Phosphorylation Of Serine Residues Research Articles

Elastin calcification in in vitro models and its prevention by MGP’s N-terminal peptide

Medial calcification has been associated with diabetes, chronic kidney disease, and genetic disorders like pseudoxanthoma elasticum. Recently, we showed that genetic reduction of arterial elastin content reduces the severity of medial calcification in matrix Gla protein (MGP)-deficient and Eln haploinsufficient Mgp−/−;Eln+/− mice. This study suggests that there might be a direct effect of elastin amount on medial calcification. We studied this using novel in vitro systems, which are based on elastin or elastin-like polypeptides. We first examined the mineral deposition properties of a transfected pigmented epithelial cell line that expresses elastin and other elastic lamina proteins. When grown in inorganic phosphate-supplemented medium, these cells deposited calcium phosphate minerals, which could be prevented by an N’-terminal peptide of MGP (m3pS) carrying phosphorylated serine residues. We next confirmed these findings using a cell-free elastin-like polypeptide (ELP3) scaffold, where the peptide prevented mineral maturation. Overall, this work describes a novel cell culture model for elastocalcinosis and examines the inhibition of mineral deposition by the m3pS peptide in this and a cell-free elastin-based scaffold. Our study provides strong evidence suggesting the critical functional roles of MGP’s phosphorylated serine residues in the prevention of elastin calcification and proposes a possible mechanism of their action.

Ovotransferrin Exhibits Osteogenic Activity Partially via Low-Density Lipoprotein Receptor-Related Protein 1 (LRP1) Activation in MC3T3-E1 Cells.

Ovotransferrin, a major protein in egg white, induces osteoblast proliferation and survival in vitro. However, it is unclear which receptor(s) drive the beneficial activities of this bioactive glycoprotein. We examined the role of the low-density lipoprotein receptor-related protein 1 (LRP1) in the actions of ovotransferrin on osteoblasts. Here, we showed that LRP1 in part regulates osteogenic action of ovotransferrin. Mouse osteoblasts, MC3T3-E1, with LRP1 deletion displayed diminished osteogenic activity. Our findings indicate that the bone-stimulatory impact of ovotransferrin on RUNX2, COL1A2, and Ca2+ signaling is LRP1-dependent. This shows that LRP1 not only acts as a scavenger receptor but also participates in ovotransferrin-mediated gene transcription. However, some of the key bone formatting factors such as ALP synthesis and serine residue phosphorylation of Akt by ovotransferrin remained independent of LRP1. Overall, this study shows that LRP1-ovotransferrin interaction might underline in part the ability of ovotransferrin to promote bone formation.

Cardio-Metabolic Effects of High-Fat Diets and Their Underlying Mechanisms-A Narrative Review.

The majority of the epidemiological evidence over the past few decades has linked high intake of fats, especially saturated fats, to increased risk of diabetes and cardiovascular disease. However, findings of some recent studies (e.g., the PURE study) have contested this association. High saturated fat diets (HFD) have been widely used in rodent research to study the mechanism of insulin resistance and metabolic syndrome. Two separate but somewhat overlapping models—the diacylglycerol (DAG) model and the ceramide model—have emerged to explain the development of insulin resistance. Studies have shown that lipid deposition in tissues such as muscle and liver inhibit insulin signaling via the toxic molecules DAG and ceramide. DAGs activate protein kinase C that inhibit insulin-PI3K-Akt signaling by phosphorylating serine residues on insulin receptor substrate (IRS). Ceramides are sphingolipids with variable acyl group chain length and activate protein phosphatase 2A that dephosphorylates Akt to block insulin signaling. In adipose tissue, obesity leads to infiltration of macrophages that secrete pro-inflammatory cytokines that inhibit insulin signaling by phosphorylating serine residues of IRS proteins. For cardiovascular disease, studies in humans in the 1950s and 1960s linked high saturated fat intake with atherosclerosis and coronary artery disease. More recently, trials involving Mediterranean diet (e.g., PREDIMED study) have indicated that healthy monounsaturated fats are more effective in preventing cardiovascular mortality and coronary artery disease than are low-fat, low-cholesterol diets. Antioxidant and anti-inflammatory effects of Mediterranean diets are potential mediators of these benefits.

Protein Phosphorylation in Serine Residues Correlates with Progression from Precancerous Lesions to Cervical Cancer in Mexican Patients.

Protein phosphorylation is a posttranslational modification that is essential for normal cellular processes; however, abnormal phosphorylation is one of the prime causes for alteration of many structural, functional, and regulatory proteins in disease conditions. In cancer, changes in the states of protein phosphorylation in tyrosine residues have been more studied than phosphorylation in threonine or serine residues, which also undergo alterations with greater predominance. In general, serine phosphorylation leads to the formation of multimolecular signaling complexes that regulate diverse biological processes, but in pathological conditions such as tumorigenesis, anomalous phosphorylation may result in the deregulation of some signaling pathways. Cervical cancer (CC), the main neoplasm associated with human papillomavirus (HPV) infection, is the fourth most frequent cancer worldwide. Persistent infection of the cervix with high-risk human papillomaviruses produces precancerous lesions starting with low-grade squamous intraepithelial lesions (LSIL), progressing to high-grade squamous intraepithelial lesions (HSIL) until CC is generated. Here, we compared the proteomic profile of phosphorylated proteins in serine residues from healthy, LSIL, HSIL, and CC samples. Our data show an increase in the number of phosphorylated proteins in serine residues as the grade of injury rises. These results provide a support for future studies focused on phosphorylated proteins and their possible correlation with the progression of cervical lesions.

Three Major Phosphoacceptor Sites in HIV-1 Capsid Protein Enhances its Structural Stability and Resistance Against the Inhibitor: Explication Through Molecular Dynamics Simulation, Molecular Docking and DFT Analysis.

Human Immunodeficiency Virus 1 (HIV-1) is a lentivirus, which causes various HIV-associated infections. The HIV-1 core dissociation is essential for viral cDNA synthesis and phosphorylation of HIV-1 capsid protein (HIV-1 CA) plays an important role in it. The aim of this study was to explicate the role of three phosphoserine sites i.e. Ser109, Ser149 and Ser178 in the structural stability of HIV-1 CA, and it's binding with GS-CA1, a novel potent inhibitor. Eight complexes were analyzed and Molecular Dynamics (MD) simulations were performed to observe the stability of HIV-1 CA in the presence and absence of phosphorylation of serine residues at four different temperatures i.e. 300K, 325K, 340K and 350K, along with molecular docking and DFT analysis. The structures showed maximum stability in the presence of phosphorylated serine residue. However, GS-CA1 docked most strongly with the native structure of HIV-1 CA i.e. binding affinity was -8.5 kcal/mol (Ki = 0.579 µM). These results suggest that the phosphorylation of these three serine residues weakens the binding of GS-CA1 with CA and casts derogatory effect on inhibition potential of this inhibitor, but it supports the stability of HIV-1 CA structure that can enhance regulation and replication of HIV-1 in host cells.

Rnd3 interacts with TAO kinases and contributes to mitotic cell rounding and spindle positioning.

Rnd3 is an atypical Rho family protein that is constitutively GTP bound, and acts on membranes to induce loss of actin stress fibers and cell rounding. Phosphorylation of Rnd3 promotes 14-3-3 binding and its relocation to the cytosol. Here, we show that Rnd3 binds to the thousand-and-one amino acid kinases TAOK1 and TAOK2 in vitro and in cells. TAOK1 and TAOK2 can phosphorylate serine residues 210, 218 and 240 near the C-terminus of Rnd3, and induce Rnd3 translocation from the plasma membrane to the cytosol. TAOKs are activated catalytically during mitosis and Rnd3 phosphorylation on serine 210 increases in dividing cells. Rnd3 depletion by RNAi inhibits mitotic cell rounding and spindle centralization, and delays breakdown of the intercellular bridge between two daughter cells. Our results show that TAOKs bind, phosphorylate and relocate Rnd3 to the cytosol and that Rnd3 contributes to mitotic cell rounding, spindle positioning and cytokinesis. Rnd3 can therefore participate in the regulation of early and late mitosis and may also act downstream of TAOKs to affect the cytoskeleton.

Identification of hot spot residues on serine-arginine protein kinase-1 by molecular dynamics simulation studies

Serine-arginine protein kinase-1 (SRPK1) is a highly specific kinase that recognizes serine-arginine dipeptide repeats and phosphorylates SR rich splicing factor ASF/SF2 in a cell-cycle regulated manner. SRPK1 processively phosphorylates serine residues on its substrate ASF/SF2. Elevated expression pattern of both SRPK1 and ASF/SF2 and their association with various carcinomas have established SRPK1 as a potent target for drug design against cancers. In order to develop specific inhibitors the binding of ASF/SF2 to SRPK1 is desired to be selectively interrupted. We have performed molecular dynamics simulation studies on crystal structure of SRPK1 complex with ASF/SF2. The ASF/SF2 acquired a stable binding on the surface of SRPK1 with strong attractive forces. Analysis revealed that there was no major position shifting of the core β-sheet region within the catalytic site of SRPK1 when present in the state of ASF/SF2 bound in comparison to apo form. Global motions of SRPK1 indicated that major stable structural changes occurred after the substrate binding. The interactions between SRPK1 and ASF/SF2 were examined and calculated during molecular dynamics simulation of 1 µs. Molecular dynamics study indicated Arg84, Lys85, Leu86, Lys174, Tyr227 and Leu479 residues of SRPK1 as essential hot spots involved in the stable binding with substrate. Structural analysis of the binding affinity and hot spot investigation provided significant information on ASF/SF2 binding which may also be considered for designing of the novel specific inhibitors of SRPK1 for the applications in cancer therapy. Communicated by Ramaswamy H. Sarma

Tyrosine phosphorylation is not a relevant mechanism to modulate aquaporin 2 activity in gestational queen endometrium and placenta.

Aquaporins have been shown to be regulated by phosphorylation of serine residues, but the possible role of tyrosine residues phosphorylation has not been evaluated. Changes in the localization of aquaporin 2 (AQP2) in the queen endometrium have been related to serum progesterone levels. The aim of this study was to determine whether these AQP2-localization changes are mediated by variations in its tyrosine phosphorylation levels. Twelve queens were included in the study and divided into (a) non-macroscopically pregnant with low levels of progesterone; (b) non-macroscopically pregnant with high levels of progesterone; (c) 30days of pregnancy; and (d) 60days of pregnancy. Samples from endometrium and placental transference zone were obtained, immunoprecipitated and analysed by immunoblotting to determine the abundance of AQP2 and its relative levels of tyrosine phosphorylation. No significant differences in the tyrosine phosphorylation levels of immunoprecipated-AQP2 were observed between groups. We can thus conclude that changes in the localization of AQP2 in the queen endometrium are not modulated by tyrosine phosphorylation.

The sequential phosphorylation of PHF10 subunit of the PBAF chromatin-remodeling complex determines different properties of the PHF10 isoforms.

ABSTRACTThe mammalian PBAF subfamily of SWI/SNF chromatin remodeling complexes plays a wide role in the regulation of gene expression. PHF10 is a subunit of the signature module of PBAF, responsible for its interaction with chromatin. PHF10 is represented by four different isoforms, which are alternatively incorporated in the complex. Two of PHF10 isoforms lacking C-terminal PHD domains contain a cluster of phosphorylated serine residues, designated as X-cluster. In the present study, we explore the phosphorylation of the X-cluster in detail. We identified additional phosphorylated serine residues and designated them as either frequently or rarely phosphorylated. The X-cluster consists of two independently phosphorylated subclusters. Phosphorylation of the second subcluster depends on phosphorylation of a primary serine 327. These two subclusters surround a sequence, which is predicted to be a nuclear localization sequence (NLS3). The NLS3 does not affect localization of PHF10 isoforms. However, it is essential for X-cluster phosphorylation and increased stability of isoforms that lack PHD. Conversely, the presence of NLS3 signal in isoforms that contain C-terminal PHD domains reduces their stability. Thus, phosphorylation of PHF10 isoforms regulates their cell level, determining the rate of incorporation in PBAF. This may alter the pattern of PBAF regulated genes.

Molecular Insights into the Trapping Effect of Ca2+ in Protein Kinase A: A Molecular Dynamics Study.

Protein kinase A has become a model system for the study of kinases, and therefore, a comprehensive understanding of the underlying molecular mechanisms in its catalytic cycle is of crucial importance. One of the aspects that has received recent attention is the role that metal cofactors play in the catalytic cycle. Although Mg2+ is the well-known physiological ion used by protein kinases, Ca2+ ions can also assist the phosphoryl transfer reaction but with lower catalytic activities. This inhibitory effect has been attributed to the ability of Ca2+ to trap the reaction products at the active site, and it has been proposed as a possible regulatory mechanism of the enzyme. Thus, in order to get a clearer understanding of these molecular events, computational simulations in the product state of PKA, in the presence of Mg2+ and Ca2+ ions, were performed through molecular dynamics (MD). Different protonation states of the active site were considered in order to model the different mechanistic pathways that have been proposed. Our results show that different protonation states of the phosphorylated serine residue at the peptide substrate (pSer21), as well as the protonation state of residue Asp166, can have a marked influence on the flexibility of regions surrounding the active site. This is the case of the glycine-rich loop, a structural motif that is directly involved in the release of the products from the PKA active site. MD simulations were capable to reproduce the crystallographic conformations but also showed other conformations not previously reported in the crystal structures that may be involved in enhancing the affinity of pSP20 to PKA in the presence of Ca2+. Hydrogen bonding interactions at the PKA-pSP20 interface were influenced whether by the protonation state of the active site or by the metal cofactor used by the enzyme. Altogether, our results provide molecular aspects into the inhibitory mechanism of Ca2+ in PKA and suggest which is the most probable protonation state of the phosphorylated product at the active site.

Back-Pyrophosphorylation Assay to Detect In Vivo InsP7-Dependent Protein Pyrophosphorylation in Mammalian Cells.

Protein pyrophosphorylation involves the transfer of a high-energy β-phosphate from inositol pyrophosphates, such as diphosphoinositol pentakisphosphate (InsP7) to phosphorylated serine residues. Over a decade of research has established several proteins, involved in diverse physiological processes, as substrates of InsP7-mediated pyrophosphorylation. However, the need for detection of this posttranslational modification on endogenous proteins is paramount. "Back-pyrophosphorylation" is a simple technique to test whether a native protein undergoes InsP7-mediated pyrophosphorylation inside cells. The basis of this technique relies on the fact that a target protein isolated from cells with lower InsP7 levels exists in a hypo-pyrophosphorylated form as compared to the same protein isolated from cells with normal InsP7 levels. Hence, when radiolabeled InsP7 is added to a target protein immunoprecipitated from both these cell types, the hypopyrophosphorylated protein accepts a higher amount of radiolabeled phosphate when compared to the protein isolated from wild-type cells. This chapter provides detailed methods to identify an InsP7 target protein and conduct a back-pyrophosphorylation assay on a target protein immunoprecipitated from cells with normal versus reduced InsP7 levels, to confirm its endogenous pyrophosphorylation status.

Insights into the Effects of Cancer Associated Mutations at the UPF2 and ATP-Binding Sites of NMD Master Regulator: UPF1.

Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that recognizes post-transcriptionally abnormal transcripts and mediates their degradation. The master regulator of NMD is UPF1, an enzyme with intrinsic ATPase and helicase activities. The cancer genomic sequencing data has identified frequently mutated residues in the CH-domain and ATP-binding site of UPF1. In silico screening of UPF1 stability change as a function over 41 cancer mutations has identified five variants with significant effects: K164R, R253W, T499M, E637K, and E833K. To explore the effects of these mutations on the associated energy landscape of UPF1, molecular dynamics simulations (MDS) were performed. MDS identified stable H-bonds between residues S152, S203, S205, Q230/R703, and UPF2/AMPPNP, and suggest that phosphorylation of Serine residues may control UPF1-UPF2 binding. Moreover, the alleles K164R and R253W in the CH-domain improved UPF1-UPF2 binding. In addition, E637K and E833K alleles exhibited improved UPF1-AMPPNP binding compared to the T499M variant; the lower binding is predicted from hindrance caused by the side-chain of T499M to the docking of the tri-phosphate moiety (AMPPNP) into the substrate site. The dynamics of wild-type/mutant systems highlights the flexible nature of the ATP-binding region in UPF1. These insights can facilitate the development of drug discovery strategies for manipulating NMD signaling in cell systems using chemical tools.

Determination of Phosphorylation and Deprotonation Induced Higher Order Structural Transitions in αs-Caseins.

One extremely sensitive and highly successful application of Raman spectroscopy is the structural characterization of proteins. Understanding higher order structure and its effect on protein stability is essential not only for biopharmaceutical and food manufacturing but also for the understanding of diseases that result from the misfolding of proteins including diabetes type II, Alzheimer's, and Parkinson's disease. Due to the large amount of structural information available in Raman spectra, even small alterations in protein conformations including increased exposure of binding regions or changes in geometry of secondary structural elements can be identified. In this study, we demonstrate the unique sensitivity of Raman spectroscopy to subtle structural transitions in an intrinsically open, flexible protein, αs-casein, in response to phosphorylation and deprotonation. Through the application of 2D correlation analysis two separate transition phases have been identified from pH 6-9 and pH 10-12 for both phosphorylated and dephosphorylated αs-casein. However, the actual structural changes observed in each pH range differed considerably between the phosphorylated and dephosphorylated αs-casein. Furthermore, the presence of the phosphorylated serine residues is demonstrated to have a shielding effect during deprotonation of the protein.

All-Trans Retinoic Acid Attenuates Fibrotic Processes by Downregulating TGF-β1/Smad3 in Early Diabetic Nephropathy.

Diabetic nephropathy (DN) involves damage associated to hyperglycemia and oxidative stress. Renal fibrosis is a major pathologic feature of DN. The aim of this study was to evaluate anti-fibrogenic and renoprotective effects of all-trans retinoic acid (ATRA) in isolated glomeruli and proximal tubules of diabetic rats. Diabetes was induced by single injection of streptozotocin (STZ, 60 mg/Kg). ATRA (1 mg/Kg) was administered daily by gavage, from days 3–21 after STZ injection. ATRA attenuated kidney injury through the reduction of proteinuria, renal hypertrophy, increase in natriuresis, as well as early markers of damage such as β2-microglobulin, kidney injury molecule-1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL). The following parameters increased: macrophage infiltration, localization of alpha-smooth muscle actin (αSMA)-positive cells in renal tissue, and pro-fibrotic proteins such as transforming growth factor-β (TGF-β1), laminin beta 1 (LAM-β1), and collagens IV and I. Remarkably, ATRA treatment ameliorated these alterations and attenuated expression and nuclear translocation of Smad3, with increment of glomerular and tubular Smad7. The diabetic condition decreased expression of retinoic acid receptor alpha (RAR-α) through phosphorylation in serine residues mediated by the activation of c-Jun N-terminal kinase (JNK). ATRA administration restored the expression of RAR-α and inhibited direct interactions of JNK/RAR-α. ATRA prevented fibrogenesis through down-regulation of TGF-β1/Smad3 signaling.

The human transmembrane mucin MUC17 responds to TNFα by increased presentation at the plasma membrane.

Transmembrane mucin MUC17 is an integral part of the glycocalyx as it covers the brush border membrane of small intestinal enterocytes and presents an extended O-glycosylated mucin domain to the intestinal lumen. Here, we identified two unknown phosphorylated serine residues, S4428 and S4492, in the cytoplasmic tail of human MUC17. We have previously demonstrated that MUC17 is anchored to the apical membrane domain via an interaction with the scaffolding protein PDZK1. S4492, localized in the C-terminal PDZ binding motif of MUC17, was mutated to generate phosphomimetic and phosphodeficient variants of MUC17. Using Caco-2 cells as a model system, we found that induction of an inflammatory state by long-term stimulation with the proinflammatory cytokine TNFα resulted in an increase of MUC17 protein levels and enhanced insertion of MUC17 and its two phospho-variants into apical membranes. Up-regulation and apical insertion of MUC17 was followed by shedding of MUC17-containing vesicles. Transmembrane mucins have previously been shown to play a role in the prevention of bacterial colonization by acting as sheddable decoys for encroaching bacteria. Overexpression and increased presentation at the plasma membrane of wild-type MUC17 and its phosphodeficient variant MUC17 S-4492A protected Caco-2 cells against adhesion of enteropathogenic Escherichia coli, indicating that C-terminal phosphorylation of MUC17 may play a functional role in epithelial cell protection. We propose a new function for MUC17 in inflammation, where MUC17 acts as a second line of defense by preventing attachment of bacteria to the epithelial cell glycocalyx in the small intestine.

Nutrition, epigenetics and health

Nutrition, epigenetics and health

Top-down mass spectrometry of intact phosphorylated β-casein: Correlation between the precursor charge state and internal fragments.

Phosphorylated proteins play essential roles in many cellular processes, and identification and characterization of the relevant phosphoproteins can help to understand underlying mechanisms. Herein, we report a collision-induced dissociation top-down approach for characterizing phosphoproteins on a quadrupole time-of-flight mass spectrometer. β-casein, a protein with two major isoforms and five phosphorylatable serine residues, was used as a model. Peaks corresponding to intact β-casein ions with charged states up to 36+ were detected. Tandem mass spectrometry was performed on β-casein ions of different charge states (12+ , and 15+ to 28+ ) in order to determine the effects of charge state on dissociation of this protein. Most of the abundant fragments corresponded to y, b ions, and internal fragments caused by cleavage of the N-terminal amide bond adjacent to proline residues (Xxx-Pro). The abundance of internal fragments increased with the charge state of the protein precursor ion; these internal fragments predominantly arose from one or two Xxx-Pro cleavage events and were difficult to accurately assign. The presence of abundant sodium adducts of β-casein further complicated the spectra. Our results suggest that when interpreting top-down mass spectra of phosphoproteins and other proteins, researchers should consider the potential formation of internal fragments and sodium adducts for reliable characterization.

Heterologous regulation of CXCR4 lysosomal trafficking

G protein-coupled receptor (GPCR) signaling is regulated by members of the protein kinase C (PKC) and GPCR kinase (GRK) families, although the relative contribution of each to GPCR function varies among specific GPCRs. The CXC motif receptor 4 (CXCR4) is a member of the GPCR superfamily that binds the CXC motif chemokine ligand 12 (CXCL12), initiating signaling that is subsequently terminated in part by internalization and lysosomal degradation of CXCR4. The purpose of this study is to define the relative contribution of PKC and GRK to CXCR4 signaling attenuation by studying their effects on CXCR4 lysosomal trafficking and degradation. Our results demonstrate that direct activation of PKC via the phorbol ester phorbol 12-myristate 13-acetate (PMA) mimics CXCL12-mediated desensitization, internalization, ubiquitination, and lysosomal trafficking of CXCR4. In agreement, heterologous activation of PKC by stimulating the chemokine receptor CXCR5 with its ligand, CXCL13, also mimics CXCL12-mediated desensitization, internalization, ubiquitination, and lysosomal degradation of CXCR4. Similar to CXCL12, PMA promotes PKC-dependent phosphorylation of serine residues within CXCR4 C-tail that are required for binding and ubiquitination by the E3 ubiquitin ligase AIP4 (atrophin-interacting protein 4). However, inhibition of PKC activity does not alter CXCL12-mediated ubiquitination and degradation of CXCR4, suggesting that other kinases are also required. Accordingly, siRNA-mediated depletion of GRK6 results in decreased degradation and ubiquitination of CXCR4. Overall, these results suggest that PKC and GRK6 contribute to unique aspects of CXCR4 phosphorylation and lysosomal degradation to ensure proper signal propagation and termination.

Phosphorylation Properties of the N-Terminal Region of Twitchin from Molluscan Catch Muscle

Molluscan smooth muscles, such as the bivalve adductor muscles and the mussel anterior byssus retractor muscles (ABRM), exhibit a unique contraction called “catch”. Catch contraction is regulated through twitchin phosphorylation and dephosphorylation. Twitchin from the ABRM of the Mediterranean mussel, Mytilus galloprovincialis, is phosphorylated by cAMP-dependent protein kinase (PKA), and PKA phosphorylation sites are located in both the N- and C-terminal regions of the twitchin molecule. The D2 site, which is adjacently located to the C-terminus, participates in forming a myosin, actin, and twitchin complex that is thought to contribute towards the maintenance of tension in the catch state. In contrast, although it has been reported to interact with thin-filaments, the molecular function of the region including the D1 site has remained largely unstudied. Three additional PKA consensus sequences were identified near the D1 site; however, it was not known if these sites could be directly phosphorylated by PKA. Here, we performed phosphorylation assays to identify phosphorylation sites near the D1 site using recombinant protein variants (TWD1-SSSS, TWD1-AAAS, TWD1-AASA, TWD1-ASAA, TWD1-SAAA, and TWD1-AAAA). All variants, except TWD1-AAAA (where all phosphorylatable serine residues were replaced by alanines), were phosphorylated by PKA. The four phosphorylation sites were named D1-1, D1-2, D1-3, and D1-4 (the originally identified D1) in order from the N-terminus. Phosphorylation assays using a 1/12.5 weight ratio of PKA to each TWD1 variant revealed that D1-4 was the most rapidly phosphorylated, closely followed by D1-1. However, D1-2 and D1-3 were phosphorylated at a lower level under equivalent conditions and were not phosphorylated when PKA was incubated with each TWD1 variant at a 1/100 weight ratio. Furthermore, we observed that TWD1-SSSS was phosphorylated in a stepwise fashion. These findings contribute towards the elucidation of the function of the twitchin D1 region in the regulatory system of catch contraction.

HipA-Mediated Phosphorylation of SeqA Does not Affect Replication Initiation in Escherichia coli.

The SeqA protein of Escherichia coli is required to prevent immediate re-initiation of chromosome replication from oriC. The SeqA protein is phosphorylated at the serine-36 (Ser36) residue by the HipA kinase. The role of phosphorylation was addressed by mutating the Ser36 residue to alanine, which cannot be phosphorylated and to aspartic acid, which mimics a phosphorylated serine residue. Both mutant strains were similar to wild-type with respect to origin concentration and initiation synchrony. The minimal time between successive initiations was also unchanged. We therefore suggest that SeqA phosphorylation at the Ser36 residue is silent, at least with respect to SeqA's role in replication initiation.