type compounds, from addition to elution, requires less than 12 min per sample if the buffers and the zinc(II)-bound Phos-tag magnetic beads have been prepared in advance. The phosphate-affinity magnetic beads are reusable at least 15 times without a decrease in their phosphate-binding ability and they are stable for three months in propan-2-ol.

Regulation of the Na+/H+ Exchanger (NHE1) in Breast Cancer Metastasis

The pH gradient in normal cells is tightly controlled by the activity of various pH-regulatory membrane proteins including the isoform protein of the Na(+)/H(+) exchanger (NHE1). NHE1 is constitutively active in a neoplastic microenvironment, dysregulating pH homeostasis and altering the survival, differentiation, and proliferation of cancer cells, thereby causing them to become tumorigenic. Cytoplasmic alkalinization in breast cancer cells occurs as a result of increased NHE1 activity and, while much is known about the pathophysiologic role of NHE1 in tumor progression with regard to ion flux, the regulation of its activity on a molecular level is only recently becoming evident. The membrane domain of NHE1 is sufficient for ion exchange. However, its activity is regulated through the phosphorylation of key amino acids in the cytosolic domain as well as by its interaction with other intracellular proteins and lipids. Here, we review the importance of these regulatory sites and what role they may play in the disrupted functionality of NHE1 in breast cancer metastasis.

HIPK2 kinase activity depends on cis-autophosphorylation of its activation loop

The multitude of mechanisms regulating the activity of protein kinases includes phosphorylation of amino acids contained in the activation loop. Here we show that the serine/threonine kinase HIPK2 (homeodomain-interacting protein kinase 2) is heavily modified by autophosphorylation, which occurs by cis-autophosphorylation at the activation loop and by trans-autophosphorylation at other phosphorylation sites. Cis-autophosphorylation of HIPK2 at Y354 and S357 in the activation loop is essential for its kinase function and the binding to substrates and the interaction partner Pin1. HIPK2 activation loop phosphorylation is also required for its biological activity as a regulator of gene expression and cell proliferation. Phosphorylation of HIPK2 at Y354 alone is not sufficient for full HIPK2 activity, which is in marked contrast to some dual-specificity tyrosine-phosphorylated and regulated kinases where tyrosine phosphorylation is absolutely essential. This study shows that differential phosphorylation of HIPK2 provides a mechanism for controlling and specifying the signal output from this kinase.

Thermochemistry of Heteroatomic Compounds: Analysis and Calculation of Thermody-namic Functions of Amino Acids and Some Peptides of Different Space Structure

The values of free energies, the heats of combustion and formation in condensed and gaseous phases over 30 amino acids of different structure were analyzed. Entropies and heat capacities in standard conditions were considered too. Twenty one equations of such type as Yо = i ± f (N – g) +Σ(hc)j, in which Yо is thermodynamic function or heat capaci-ty, i and f are stoichiometric coefficiens, Σ(hc)j are the sums of the number (j) of the heat corrections for the mentioned thermodynamic magnitudes, N is the number of valence electrons, from which a number of lone electron pairs (g) is ex-cepted for. The obtained equations were used for the calculations of a new such parameters for four phosphorylated amino acids, two enkephalines and two human peptides of low molecular weights.

The Protein Phosphorylation Profile in Oridonin-Treated Kasumi-1 Cells

Leukemia is one of the most common type cancer, and it is seriously concerned about the prevention and treatment in our country. Oridonin, a natural diterpenoid isolated from Rabdosia rubescens,has been reported to have potential antitumor activities. However, the mechanism is not fully understood.Here, changes in the phosphorylation level of a particular set of proteins of Kasumi-1 cells treated with oridonin were systematically analyzed. To investigate more information of the phosphorylation signals induced by oridonin, the authors constructed a correlation network by using stringent P-value criteria to show correlation linkages between changes in the phosphorylation levels of amino acid sites. The correlation network presented a band-type structure, which was different from gene regulatory network, showing the characteristics of signal propagation through the cascade which resulted in more phosphorylated amino acid sites. Proteins with changes in phosphorylation levels significantly enriched in cellular proliferation, kinase binding functions and cancer associated pathways. The analysis showes that oridonin plays important roles in adjusting the phosphorylation levels and some key characteristics of its effects.

Phosphoproteome dynamics reveal novel ERK1/2 MAP kinase substrates with broad spectrum of functions

The ERK1/2 MAP kinase pathway is an evolutionarily conserved signaling module that controls many fundamental physiological processes. Deregulated activity of ERK1/2 MAP kinases is associated with developmental syndromes and several human diseases. Despite the importance of this pathway, a comprehensive picture of the natural substrate repertoire and biochemical mechanisms regulated by ERK1/2 is still lacking. In this study, we used large-scale quantitative phosphoproteomics and bioinformatics analyses to identify novel candidate ERK1/2 substrates based on their phosphorylation signature and kinetic profiles in epithelial cells. We identified a total of 7936 phosphorylation sites within 1861 proteins, of which 155 classify as candidate ERK1/2 substrates, including 128 new targets. Candidate ERK1/2 substrates are involved in diverse cellular processes including transcriptional regulation, chromatin remodeling, RNA splicing, cytoskeleton dynamics, cellular junctions and cell signaling. Detailed characterization of one newly identified substrate, the transcriptional regulator JunB, revealed that ERK1/2 phosphorylate JunB on a serine adjacent to the DNA-binding domain, resulting in increased DNA-binding affinity and transcriptional activity. Our study expands the spectrum of cellular functions controlled by ERK1/2 kinases.

Comprehensive Mutational Analysis Reveals p6 Gag Phosphorylation To Be Dispensable for HIV-1 Morphogenesis and Replication

The structural polyprotein Gag of human immunodeficiency virus type 1 (HIV-1) is necessary and sufficient for formation of virus-like particles. Its C-terminal p6 domain harbors short peptide motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. p6 has been shown to be the major viral phosphoprotein in HIV-1-infected cells and virions, but the sites and functional relevance of p6 phosphorylation are not clear. Here, we identified phosphorylation of several serine and threonine residues in p6 in purified virus preparations using mass spectrometry. Mutation of individual candidate phosphoacceptor residues had no detectable effect on virus assembly, release, and infectivity, however, suggesting that phosphorylation of single residues may not be functionally relevant. Therefore, a comprehensive mutational analysis was conducted changing all potentially phosphorylatable amino acids in p6, except for a threonine that is part of an essential peptide motif. To avoid confounding changes in the overlapping pol reading frame, mutagenesis was performed in a provirus with genetically uncoupled gag and pol reading frames. An HIV-1 derivative carrying 12 amino acid changes in its p6 region, abolishing all but one potential phosphoacceptor site, showed no impairment of Gag assembly and virus release and displayed only very subtle deficiencies in viral infectivity in T-cell lines and primary lymphocytes. All mutations were stable over 2 weeks of culture in primary cells. Based on these findings, we conclude that phosphorylation of p6 is dispensable for HIV-1 assembly, release, and infectivity in tissue culture.

Circulation: Clinical Summaries

<i>Circulation:</i> Clinical Summaries

Targeted Proteomics for Determining Phosphorylation Site-Specific Associations in Cardiovascular Disease

Post-translational modifications (PTMs) are chemical or physical changes to proteins that can alter structure/function relationships and thus influence catalysis, cell localization and interactions with other proteins and biomolecules. There are over 200 recognized PTMs, generating a vast scope for altering protein function and increasing the potential for complex regulatory cross-talk between diverse modifications. Protein phosphorylation is of major significance since 30-50% of all cellular proteins may be targeted1,2. Phosphorylation is a transient means of amplifying environmental signals to rapidly alter protein structure, interactions and ultimately, function.

Effect of nitric oxide synthase enzyme inhibition and a nitric oxide scavenger on tyrosine amino acid phosphorylation and acrosome reaction of cryopreserved equine sperm

Effect of nitric oxide synthase enzyme inhibition and a nitric oxide scavenger on tyrosine amino acid phosphorylation and acrosome reaction of cryopreserved equine sperm