Mechanistic studies of Ser/Thr dehydration catalyzed by a member of the LanL lanthionine synthetase family

Members of the LanL family of lanthionine synthetases consist of three catalytic domains, an N-terminal pSer/pThr lyase domain, a central Ser/Thr kinase domain, and a C-terminal lanthionine cyclase domain. The N-terminal lyase domain has sequence homology with members of the OspF family of effector proteins. In this study, the residues in the lyase domain of VenL that are conserved in the active site of OspF proteins were mutated to evaluate their importance for catalysis. In addition, residues that are fully conserved in the LanL family but not in the OspF family were mutated. Activity assays with these mutant proteins are consistent with a model in which Lys80 in VenL deprotonates the α-proton of pSer/pThr residues to initiate the elimination reaction. Lys51 is proposed to activate this proton by coordination to the carbonyl of the pSer/pThr, and His53 is believed to protonate the phosphate leaving group. These functions are very similar to the corresponding homologous residues in OspF proteins. On the other hand, recognition of the phosphate group of pSer/pThr appears to be achieved differently in VenL than in the OspF proteins. Arg156 and Lys103 are thought to interact with the phosphate group on the basis of a structural homology model.     

Phosphorylation of the human full-length protein kinase Ciota

Atypical protein kinases C, including protein kinase Ciota (PKCiota), play critical roles in signaling pathways that control cell growth, differentiation and survival. This qualifies them as attractive targets for development of novel therapeutics for the treatment of various human diseases. In this study, the full-length PKCiota was expressed in Sf9 insect cells, purified, and digested with trypsin and endoproteinase Asp-N, and its phosphorylation analyzed by liquid chromatography-high accuracy mass spectrometry. This strategy mapped 97% of the PKCiota protein sequence and revealed seven new Ser/Thr phosphorylation sites, in addition to the two previously known, pThr403 in the activation loop and pThr555 in the turn motif of the kinase domain. Most of the newly identified phosphorylation sites had low estimated occupancies (below 2%). Two phosphorylation sites were located in domain connecting amino acid sequence stretches (pSer217 and pSer237/pSer238) and may contribute to an improved stability and solubility of the protein. The most interesting new phosphorylation site was detected in a well-accessible loop of the PB1 domain (pSer35/pSer37) and may be involved in the interactions of the PB1 domain with different partners in the relevant signaling pathways.     

Synthesis of fluorine-containing bioisosteres corresponding to phosphoamino acids and dipeptide units

It has been shown that fluorinated analogues of naturally occurring biological active compounds including amino acids often exhibit unique physiological activity. Among wide varieties of fluorine-containing amino acids, nonhydrolyzable phosphoamino acids possessing a substituent of the difluoromethylene (CF(2)) unit for the phosphoryl ester oxygen are of value in the medicinal and biological fields. We have engaged in the synthesis of these classes of nonhydrolyzable phosphoamino acids corresponding to pTyr 3, pSer 4, and pThr 5 with their incorporation into peptides using newly developed deprotecting procedures. In this article, stereoselective synthesis of the CF(2)-substituted pThr mimetics and development of a two-step deprotecting methodology for the nonhydrolyzable analogues are reviewed. In the course of the above synthetic study, we found that gamma,gamma-difluoro-alpha,beta-enoates were reduced to gamma-fluoro-beta,gamma-enoates by organocopper reagents and then applied to the synthesis of (Z)-fluoroalkene dipeptide isosteres, which have served as potential dipeptide mimetics having structural as well as electrostatic similarity to the parent peptide bonds. Furthermore, mechanistic investigation of the organocopper-mediated reduction led us to development of a SmI(2)-mediated approach toward the synthesis of the fluoroalkene isosteres.     

Proteomic screening method for phosphopeptide motif binding proteins using peptide libraries

Phosphopeptide binding domains mediate the directed and localized assembly of protein complexes essential to intracellular kinase signaling. To identify phosphopeptide binding proteins, we developed a proteomic screening method using immobilized partially degenerate phosphopeptide mixtures combined with SILAC and microcapillary LC-MS/MS. The method was used to identify proteins that specifically bound to phosphorylated peptide library affinity matrices, including pTyr, and the motifs pSer/pThr-Pro, pSer/pThr-X-X-X-pSer/pThr, pSer/pThr-Glu/Asp, or pSer/pThr-pSer/pThr in degenerate sequence contexts. Heavy and light SILAC lysates were applied to columns containing these phosphorylated and nonphosphorylated (control) peptide libraries respectively, and bound proteins were eluted, combined, digested, and analyzed by LC-MS/MS using a hybrid quadrupole-TOF mass spectrometer. Heavy/light peptide ion ratios were calculated, and peptides that yielded ratios greater than ～3:1 were considered as being from potential phosphopeptide binding proteins since this ratio represents the lowest ratio from a known positive control. Many of those identified were known phosphopeptide-binding proteins, including the SH2 domain containing p85 subunit of PI3K bound to pTyr, 14-3-3 bound to pSer/pThr-Asp/Glu, polo-box domain containing PLK1 and Pin1 bound to pSer/pThr-Pro, and pyruvate kinase M2 binding to pTyr. Approximately half of the hits identified by the peptide library screens were novel. Protein domain enrichment analysis revealed that most pTyr hits contain SH2 domains, as expected, and to a lesser extent SH3, C1, STAT, Tyr phosphatase, Pkinase, C2, and PH domains; however, pSer/pThr motifs did not reveal enriched domains across hits.     

Sequential phosphorylation and multisite interactions characterize specific target recognition by the FHA domain of Ki67

The forkhead-associated (FHA) domain of human Ki67 interacts with the human nucleolar protein hNIFK, recognizing a 44-residue fragment, hNIFK226-269, phosphorylated at Thr234. Here we show that high-affinity binding requires sequential phosphorylation by two kinases, CDK1 and GSK3, yielding pThr238, pThr234 and pSer230. We have determined the solution structure of Ki67FHA in complex with the triply phosphorylated peptide hNIFK226-269(3P), revealing not only local recognition of pThr234 but also the extension of the beta-sheet of the FHA domain by the addition of a beta-strand of hNIFK. The structure of an FHA domain in complex with a biologically relevant binding partner provides insights into ligand specificity and potentially links the cancer marker protein Ki67 to a signaling pathway associated with cell fate specification.     

Conformational preferences and prolyl cis–trans isomerization of phosphorylated Ser/Thr‐Pro motifs

The conformational study on Ac‐pSer‐Pro‐NHMe and Ac‐pThr‐Pro‐NHMe peptides has been carried out using hybrid density functional methods with the implicit solvation reaction field theory at the B3LYP/ 6‐311++G(d,p)//B3LYP/6‐31+G(d) level of theory in the gas phase and in solution (chloroform and water). For both pSer‐Pro and pThr‐Pro peptides in the gas phase and in chloroform, the most preferred conformation has the α‐helical structure for the pSer/pThr residue, the down‐puckered polyproline I structure for the Pro residue, and the cis prolyl peptide bond between the two residues, in which two hydrogen bonds between the phosphate oxygens with the backbone N? H groups seem to play a role. However, the trans conformations that have a single hydrogen bond of the phosphate oxygen with either of two backbone N? H groups become most preferred for both peptides in water. This is because the hydration free energy of the anionic oxygen of the phosphate group is expected to dramatically decrease for the cis conformation upon formation of the hydrogen bond with the backbone N? H groups. These calculated results are consistent with the observations by NMR and IR experiments, suggesting the existence of hydrogen bonds between the charged phosphoryl group and the backbone amide protons in solution. The calculated cis populations of 14.7 and 14.2% and rotational barriers of 19.87 and 20.57 kcal/mol to the cis‐to‐trans isomerization for pSer‐Pro and pThr‐Pro peptides in water, respectively, are consistent with the observed values for pSer‐Pro and pThr‐Pro containing peptides from NMR experiments. However, the hydrogen bond between the prolyl nitrogen and the following amide N? H group, which was suggested to be capable of catalyzing the prolyl isomerization, does not play a role in stabilizing the preferred transition state for the pSer/pThr‐Pro peptides in water. Instead, the amide hydrogen of the NHMe group is involved in a bifurcated hydrogen bond with the anionic oxygen and phosphoester oxygen of the phosphate group. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 330–339, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Receptor‐Like Kinase Phosphorylation of Arabidopsis Heterotrimeric G‐Protein Gα ‐Subunit AtGPA1

As molecular on–off switches, heterotrimeric G protein complexes, comprised of a Gα subunit and an obligate Gβγ dimer, transmit extracellular signals received by G protein–coupled receptors (GPCRs) to cytoplasmic targets that respond to biotic and abiotic stimuli. Signal transduction is modulated by phosphorylation of GPCRs and G protein complexes. In Arabidopsis thaliana, the Gα subunit AtGPA1 is phosphorylated by the receptor‐like kinase (RLK) BRI1‐associated Kinase 1 (BAK1), but the extent that other RLKs phosphorylates AtGPA1 is unknown. Twenty‐two trans‐phosphorylation sites on AtGPA1 are mapped by 12 RLKs hypothesized to act in the Arabidopsis G protein signaling pathway. Cis‐phosphorylation sites are also identified on these RLKs, some newly shown to be dual specific kinases. Multiple sites are present in the core AtGPA1 functional units, including pSer52 and/or pThr53 of the conserved P‐loop that directly binds nucleotide/phosphate, pThr164, and pSer175 from αE helix in the intramolecular domain interface for nucleotide exchange and GTP hydrolysis, and pThr193 and/or pThr194 in Switch I (SwI) that coordinates nucleotide exchange and protein partner binding. Several AtGPA1 S/T phosphorylation sites are potentially nucleotide‐dependent phosphorylation patterns, such as Ser52/Thr53 in the P‐loop and Thr193 and/or Thr194 in SwI.     

ATM activation is accompanied with earlier stages of prostate tumorigenesis

The ATM (ataxia telangiectasia mutated) kinase plays an essential role in maintaining genome integrity by coordinating cell cycle arrest, apoptosis, and DNA damage repair. Phosphorylation of ATM at serine 1981 (ATMpSer1981) by DNA damage activates ATM, which subsequently phosphorylates H2AX Ser139 (gammaH2AX), Chk2 Thr68 (Chk2pThr68), and p53 Ser15 (p53pSer15). To determine the role of the ATM pathway in prostate cancer tumorigenesis, we have analyzed 35 primary prostate cancer specimens for ATMpSer1981 (ATM activation), Chk2pThr68, gammaH2AX, and p53pSer15 by immunohistochemistry (IHC) in normal glands, prostatic intraepithelial neoplasias (PINs), and carcinomas. Increases in the intensities of ATMpSer1981, Chk2pThr68, and gammaH2AX and in the percentage of cells that are positive for ATMpSer1981, Chk2pThr68, or gammaH2AX were observed in PINs (p<0.001) compared to normal prostatic glands and carcinoma. However, this pattern of immunostaining was not seen for p53pSer15. Thus, ATM and Chk2 are specifically activated in PINs. As PINs are generally regarded as precursors of prostatic carcinoma, our results suggest that ATM and Chk2 activation at earlier stages of prostate tumorigenesis suppresses tumor progression, with attenuation of ATM activation leading to cancer progression.     

Mapping of phosphorylation-dependent anti-tau monoclonal antibodies in immunoblots using human tau-constructs synthesized by native chemical ligation

In Alzheimer’s disease (AD) neurofibrillary tangles (NFT) are formed by hyperphosphorylated microtubule-associated tau protein. It is still a matter of controversy which phosphorylation sites are AD-specific and how these might be linked to the cause or progress of the disease. Whereas most research projects in this field rely on phosphorylation-dependent tau-specific monoclonal antibodies (mAbs), the phosphorylation patterns recognized by these mAbs are often not characterized in detail. Therefore, we synthesized unphosphorylated, two monophosphorylated (pThr231, pSer235), and the bisphosphorylated (pThr231 + pSer235) tau226-240 peptides. The phosphopeptides were ligated via an N-terminal cysteine to the thioester-activated C-terminus of human aldo/keto reductase AKR1A1. After purification by preparative gel electrophoresis, the ligation products were analyzed by Western blotting and probed with phosphorylation-dependent anti-tau mAbs HPT-101, HPT-103, HPT-104, and HPT-110. The obtained specificities were very similar to the data obtained by ELISA, showing that ELISA-based epitope mapping studies are also valid for immunoblot analyses.     

Characterization of the AT180 epitope of phosphorylated Tau protein by a combined nuclear magnetic resonance and fluorescence spectroscopy approach

We present here the characterization of the epitope recognized by the AT180 monoclonal antibody currently used to define an Alzheimer’s disease (AD)-related pathological form of the phosphorylated Tau protein. Some ambiguity remains as to the exact phospho-residue(s) recognized by this monoclonal: pThr231 or both pThr231 and pSer235. To answer this question, we have used a combination of nuclear magnetic resonance (NMR) and fluorescence spectroscopy to characterize in a qualitative and quantitative manner the phospho-residue(s) essential for the epitope recognition. Data from the first step of NMR experiments are used to map the residues bound by the antibodies, which were found to be limited to a few residues. A fluorophore is then chemically attached to a cystein residue introduced close-by the mapped epitope, at arginine 221, by mutagenesis of the recombinant protein. The second step of Förster resonance energy transfer (FRET) between the AT180 antibody tryptophanes and the phospho-Tau protein fluorophore allows to calculate a dissociation constant Kd of 30 nM. We show that the sole pThr231 is necessary for the AT180 recognition of phospho-Tau and that phosphorylation of Ser235 does not interfere with the binding.     

Electrochemical investigations of tau protein phosphorylations and interactions with pin1

Phosphorylation of Tau by the protein kinase GSK-3β was monitored by electrochemical impedance spectroscopy of immobilized Tau on gold surfaces. As a result of Tau phosphorylation, the film resistance decreases significantly due to conformational changes and reorganization of the immobilized phosphorylated Tau (pTau) protein, which in turn enables the interactions of pTau with the peptidyl-prolyl cis/trans isomerase, Pin1. Interactions are specific to phospho-Ser (pSer) and phospho-Thr (pThr) residues of pTau. Impedance changes occurred as a function of pTau?Pin1 interactions and are related to the amount of Pin1 bound, which resulted in an increase of the charge-transfer resistance, R(CT) . Our results clearly indicate that the isomerase Pin1 interacts favorably with pSer/pThr-Pro residues in Tau, but does not bind non-phosphorylated Tau or phospho-Tyr residues in Tau films. Our study demonstrates the utility of electrochemical impedance studies to probe protein modifications and biomolecular interactions.     

Identification of Ser/Thr kinase and Forkhead Associated Domains in Mycobacterium ulcerans: Characterization of Novel Association between Protein Kinase Q and MupFHA

Background

Mycobacterium ulcerans, the causative agent of Buruli ulcer in humans, is unique among the members of Mycobacterium genus due to the presence of the virulence determinant megaplasmid pMUM001. This plasmid encodes multiple virulence-associated genes, including mup011, which is an uncharacterized Ser/Thr protein kinase (STPK) PknQ.

Methodology/Principal Findings

In this study, we have characterized PknQ and explored its interaction with MupFHA (Mup018c), a FHA domain containing protein also encoded by pMUM001. MupFHA was found to interact with PknQ and suppress its autophosphorylation. Subsequent protein-protein docking and molecular dynamic simulation analyses showed that this interaction involves the FHA domain of MupFHA and PknQ activation loop residues Ser¹⁷⁰ and Thr¹⁷⁴. FHA domains are known to recognize phosphothreonine residues, and therefore, MupFHA may be acting as one of the few unusual FHA-domain having overlapping specificity. Additionally, we elucidated the PknQ-dependent regulation of MupDivIVA (Mup012c), which is a DivIVA domain containing protein encoded by pMUM001. MupDivIVA interacts with MupFHA and this interaction may also involve phospho-threonine/serine residues of MupDivIVA.

Conclusions/Significance

Together, these results describe novel signaling mechanisms in M. ulcerans and show a three-way regulation of PknQ, MupFHA, and MupDivIVA. FHA domains have been considered to be only pThr specific and our results indicate a novel mechanism of pSer as well as pThr interaction exhibited by MupFHA. These results signify the need of further re-evaluating the FHA domain –pThr/pSer interaction model. MupFHA may serve as the ideal candidate for structural studies on this unique class of modular enzymes.     

Structure of a Fbw7-Skp1-cyclin E complex: multisite-phosphorylated substrate recognition by SCF ubiquitin ligases

The ubiquitin-mediated proteolysis of cyclin E plays a central role in cell-cycle progression, and cyclin E accumulation is a common event in cancer. Cyclin E degradation is triggered by multisite phosphorylation, which induces binding to the SCF(Fbw7) ubiquitin ligase complex. Structures of the Skp1-Fbw7 complex bound to cyclin E peptides identify a doubly phosphorylated pThr380/pSer384 cyclin E motif as an optimal, high-affinity degron and a singly phosphorylated pThr62 motif as a low-affinity one. Biochemical data indicate that the closely related yeast SCF(Cdc4) complex recognizes the multisite phosphorylated Sic1 substrate similarly and identify three doubly phosphorylated Sic1 degrons, each capable of high-affinity interactions with two Cdc4 phosphate binding sites. A model that explains the role of multiple cyclin E/Sic1 degrons is provided by the findings that Fbw7 and Cdc4 dimerize, that Fbw7 dimerization enhances the turnover of a weakly associated cyclin E in vivo, and that Cdc4 dimerization increases the rate and processivity of Sic1 ubiquitination in vitro.     

Role of myosin regulatory light chain and Rac1 in the migration of polyamine-depleted intestinal epithelial cells

We have previously shown that polyamine depletion decreased migration, Rac activation, and protein serine threonine phosphatase 2A activity. We have also shown that polyamine depletion increased cortical F-actin and decreased lamellipodia and stress fibers. In this study, we used staurosporine (STS), a potent, cell-permeable, and broad-spectrum serine/threonine kinase inhibitor, and studied migration. STS concentrations above 100 nM induced apoptosis. However, in polyamine-depleted cells, a lower concentration of STS (5 nM) increased attachment, spreading, Rac1 activation, and, subsequently, migration without causing apoptosis. STS-induced migration was completely prevented by a Rac1 inhibitor (NSC-23766) and dominant negative Rac1. These results imply that STS restores migration in polyamine-depleted cells through Rac1. The most important finding in this study was that polyamine depletion increased the association of phosphorylated myosin regulatory light chain (pThr(18)/Ser(19)-MRLC) at the cell periphery, which colocalized with thick cortical F-actin. Localization of pThr(18)- and pSer(19)-MRLC was found with stress fibers and nuclei, respectively. STS decreased the phosphorylation of cellular and peripheral pThr(18)-MRLC without any effect on nuclear pSer(19)-MRLC, dissolved thick cortical F-actin, and increased lamellipodia and stress fiber formation in polyamine-depleted cells. In control and polyamine-depleted cells, focal adhesion kinase (FAK) colocalized with stress fibers and the actin cortex, respectively. STS reorganized FAK, paxillin, and the cytoskeleton. These results suggest that polyamine depletion prevents the dephosphorylation of MRLC and thereby prevents the dynamic reorganization of the actin cytoskeleton and decreases lamellipodia formation resulting in the inhibition of migration.     

Complementary regulation of TBC1D1 and AS160 by growth factors, insulin and AMPK activators

AS160 (Akt substrate of 160 kDa) and TBC1D1 are related RabGAPs (Rab GTPase-activating proteins) implicated in regulating the trafficking of GLUT4 (glucose transporter 4) storage vesicles to the cell surface. All animal species examined contain TBC1D1, whereas AS160 evolved with the vertebrates. TBC1D1 has two clusters of phosphorylated residues, either side of the second PTB (phosphotyrosine-binding domain). Each cluster contains a 14-3-3-binding site. When AMPK (AMP-activated protein kinase) is activated in HEK (human embryonic kidney)-293 cells, 14-3-3s bind primarily to pSer237 (where pSer is phosphorylated serine) in TBC1D1, whereas 14-3-3 binding depends primarily on pThr596 (where pThr is phosphorylated threonine) in cells stimulated with IGF-1 (insulin-like growth factor 1), EGF (epidermal growth factor) and PMA; and both pSer237 and pThr596 contribute to 14-3-3 binding in cells stimulated with forskolin. In HEK-293 cells, LY294002 inhibits phosphorylation of Thr596 of TBC1D1, and promotes phosphorylation of AMPK and Ser237 of TBC1D1. In vitro phosphorylation experiments indicated regulatory interactions among phosphorylated sites, for example phosphorylation of Ser235 prevents subsequent phosphorylation of Ser237. In rat L6 myotubes, endogenous TBC1D1 is strongly phosphorylated on Ser237 and binds to 14-3-3s in response to the AMPK activators AICAR (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside), phenformin and A-769662, whereas insulin promotes phosphorylation of Thr596 but not 14-3-3 binding. In contrast, AS160 is phosphorylated on its 14-3-3-binding sites (Ser341 and Thr642) and binds to 14-3-3s in response to insulin, but not A-769662, in L6 cells. These findings suggest that TBC1D1 and AS160 may have complementary roles in regulating vesicle trafficking in response to insulin and AMPK-activating stimuli in skeletal muscle.     

Protein phosphorylation influences proteolytic cleavage and kinase substrate properties exemplified by analysis of in vitro phosphorylated Plasmodium falciparum glideosome-associated protein 45 by nano-ultra performance liquid chromatography-tandem mass spectrometry

Plasmodium falciparum glideosome-associated protein 45 (PfGAP45) was in vitro phosphorylated by P. falciparum calcium-dependent protein kinase (PfCDPK1) and digested using the four proteases trypsin, chymotrypsin, AspN, and elastase. Subsequently, phosphopeptide enrichment using Ga(III) immobilized metal affinity chromatography (IMAC) was performed. The resulting fractions were analyzed using ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS), resulting in the identification of a total of nine phosphorylation sites: Ser31, Ser89, Ser103, Ser109, Ser121, Ser149, Ser156, Thr158, and Ser173. During in-depth analyses of the detected phosphopeptides, it was observed that phosphorylation alters the properties of PfGAP45 as kinase and protease substrate. The closely adjacent phosphorylation sites Ser156 (major site) and Thr158 (minor site) were analyzed in detail because at first glance the specific proteases gave highly variable results with respect to the relative abundance of these sites. It was observed that (i) formation of pSer156 and pThr158 was mutually exclusive and (ii) phosphorylation at Ser156 or Thr158 interfered specifically with proteolysis by chymotrypsin or trypsin, respectively. The latter effect was studied in detail using synthetic phosphopeptides carrying either pSer156 or pThr158 as substrate for chymotrypsin or trypsin, respectively.     

Molecular dynamics simulation of the phosphorylation‐induced conformational changes of a tau peptide fragment

Aggregation of the microtubule associated protein tau (MAPT) within neurons of the brain is the leading cause of tauopathies such as Alzheimer's disease. MAPT is a phospho‐protein that is selectively phosphorylated by a number of kinases in vivo to perform its biological function. However, it may become pathogenically hyperphosphorylated, causing aggregation into paired helical filaments and neurofibrillary tangles. The phosphorylation induced conformational change on a peptide of MAPT (htau_225?250) was investigated by performing molecular dynamics simulations with different phosphorylation patterns of the peptide (pThr231 and/or pSer235) in different simulation conditions to determine the effect of ionic strength and phosphate charge. All phosphorylation patterns were found to disrupt a nascent terminal β‐sheet pattern (²²⁶VAVVR²³⁰ and ²⁴⁴QTAPVP²⁴⁹), replacing it with a range of structures. The double pThr231/pSer235 phosphorylation pattern at experimental ionic strength resulted in the best agreement with NMR structural characterization, with the observation of a transient α‐helix (²³⁹AKSRLQT²⁴⁵). PPII helical conformations were only found sporadically throughout the simulations. Proteins 2014; 82:1907–1923. © 2014 Wiley Periodicals, Inc.     

Genetic and biochemical characterization of a protein phosphatase with dual substrate specificity in Streptomyces coelicolor A3(2)

A gene encoding a protein phosphatase (SppA) with a phosphoesterase motif, which was predicted by the genome project of the Gram-positive bacterium Streptomyces coelicolor A3(2), was cloned by PCR in pET32a(+) and expressed in Escherichia coli. SppA fused to thioredoxin (TRX-SppA) showed distinct heat-stable phosphatase activity toward p-nitrophenyl phosphate with optimal pH 8.0 and optimal temperature 55 degrees C. Mn2+ greatly enhanced enzyme activity, as is found with other protein Ser/Thr phosphatases. TRX-SppA was not inhibited by sodium orthovanadate or okadaic acid, both of which are known to be specific inhibitors of protein phosphatases. TRX-SppA showed phosphatase activity toward not only phosphoThr (pThr) and pTyr but also oligopeptides containing pSer, pThr, and pTyr, indicating that SppA is a protein phosphatase with dual substrate specificity. Disruption of the chromosomal sppA gene resulted in severe impairment of vegetative growth. All of these observations show that SppA, a protein phosphatase with dual specificity, plays an important, but not essential, role in vegetative growth of S. coelicolor A3(2). The presence of a single copy of sppA in all the 13 Streptomyces species examined, as determined by Southern hybridization, suggests a common role of SppA in general in Streptomyces species.     

Structure and substrate recognition of the Staphylococcus aureus protein tyrosine phosphatase PtpA

Phosphosignaling through pSer/pThr/pTyr is emerging as a common signaling mechanism in prokaryotes. The human pathogen Staphylococcus aureus produces two low-molecular-weight protein tyrosine phosphatases (PTPs), PtpA and PtpB, with unknown functions. To provide the structural context for understanding PtpA function and substrate recognition, establish PtpA's structural relations within the PTP family, and provide a framework for the design of specific inhibitors, we solved the crystal structure of PtpA at 1 Å resolution. While PtpA adopts the common, conserved PTP fold and shows close overall similarity to eukaryotic PTPs, several features in the active site and surface organization are unique and can be explored to design selective inhibitors. A peptide bound in the active site mimics a phosphotyrosine substrate, affords insight into substrate recognition, and provides a testable substrate prediction. Genetic deletion of ptpA or ptpB does not affect in vitro growth or cell wall integrity, raising the possibility that PtpA and PtpB have specialized functions during infection.     

Cis phosphorylated tau as the earliest detectable pathogenic conformation in Alzheimer disease,offering novel diagnostic and therapeutic strategies

After protein phosphorylation on certain serine or threonine residues preceding a proline (pSer/Thr-Pro), the function of certain phosphorylated protein is further regulated by cis-trans conformational change. Due to the lack of any tool to detect such two conformations in cells, however, it is not even known whether any cis or trans conformation exists in vivo, not to mention their conformation-specific functions or regulation. We developed a novel peptide chemistry technology to generate the first pair of antibodies that can distinguish cis from trans pThr231-Pro tau. Cis, but not trans, pThr231-tau appears early in mild cognitive impairment (MCI) neurons and further accumulates in only degenerating neurons as Alzheimer disease (AD) progresses, localizing to dystrophic neurites, which are known to correlate well with memory loss. Unlike trans p-tau, the cis cannot promote microtubule assembly, and is more resistant to dephosphorylation and degradation and more prone to aggregation. Pin1 accelerates cis to trans isomerization to prevent tau pathology in AD. Thus, during MCI and AD development, cis pThr231-Pro tau is the earliest detectable pathogenic tau conformation and antibodies and vaccines against the pathogenic cis p-tau may be used for the early diagnosis and treatment of AD. These findings offer in vivo approach to study conformational regulation of Pro-directed phosphorylation signaling.