Dephosphorylation Of Peptide Research Articles

Effect of phosphorylation of protamine-like cationic peptide on the binding affinity to DNA

Protamines are more arginine-rich and more basic than histones and are responsible for providing a highly compacted shape to the sperm heads in the testis. Phosphorylation and dephosphorylation are two events that occur in the late phase of spermatogenesis before the maturation of sperms. In this work, we have studied the effect of phosphorylation of protamine-like cationic peptides using all-atom molecular dynamics simulations. Through thermodynamic analyses, we found that phosphorylation reduces the binding efficiency of such cationic peptides on DNA duplexes. Peptide phosphorylation leads to a less efficient DNA condensation, due to a competition between DNA-peptide and peptide-peptide interactions. We hypothesize that the decrease of peptide bonds between DNA together with peptide self-assembly might allow an optimal re-organization of chromatin and an efficient condensation through subsequent peptide dephosphorylation. Based on the globular and compact conformations of phosphorylated peptides mediated by arginine-phosphoserine H-bonding, we furthermore postulate that phosphorylated protamines could more easily intrude into chromatin and participate to histone release through disruption of histone-histone and histone-DNA binding during spermatogenesis.

Partial-, Double-Enzymatic Dephosphorylation and EndoGluC Hydrolysis as an Original Approach to Enhancing Identification of Casein Phosphopeptides (CPPs) by Mass Spectrometry.

The identification of phosphopeptides is currently a challenge when they are part of a complex matrix of peptides, such as a milk protein enzymatic hydrolysate. This challenge increases with both the number of phosphorylation sites on the phosphopeptides and their amino acid length. Here, this paper reports a four-phase strategy from an enzymatic casein hydrolysate before a mass spectrometry analysis in order to enhance the identification of phosphopeptides and phosphosites: (i) the control protein hydrolysate, (ii) a two-step enzymatic dephosphorylation of the latter, allowing for the almost total dephosphorylation of peptides, (iii) a one-step enzymatic dephosphorylation, allowing for the partial dephosphorylation of the peptides and (iv) an additional endoGluC enzymatic hydrolysis, allowing for the cleavage of long-size peptides into shorter ones. The reverse-phase high-pressure liquid chromatography–tandem mass spectrometry (RP-HPLC-MS/MS) analyses of hydrolysates that underwent this four-phase strategy allowed for the identification of 28 phosphorylation sites (90%) out of the 31 referenced in UniprotKB/Swiss-Prot (1 June 2021), compared to 17 sites (54%) without the latter. The alpha-S2 casein phosphosites, referenced by their similarity in the UniProt database, were experimentally identified, whereas pSer148, pThr166 and pSer187 from a multiphosphorylated long-size kappa-casein were not. Data are available via ProteomeXchange with identifier PXD027132.

Phosphorylation-Responsive Membrane Transport of Peptides.

Phosphorylation and dephosphorylation of peptides by kinases and phosphatases is essential for signal transduction in biological systems, and many diseases involve abnormal activities of these enzymes. Herein, we introduce amphiphilic calixarenes as key components for supramolecular, phosphorylation-responsive membrane transport systems. Dye-efflux experiments with liposomes demonstrated that calixarenes are highly active counterion activators for established cell-penetrating peptides, with EC50 values in the low nanomolar range. We have now found that they can even activate membrane transport of short peptide substrates for kinases involved in signal transduction, whereas the respective phosphorylated products are much less efficiently transported. This allows regulation of membrane transport activity by protein kinase A (PKA) and protein kinase C (PKC), as well as monitoring of their activity in a label-free kinase assay.

Small peptide substrates and high resolution peptide gels for the analysis of site-specific protein phosphorylation and dephosphorylation.

Protein phosphorylation and dephosphorylation reactions play key regulatory roles in many fundamental cellular processes. Due to the large number of kinases and phosphatases in the genome, the identification of the specific enzymes responsible for a given site in a given protein is immensely challenging. However, because protein kinases and phosphatases recognize local specificity determinants within proteins, it is possible to use small peptides to study the characteristics of site-specific phosphorylation. In addition, phosphorylation usually causes retardation in gel mobility, providing an opportunity to investigate peptide phosphorylation and dephosphorylation by monitoring migration on high resolution peptide gels. In this study, we demonstrate the utility of such a technique using small peptides corresponding to cyclin-dependent kinase-1 (Cdk1)/cyclin B1 sites in two important apoptotic regulatory proteins, Bcl-xL and caspase-9. We show that the mobility of the peptides is retarded following Cdk1-mediated phosphorylation, and that peptide dephosphorylation, catalyzed either by purified phosphatase or by crude cell extracts, is readily observable by increased peptide gel mobility. Furthermore, the procedure can be conducted without the use of radioactive adenosine triphosphate (ATP), and does not require any specialized reagents or apparatus. The method can be used to identify and characterize specific kinase and phosphatases responsible for phosphorylation and dephosphorylation of specific sites in any protein of interest.

Phosphotyrosine Substrate Sequence Motifs for Dual Specificity Phosphatases.

Protein tyrosine phosphatases dephosphorylate tyrosine residues of proteins, whereas, dual specificity phosphatases (DUSPs) are a subgroup of protein tyrosine phosphatases that dephosphorylate not only Tyr(P) residue, but also the Ser(P) and Thr(P) residues of proteins. The DUSPs are linked to the regulation of many cellular functions and signaling pathways. Though many cellular targets of DUSPs are known, the relationship between catalytic activity and substrate specificity is poorly defined. We investigated the interactions of peptide substrates with select DUSPs of four types: MAP kinases (DUSP1 and DUSP7), atypical (DUSP3, DUSP14, DUSP22 and DUSP27), viral (variola VH1), and Cdc25 (A-C). Phosphatase recognition sites were experimentally determined by measuring dephosphorylation of 6,218 microarrayed Tyr(P) peptides representing confirmed and theoretical phosphorylation motifs from the cellular proteome. A broad continuum of dephosphorylation was observed across the microarrayed peptide substrates for all phosphatases, suggesting a complex relationship between substrate sequence recognition and optimal activity. Further analysis of peptide dephosphorylation by hierarchical clustering indicated that DUSPs could be organized by substrate sequence motifs, and peptide-specificities by phylogenetic relationships among the catalytic domains. The most highly dephosphorylated peptides represented proteins from 29 cell-signaling pathways, greatly expanding the list of potential targets of DUSPs. These newly identified DUSP substrates will be important for examining structure-activity relationships with physiologically relevant targets.

Label-free electrochemical impedance detection of kinase and phosphatase activities using carbon nanofiber nanoelectrode arrays

We demonstrate the feasibility of a label-free electrochemical method to detect the kinetics of phosphorylation and dephosphorylation of surface-attached peptides catalyzed by kinase and phosphatase, respectively. The peptides with a sequence specific to c-Src tyrosine kinase and protein tyrosine phosphatase 1B (PTP1B) were first validated with ELISA-based protein tyrosine kinase assay and then functionalized on vertically aligned carbon nanofiber (VACNF) nanoelectrode arrays (NEAs). Real-time electrochemical impedance spectroscopy (REIS) measurements showed reversible impedance changes upon the addition of c-Src kinase and PTP1B phosphatase. Only a small and unreliable impedance variation was observed during the peptide phosphorylation, but a large and fast impedance decrease was observed during the peptide dephosphorylation at different PTP1B concentrations. The REIS data of dephosphorylation displayed a well-defined exponential decay following the Michaelis–Menten heterogeneous enzymatic model with a specific constant, kcat/Km, of (2.1±0.1)×107M−1s−1. Consistent values of the specific constant was measured at PTP1B concentration varying from 1.2 to 2.4nM with the corresponding electrochemical signal decay constant varying from 38.5 to 19.1s. This electrochemical method can be potentially used as a label-free method for profiling enzyme activities in fast reactions.

Measurement of Protein Phosphorylation Stoichiometry by SRM‐MS

AbstractProtein phosphorylation is a major post‐translational modification involved in most biological processes and numerous diseases. Much biological relevance lies in the knowledge of the extent of phosphorylation at any given protein phosphorylation site, i.e., protein phosphorylation stoichiometry. Major progress in quantitative mass spectrometry has allowed the establishment of protocols that permit accurate determination of protein phosphorylation stoichiometry. The Basic Protocol presented herein describes a label‐free mass spectrometry approach for determining phosphorylation stoichiometry using electrospray nano‐liquid chromatography coupled to selected reaction monitoring (SRM). This entails the calculation of the respective response rates of the phosphorylated and unphosphorylated cognate peptide species using a peptide dephosphorylation reaction. The ratio of these response rates is then applied as a correction factor to the LC‐SRM‐MS signal intensity ratio of the corresponding endogenous phosphopeptide/nonphosphopeptide pair in a biological sample of interest. An Alternate Protocol is also presented that describes how heavy isotope‐labeled synthetic peptides can be used as internal standards to determine the stoichiometry of phosphorylation. Curr. Protoc. Chem. Biol. 4:65‐81 © 2012 by John Wiley & Sons, Inc.

A 2D NMR method to study peptide phosphorylation

We demonstrate a 2D NMR method which distinguishes between phosphorylated and non-phosphorylated amino acids. The method is capable of monitoring the amino acid and site-specific enzymatic phosphorylation and dephosphorylation of peptides. The method was developed using O-phosphorylated amino acids and its potential is shown with a peptide fragment of the myelin basic protein (MBP).

Phosphorylation State-Responsive Lanthanide Peptide Conjugates: A Luminescence Switch Based on Reversible Complex Reorganization

[reaction: see text] A luminogenic probe for peptide dephosphorylation has been developed. It consists of a serine-/tyrosine-containing peptide modified on the N-terminus with a tryptophan residue and a DTPA chelate capable of binding Tb(3+). We propose a mechanistic model for the luminescence enhancement based on the interconversion of monomeric and dimeric lanthanide species, which is affected by the phosphorylation state of the serine or tyrosine residue. The optical switch reports effectively on phosphatase-catalyzed dephosphorylation in vitro.

Binding of HTm4 to Cyclin-dependent Kinase (Cdk)-associated Phosphatase (KAP)·Cdk2·Cyclin A Complex Enhances the Phosphatase Activity of KAP, Dissociates Cyclin A, and Facilitates KAP Dephosphorylation of Cdk2

Cyclin-dependent kinase 2 (cdk2) activation requires phosphorylation of Thr160 and dissociation from cyclin A. The T-loop of cdk2 contains a regulatory phosphorylation site at Thr160. An interaction between cdc-associated phosphatase (KAP) and cdk2 compromises the interaction between cdk2 and cyclin A, which permits access of KAP, a Thr160-directed phosphatase, to its substrate, cdk2. We have reported that KAP is bound and activated by a nuclear membrane protein, HTm4. Here, we present in vitro data showing the direct interaction between the HTm4 C terminus and KAP Tyr141. We show that this interaction not only facilitates access of KAP to Thr160 and accelerates KAP kinetics, but also forces exclusion of cyclin A from the KAP.cdk2 complex.

Metformin (Glucophage) inhibits tyrosine phosphatase activity to stimulate the insulin receptor tyrosine kinase

Metformin is a commonly used anti-diabetic but whether its mechanism involves action on the insulin receptor or on downstream events is still controversial. With a time course that was slow compared with insulin action, metformin increased tyrosine phosphorylation of the regulatory domain of the insulin receptor (specifically, tyrosine residues 1150 and 1151). In a direct action, therapeutic levels of metformin stimulated the tyrosine kinase activity of the soluble intracellular portion of the beta subunit of the human insulin receptor toward a substrate derived from the insulin receptor regulatory domain. However, metformin did not alter the order of substrate phosphorylation by the insulin receptor kinase. Using a Xenopus oocyte preparation, we simultaneously recorded tyrosine kinase and phosphatase activities that regulate the insulin receptor by measuring the tyrosine phosphorylation and dephosphorylation of peptides derived from the regulatory domain of the human insulin receptor. In an indirect stimulation of the insulin receptor, metformin inhibited endogenous tyrosine phosphatases and purified human protein tyrosine phosphatase 1B that dephosphorylate and inhibit the insulin receptor kinase. Thus, there was evidence that metformin acted directly upon the insulin receptor and indirectly through inhibition of tyrosine phosphatases.

Dephosphorylation of distinct sites in myosin light chain by two types of phosphatase in aortic smooth muscle

Two types of myosin light chain phosphatase from aortic smooth muscle extract were separated by chromatography on heparin-agarose. The phosphatase which appeared in the flow-through fractions had low activity on actomyosin, its apparent molecular mass was 260 kDa and upon ethanol treatment it generated a catalytic subunit with an apparent molecular mass of 36–39 kDa as determined by gel filtration. This phosphatase preferentially dephosphorylated the α-subunit of phosphorylase kinase and its phosphorylase phosphatase activity was not inhibited by heparin, inhibitor-1 or inhibitor-2. The phosphatase retained by heparin-agarose had high activity on actomyosin, its apparent molecular mass was 150 kDa and upon ethanol treatment it generated a catalytic subunit with an apparent molecular mass of 39–42 kDa. It preferentially dephosphorylated the β-subunit of phosphorylase kinase and its phosphorylase phosphatase activity was not inhibited by heparin, inhibitor-1 or inhibitor-2. Myosin light chain was phosphorylated by myosin light chain kinase in peptides AB (Ser-P) and CD (Thr-P), and/or by protein kinase C in peptides E (Ser-P) and F (Thr.P) as determined by one-dimensional phosphopeptide mapping. The catalytic subunit of heparin-agarose flow-through phosphatase preferentially dephosphorylated peptide F over peptides AB, CD and E in both isolated light chain and actomyosin. The catalytic subunit of heparin-agarose bound phosphatase could effectively dephosphorylate all sites in isolated light chain, whereas it was less effective on dephosphorylation of peptide E in actomyosin.

Comparative free radical chemistry in the radiolytic deamination and dephosphorylation of bio-organic molecules. II. Oxygenated solutions

The radiolytic deamination and dephosphorylation of peptides and phosphate esters in oxygenated solutions are OH-induced and involve the formation and subsequent degradation of the peroxyl radicals RCONHC(Ȯ 2)R 2 and £OC(Ȯ 2)R 2 respectively. Reaction analogues in the degradation chemistry of these peroxyl intermediates from various peptides and phosphate esters including protein and DNA are evaluated in detail. The evidence is that the reaction mechanisms recently proposed for removal of £OC(Ȯ 2)R 2 radicals in the radiolytic scission of the DNA strand are identical in form with the mechanisms identified in earlier studies of RCONHC(Ȯ 2)R 2 radicals in the radiolytic cleavage of the peptide chain. The analogy includes the chemical forms of the final products and their specific yields.

Insulin stimulates the dephosphorylation of phosphothreonine from FAT-PAD ATP-citrate lyase

ATP-citrate lyase is phosphorylated in vivo at three amino acid residues on two peptide sequences (peptides a and b). Insulin action is known to increase the phosphorylation of peptide a. To study the effect of insulin on peptide b phosphorylation ATP-citrate lyase was radiolabeled in vivo by incubating fat pads with 32Pi. Following “cold chase”, insulin action decreased the calculated specific radioactivity of peptide b to less than 30% of control whereas the specific radioactivity of peptide a increased 5–6 fold. The insulin induced decrease in peptide b phosphorylation was mainly due to a decrease in phosphothreonine phosphoryaltion. Isoproterenol treatment increased peptide a phosphorylation 4–6 fold but did not decrease peptide b phosphorylation. Specific radioactivity of ATP did not change significantly with hormone treatment. These results suggest that insulin action increases the dephosphorylation of peptide b by increasing the activity of a putative phosphothreonine phosphatase.