Phos-tag Gels Research Articles

Abstract Or105: Constitutive Deletion of the Obscurin Ig58/59 Domains Induces Structural and Electrophysiological Remodeling in Atria

Introduction: Obscurin is a giant cytoskeletal protein that supports muscle development, tethers intracellular compartments and regulates contraction. In the ObscnΔIg58/59 mouse model, expressing obscurin lacking Immunoglobulin (Ig) domains 58 and 59, aging males exhibit irregular heart rhythm with prominent atrial fibrillation, atrial enlargement, and progressive remodeling of the ventricles. A mechanistic basis for the emergence of arrhythmia early on could not be identified in ObscnΔIg58/59 ventricles, suggesting that the atria are preferentially impacted by deletion of the obscurin Ig58/59 module prior to the ventricles. Hypothesis: We hypothesize that Ig58/59 deletion elicits unique structural, electrical, and functional consequences in the atria preceding ventricular maladaptation. Aims: I. Examine the structural organization of ObscnΔIg58/59 atria II. Assess Ca 2+ handling in isolated atrial cardiomyocytes and determine molecular alterations. Methods: Electron microscopy and super resolution microscopy were utilized to visualize sarcomeric ultrastructure and the transverse-axial tubule (TAT) network, respectively. Ca 2+ handling activity and synchrony were also resolved, while Phos-Tag gels were used to quantify T-cap phospho-species. Results: ObscnΔIg58/59 atria exhibited misalignment of Z-disks. Spontaneous and stimulated Ca 2+ cycling behavior were [AB1] differentially disrupted in atrial cardiomyocytes in 6- and 12-month ObscnΔIg58/59 males. Relatedly, atrial cells showed an age-dependent deterioration in TAT architecture. Finally, as a function of aging, ObscnΔIg58/59 atria displayed alterations in the expression and phosphorylation of T-cap, a Z-disk protein implicated in the integration of t-tubules with the sarcomeric cytoskeleton. Conclusions: The structural and electrical alterations that arise in ObscnΔIg58/59 atria precede ventricular dysfunction and coincide with the emergence of atrial fibrillation. Collectively, our work indicates that the atria are principally affected by Ig58/59 elimination, instigating arrhythmias and morphological alterations with age. The ObscnΔIg58/59 mouse model has thus emerged as a proxy for atrial cardiomyopathy.

Phosphorylation of RGS16 at Tyr168 promote HBeAg-mediated macrophage activation by ERK pathway to accelerate liver injury.

Liver injury is closely associated with macrophage activation following HBV infection. Our previous study showed that only HBeAg, but not HBsAg and HBcAg, stably enhances inflammatory cytokine production in macrophages. And we also indicated that HBeAg could induce macrophage activation via TLR2 and thus aggravate the progression of liver fibrosis. However, the specific molecular mechanism of HBeAg in macrophage activation is not clear. We screened significantly overexpressed RGS16 from RNASeq results of HBeAg-stimulated macrophages and validated them with cellular assays, GSE83148 microarray dataset, and in clinical samples. Meanwhile, small interference, plasmid, and lentivirus transfection assays were used to establish cell models for knockdown and overexpression of RGS16, and q-PCR, ELISA, Transwell, and CCK-8 assays were used to analyze the role of RGS16 in HBeAg-induced macrophage activation. In addition, the upstream and downstream mechanisms of RGS16 in HBeAg-treated macrophage activation were explored using inhibitors, phostag gels, and RGS16 phosphorylation mutant plasmids. Finally, the effect of RGS16 on hepatic inflammation in murine tissues was evaluated by H&E staining, liver enzyme assay and immunofluorescence. RGS16 was significantly upregulated in HBeAg-induced macrophage activation, and its expression was enhanced with increasing HBeAg content and treatment time. Functional experiments showed that overexpression of RGS16 promoted the production of inflammatory factors TNF-α and IL-6 and boosted macrophage proliferation and migration, while knockdown of RGS16 exhibited the opposite effect. Mechanistically, we discovered that RGS16 is regulated by the TLR2/P38/STAT5 signaling pathway. Meanwhile, RGS16 enhanced ERK phosphorylation via its own Tyr168 phosphorylation to contribute to macrophage activation, thereby accelerating liver injury. Finally, in mice, overexpression of RGS16 markedly strengthened liver inflammation. HBeAg upregulates RGS16 expression through the TLR2-P38-STAT5 axis, and the upregulated expression of RGS16 enhances macrophage activation and accelerates liver injury by promoting ERK phosphorylation. In this process, phosphorylation of Tyr168 is necessary for RGS16 to function. KEY MESSAGES: RGS16 boosted HBeAg-induced macrophage inflammation, proliferation, and migration. Tyr168 phosphorylation of RGS16 affected by ERK promoted macrophage activation. HBeAg upregulated the expression of RGS16 through TLR2/P38/STAT5 signal pathway. RGS16 promoted liver injury by regulating macrophage functions in mouse model.

Analysis of protein phosphorylation using Phos-tag gels

Phosphorylation is a major regulatory mechanism controlling protein and cell function. Phosphoproteomics is continuing to reveal the extent and complexity of protein phosphorylation. In particular, most proteins are emerging to contain multiple phosphorylation sites. However, phosphoproteomics has outpaced current understanding of the functional roles of individual phospho-sites. In this paper the Phos-tag gel method is presented and discussed in the context of other available tools for phosphorylation research. Strengths and weaknesses of Phos-tag gels are outlined and an integrated approach to phosphorylation research is proposed. SignificanceThe Phos-tag gel method has unique strengths especially regarding the analysis of multi-site phosphorylation. A combined approach including Phos-tag gels together with other methods like isotope labelling, phospho-specific antibodies, and mass spectrometry is required to advance current understanding of protein phosphorylation.

Assays to Study IRE1 Activation and Signaling.

The endoplasmic reticulum (ER) stress sensor IRE1 is a a major player of the unfolded protein response (UPR), the main pathway driving adaptation processes to restore proteostasis.In addition, overactivation of IRE1 signaling contributes to a variety of pathologies including diabetes, neurodegenerative diseases, and cancer. Under ER stress, IRE1 auto-transphosphorylates and oligomerizes, triggering the activation of its endoribonuclease domain located in the cytosolic region. Active IRE1 catalyzes the splicing of the mRNA encoding for the XBP1 transcription factor, in addition to degrade several RNAs through a process known asregulated IRE1-dependent decay of mRNA (RIDD). Besides its role as an UPR transducer, several posttranslational modifications and protein-protein interactions can regulate IRE1 activity and modulate its signaling in the absence of stress.Thus, investigating the function of IRE1 in physiology and disease requires the use of complementary approaches. Here, we provide detailed protocols to perform four different assays to study IRE1 activation and signaling: (i) Phos-tag gels to evaluate the phosphorylation status of IRE1, (ii) microscopy using TREX-IRE1-GFP cells to measure IRE1 oligomerization, (iii) conventional RT-PCR to assess XBP1 mRNA processing, and (iv) quantitative PCR to determine the levels of canonical UPR target genes and the degradation of several mRNAs that are target of RIDD. We propose to use these experimental strategies as "gold standards" to study IRE1 signaling.

Exploring Obscurin and SPEG Kinase Biology.

Three members of the obscurin protein family that contain tandem kinase domains with important signaling functions for cardiac and striated muscles are the giant protein obscurin, its obscurin-associated kinase splice isoform, and the striated muscle enriched protein kinase (SPEG). While there is increasing evidence for the specific roles that each individual kinase domain plays in cross-striated muscles, their biology and regulation remains enigmatic. Our present study focuses on kinase domain 1 and the adjacent low sequence complexity inter-kinase domain linker in obscurin and SPEG. Using Phos-tag gels, we show that the linker in obscurin contains several phosphorylation sites, while the same region in SPEG remained unphosphorylated. Our homology modeling, mutational analysis and molecular docking demonstrate that kinase 1 in obscurin harbors all key amino acids important for its catalytic function and that actions of this domain result in autophosphorylation of the protein. Our bioinformatics analyses also assign a list of putative substrates for kinase domain 1 in obscurin and SPEG, based on the known and our newly proposed phosphorylation sites in muscle proteins, including obscurin itself.

The Serine/Threonine Protein Phosphatase 2A (PP2A) Regulates Syk Activity in Human Platelets.

Distinct membrane receptors activate platelets by Src-family-kinase (SFK)-, immunoreceptor-tyrosine-based-activation-motif (ITAM)-dependent stimulation of spleen tyrosine kinase (Syk). Recently, we reported that platelet activation via glycoprotein (GP) VI or GPIbα stimulated the well-established Syk tyrosine (Y)-phosphorylation, but also stoichiometric, transient protein kinase C (PKC)-mediated Syk serine(S)297 phosphorylation in the regulatory interdomain-B, suggesting possible feedback inhibition. The transient nature of Syk S297 phosphorylation indicated the presence of an unknown Syk pS297 protein phosphatase. In this study, we hypothesize that the S-protein phosphatase 2A (PP2A) is responsible for Syk pS297 dephosphorylation, thereby affecting Syk Y-phosphorylation and activity in human washed platelets. Using immunoblotting, we show that specific inhibition of PP2A by okadaic acid (OA) alone leads to stoichiometric Syk S297 phosphorylation, as analyzed by Zn2+-Phos-tag gels, without affecting Syk Y-phosphorylation. Pharmacological inhibition of Syk by PRT060318 or PKC by GF109203X only minimally reduced OA-induced Syk S297 phosphorylation. PP2A inhibition by OA preceding GPVI-mediated platelet activation induced by convulxin extended Syk S297 phosphorylation but inhibited Syk Y-phosphorylation. Our data demonstrate a novel biochemical and functional link between the S-protein phosphatase PP2A and the Y-protein kinase Syk in human platelets, and suggest that PP2A represents a potential enhancer of GPVI-mediated Syk activity caused by Syk pS297 dephosphorylation.

Abstract 518: Phosphorylation of MK5 at Threonine-182 in the Activation Loop is Mediated by P38α/β in Cardiac Fibroblasts

MAP kinase-activated protein kinase-5 (MK5) is a protein serine/threonine kinase involved in fibroblast function. MK5 is activated by phosphorylation at threonine-182 (Thr182): p38α/β, ERK3, and ERK4 have been implicated. We examined the phosphorylation of MK5 in adult cardiac ventricular fibroblasts. In serum-starved cardiac myofibroblasts (fibroblasts maintained on a plastic substrate), phospho-MK5 Thr182 (pThr182) immunoreactivity was predominantly nuclear. In response to serum, sorbitol, angiotensinII, TGFβ, or H 2 O 2 , pThr182 immunoreactivity both increased in intensity and relocated to the cytoplasm and the perinuclear region. In each case, the p38α/β inhibitor, SB203580, prevented both the increase in intensity and redistribution of pThr182 immunoreactivity. Incontrast, siRNA-mediated knockdown of ERK3 resulted in a diffuse cytosolic distribution of pThr182 immunoreactivity but failed to attenuate the increase in intensity. On Phos-tag PAGE, the electrophoretic mobility of phosphorylated proteins is reduced. Phos-tag PAGE resolved MK5 immunoreactivity from actively dividing myofibroblasts into several slower-migrating bands that were absent following 1) pretreatment with phosphoprotein phosphatase or 2) including EDTA in the Phos-tag gels. In serum-stimulated myofibroblasts, SB203580 reduced both the abundance of lower-mobility forms of MK5 on Phos-tag PAGE and the abundance of MK5 immunoreactivity in ERK3 immunoprecipitates. When fibroblasts were maintained on a compliant (8-kPa) substrate, and hence quiescent, the lower mobility forms of MK5 immunoreactivity were less abundant relative to myofibroblasts. Furthermore, in whole heart lysates from micesacrificed 8 weeks after constriction of the transverse aorta (TAC), Phos-tag PAGE revealed banding patterns consistent with increased MK5 phosphorylation relative to sham hearts. Taken together, these observations suggest: 1) p38α/β are the primarymediators of MK5 phosphorylation at Thr182 in cardiac fibroblasts, 2) ERK3 may be responsible for targeting activated MK5 to specific cytosolic sites, 3) Thr182 is not the only site at which MK5 is phosphorylated in vivo , and 4) MK5 phosphorylation increases with fibroblast activation.

Determining the Phosphorylation Status of Hippo Components YAP and TAZ Using Phos-tag.

Protein phosphorylation is one of the most important posttranslational modifications in cell signaling and regulation. Protein phosphorylation can be detected by site-specific phospho-antibodies, but generating these antibodies can be costly, time-consuming, and difficult. Recently, Phos-tag technology has been developed to detect protein phosphorylation. Here, we describe our method for using Phos-tag gels to compare the phosphorylation status of YAP and TAZ, the most important downstream effectors of the Hippo pathway.

Abstract 306: Identifying Downstream Effectors of the Extracellular Signal-related Kinase 1/2-controlled Directional Growth Response in Cardiomyocytes

A better understating of the mechanisms by which cardiomyocytes grow in different directions could open up new therapeutic avenues for hypertrophic and dilated cardiomyopathies, diseases that include the lengthening and widening of cardiomyocytes, respectively. To-date the only known proteins found to control this directional growth of cardiomyocytes are MEK1, mitogen-activated protein kinase kinase 1, and ERK1/2, extracellular signal-related kinase 1/2. The only known role of MEK1 is to phosphorylate ERK1/2 after MEK1 itself becomes phosphorylated through RAS/RAF signaling. Upon phospho-activation, ERK1/2 can interact with targets in the cytoplasm or translocate to the nucleus to induce cell growth and proliferation pathways. We previously found that cardiac overexpression of activated MEK1 causes cardiac hypertrophy and cardiomyocyte widening, while genetic ablation of ERK1/2 proteins in the heart causes dilation and cardiomyocyte lengthening. To determine how these proteins may control this directional growth response, we conducted a phosphoproteomic screen using neonatal rat ventricular myocytes (NRVMs) treated with activated MEK1 adenovirus, U0126 (a MEK1 inhibitor), or GFP adenovirus (control). For proteins with the greatest change in phosphorylation upon MEK1 activation or inhibition, we have verified the screen results using Phos-tag gels and western blots. Many of these hits have cytoskeletal or actin regulatory functions, and a few have also been implicated in stress-sensing in previous literature in other cells types. Furthermore, many hits showed increased protein levels in addition to augmented phosphorylation in NRVMs treated with activated MEK1 adenovirus. Given our nearly 20 interesting candidate proteins, we are currently conducting an in vivo screen in which each gene is individually expressed in mice using AAV9. After injection of virus into the thoracic cavities of P5 wild type mice, we are assessing if cardiac hypertrophy/dilation or cardiomyocyte dimensional growth are present at 4 months of age. Any novel candidates identified in this manner will be presented.

Analysis of Protein Phosphorylation Using Phos-Tag Gels.

Phos-tag gels are recent tools to dissect protein phosphorylation that operate by inducing a shift in the electrophoretic mobility of phosphorylated proteins compared to their nonphosphorylated counterparts. This article describes the preparation and electrophoresis of Zn2+ -Phos-tag gels along with electrotransfer of the separated phospho- and nonphosphoproteins onto a PVDF membrane using either wet-tank or semidry transfer. We also discuss the theory behind the technology with critical parameters to keep in mind for its successful application. © 2018 by John Wiley & Sons, Inc.

Monophosphorylation of cardiac troponin-I at Ser-23/24 is sufficient to regulate cardiac myofibrillar Ca2+ sensitivity and calpain-induced proteolysis

The acceleration of myocardial relaxation produced by β-adrenoreceptor stimulation is mediated in part by protein kinase A (PKA)-mediated phosphorylation of cardiac troponin-I (cTnI), which decreases myofibrillar Ca2+ sensitivity. Previous evidence suggests that phosphorylation of both Ser-23 and Ser-24 in cTnI is required for this Ca2+ desensitization. PKA-mediated phosphorylation also partially protects cTnI from proteolysis by calpain. Here we report that protein kinase D (PKD) phosphorylates only one serine of cTnI Ser-23/24. To explore the functional consequences of this monophosphorylation, we examined the Ca2+ sensitivity of force production and susceptibility of cTnI to calpain-mediated proteolysis when Ser-23/24 of cTnI in mouse cardiac myofibrils was nonphosphorylated, mono-phosphorylated, or bisphosphorylated (using sequential incubations in λ-phosphatase, PKD, and PKA, respectively). Phos-tag gels, Western blotting, and high-resolution MS revealed that PKD produced >90% monophosphorylation of cTnI, primarily at Ser-24, whereas PKA led to cTnI bisphosphorylation exclusively. PKD markedly decreased the Ca2+ sensitivity of force production in detergent-permeabilized ventricular trabeculae, whereas subsequent incubation with PKA produced only a small further fall of Ca2+ sensitivity. Unlike PKD, PKA also substantially phosphorylated myosin-binding protein-C and significantly accelerated cross-bridge kinetics (ktr). After phosphorylation by PKD or PKA, cTnI in isolated myofibrils was partially protected from calpain-mediated degradation. We conclude that cTnI monophosphorylation at Ser-23/24 decreases myofibrillar Ca2+ sensitivity and partially protects cTnI from calpain-induced proteolysis. In healthy cardiomyocytes, the basal monophosphorylation of cTnI may help tonically regulate myofibrillar Ca2+ sensitivity.

Post-translational regulation of a Porphyromonas gingivalis regulator

ABSTRACT Background: Bacteria use two-component signal transduction systems (among others) to perceive and respond to environmental changes. Within the genus Porphyromonas, we observed degeneration of these systems, as exemplified by the loss of RprX, the sensor kinase partner of the RprY. Objective: The purpose of this study was to investigate modulation of RprY function by acetylation. Design: The transcriptional activity of the rprY-pat genes were measured by RT-PCR and 5ʹ-RACE. The acetylation of RprY were detected by western blotting. Electromobility shift and in vitro ChIP assays were used to measure the DNA binding activity of RprY. The expression of RprY target genes was measured by qRT-PCR. Effects of acetylation on phosphorylation of RprY were measured by Phos-tag gels. Results: The rprY gene is cotranscribed with pat. RprY is acetylated in vivo, and autoacetylated in vitro in a reaction that is enhanced by Pat; the CobB sirtuin deacetylates RprY. Acetylation reduced the DNA binding of RprY. Induced oxidative stress decreased production of RprY in vivo, increased its acetylation and increased expression of nqrA. Conclusions: We propose that to compensate for the loss of RprX, P. gingivalis has evolved a novel mechanism to inactivate RprY through acetylation.

Abstract 215: Characterization by mass spectrometry of protein kinase C substrates differentially phosphorylated in LNCaP cells in response to phorbol ester and bryostatin 1 treatment

Abstract Bryostatin 1 (bryo 1) is a natural product of therapeutic interest for cancer and Alzheimer disease. Its unique behavior as a protein kinase C activator that paradoxically antagonizes many but not all phorbol ester responses has led to intense interest in its mechanism of action. Recently, using microarray analysis in two different cellular systems (LNCaP prostate cancer and U937 leukemia cell lines) where the typical phorbol ester PMA and bryo 1 have different biology, we have shown that a core mechanism contributing to the unique biology of bryo 1 is transiency of action resulting in a variable extent of decreased or missing late responses. We have excluded that there is a class of genes whose transcription is uniquely regulated at early times by bryo 1. In continuing to explore the mechanisms underlying the variable transiency of the responses by bryo 1 we have evaluated the substrates phosphorylated after treatment with PMA and bryo 1. Since PMA and bryo 1 induce differential subcellular localization of PKCs, they should result in differential access to substrates and consequent differences in the pattern of substrate phosphorylation. Whole cell lysates of LNCaP cells treated for 30 min with fully effective doses (100 nM) of PMA and bryo 1 and with DMSO as vehicle control were subjected to mass spectroscopic analysis. The comprehensive analysis identified several thousand phosphopeptides after drug treatment including the expected phosphopeptides for ERK2 (S185, Y187) and PKCdelta (S299, S302, S304). Many of peptides were similarly phosphorylated in response to both drugs (e.g. S641/S646 of Fam129B, S310 of actin-related protein 2/3 complex subunit 1B, S876 of RhoGEF and pleckstrin domain-containing protein 2, or multiple previously unknown sites for PKD1 (S239, S247), while a limited number were differentially phosphorylated. PMA specific phosphorylations included PKCdelta at Y313, mTOR at T2471, MAP4 at S624 and/or T627, E3 ubiquitin-protein ligases ZNRF2 at S82 and HUWE1 at S3818. Bryo 1 specific phosphorylations included BCL2-like 13 at S303, TOM1L2 at S424, and MAP2K2 in the T17, T25, S26 region. Selected specific phosphorylations are being validated using phostag gels, a method that enables the separation of phosphorylated proteins from their non-phosphorylated counterparts on SDS-gels. The identification of PKC substrates that are differentially phosphorylated by bryo 1 should both facilitate screening of other ligands capturing the biological behavior of bryo 1 as well as further illuminate the specifics of the pathways downstream of PKC activation by these differently acting ligands. Citation Format: Noemi Kedei, Sudipto Das, Thorkell Andresson, Peter M. Blumberg. Characterization by mass spectrometry of protein kinase C substrates differentially phosphorylated in LNCaP cells in response to phorbol ester and bryostatin 1 treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 215. doi:10.1158/1538-7445.AM2017-215

Specific glutamic acid residues in targeted proteins induce exaggerated retardations in Phos-tag SDS-PAGE migration.

We describe two unique proteins, Escherichia coli ClpX and human histone H2A, that show extremely retarded migrations relative to their molecular weights in Phos-tag SDS-PAGE, despite being nonphosphorylated. Although ClpX separated into multiple migration bands in Phos-tag gels, the separation was not due to phosphorylation. The N-terminal 47-61 region of ClpX was responsible for producing multiple phosphorylation-independent structural variants, even under denaturing conditions, and some of these variants were detected as highly up-shifted bands. By systematic Ala-scanning mutation analysis in the N-47-61 region, we concluded that the Glu-51 or Glu-54 residue was responsible for the appearance of exaggerated mobility-shifting bands. Histone H2A showed a much slower migration in Phos-tag gels in comparison with other major histones having similar molecular weights, and we found that the Glu-62 or Glu-65 residue caused the retarded migration. In addition, Phos-tag SDS-PAGE permitted us to detect a shift in the mobility of the phosphorylated form of histone H2A from that of the nonphosphorylated one. This is the first report showing that exaggerated retardation in the migration of a certain protein in Phos-tag SDS-PAGE is induced by interactions between the Phos-tag molecule and the carboxylate group of a specific Glu residue on the target.

MARK4 inhibits Hippo signaling to promote proliferation and migration of breast cancer cells.

The Hippo pathway is a critical regulator of tissue size, and aberrations in pathway regulation lead to cancer. MST1/2 and LATS1/2 kinases comprise the core of the pathway that, in association with adaptor proteins SAV and MOB, functions in a sequential manner to phosphorylate and inhibit the transcription factors YAP and TAZ. Here we identify mammalian MARK family members as activators of YAP/TAZ. We show that depletion of MARK4 in MDA-MB-231 breast cancer cells results in the loss of nuclear YAP/TAZ and decreases the expression of YAP/TAZ targets. We demonstrate that MARK4 can bind to MST and SAV, leading to their phosphorylation, and that MARK4 expression attenuates the formation of a complex between MST/SAV and LATS, which depends on the kinase activity of MARK4. Abrogation of MARK4 expression using siRNAs and CRISPR/Cas9 gene editing attenuates the proliferation and migration of MDA-MB-231 cells. Our results show that MARK4 acts as a negative regulator of the Hippo kinase cassette to promote YAP/TAZ activity and that loss of MARK4 restrains the tumorigenic properties of breast cancer cells.

Non-radioactive in vitro PINK1 Kinase Assays Using Ubiquitin or Parkin as Substrate.

This protocol describes the in vitro phosphorylation of ubiquitin and Parkin by the kinase PINK1 using recombinant proteins. Both substrates, ubiquitin and Parkin, are phosphorylated at the conserved serine 65 residue (pS65-ubiquitin and pS65-Parkin). The protocol also includes the use of monomeric and K48- and K63-linked poly-ubiquitin chains as alternative substrates. Although there are commercially available antibodies, we have not tested their performance in this assay since, but used validated antibodies from our laboratory. An alternative antibody-independent method, the use of phostag gels to detect pS65-ubiquitin and pS65-Parkin, is described in addition.

Cyclic Nucleotide-dependent Protein Kinases Target ARHGAP17 and ARHGEF6 Complexes in Platelets

Endothelial cells release prostacyclin (PGI2) and nitric oxide (NO) to inhibit platelet functions. PGI2 and NO effects are mediated by cyclic nucleotides, cAMP- and cGMP-dependent protein kinases (PKA, PKG), and largely unknown PKA and PKG substrate proteins. The small G-protein Rac1 plays a key role in platelets and was suggested to be a target of cyclic nucleotide signaling. We confirm that PKA and PKG activation reduces Rac1-GTP levels. Screening for potential mediators of this effect resulted in the identification of the Rac1-specific GTPase-activating protein ARHGAP17 and the guanine nucleotide exchange factor ARHGEF6 as new PKA and PKG substrates in platelets. We mapped the PKA/PKG phosphorylation sites to serine 702 on ARHGAP17 using Phos-tag gels and to serine 684 on ARHGEF6. We show that ARHGAP17 binds to the actin-regulating CIP4 protein in platelets and that Ser-702 phosphorylation interferes with this interaction. Reduced CIP4 binding results in enhanced inhibition of cell migration by ARHGAP17. Furthermore, we show that ARHGEF6 is constitutively linked to GIT1, a GAP of Arf family small G proteins, and that ARHGEF6 phosphorylation enables binding of the 14-3-3 adaptor protein to the ARHGEF6/GIT1 complex. PKA and PKG induced rearrangement of ARHGAP17- and ARHGEF6-associated protein complexes might contribute to Rac1 regulation and platelet inhibition.

Phosphorylation events in the multiple gene regulator of group A Streptococcus significantly influence global gene expression and virulence.

Whole-genome sequencing analysis of ∼800 strains of group A Streptococcus (GAS) found that the gene encoding the multiple virulence gene regulator of GAS (mga) is highly polymorphic in serotype M59 strains but not in strains of other serotypes. To help understand the molecular mechanism of gene regulation by Mga and its contribution to GAS pathogenesis in serotype M59 GAS, we constructed an isogenic mga mutant strain. Transcriptome studies indicated a significant regulatory influence of Mga and altered metabolic capabilities conferred by Mga-regulated genes. We assessed the phosphorylation status of Mga in GAS cell lysates with Phos-tag gels. The results revealed that Mga is phosphorylated at histidines in vivo. Using phosphomimetic and nonphosphomimetic substitutions at conserved phosphoenolpyruvate:carbohydrate phosphotransferase regulation domain (PRD) histidines of Mga, we demonstrated that phosphorylation-mimicking aspartate replacements at H207 and H273 of PRD-1 and at H327 of PRD-2 are inhibitory to Mga-dependent gene expression. Conversely, non-phosphorylation-mimicking alanine substitutions at H273 and H327 relieved inhibition, and the mutant strains exhibited a wild-type phenotype. The opposing regulatory profiles observed for phosphorylation- and non-phosphorylation-mimicking substitutions at H273 extended to global gene regulation by Mga. Consistent with these observations, the H273D mutant strain attenuated GAS virulence, whereas the H273A strain exhibited a wild-type virulence phenotype in a mouse model of necrotizing fasciitis. Together, our results demonstrate phosphoregulation of Mga and its direct link to virulence in M59 GAS strains. These data also lay a foundation toward understanding how naturally occurring gain-of-function variations in mga, such as H201R, may confer an advantage to the pathogen and contribute to M59 GAS pathogenesis.

Phosphorylation status of human RNA-binding protein 8A in cells and its inhibitory regulation by Magoh.

The RNA-binding protein 8A (RBM8A)-mago-nashi homolog, proliferation-associated (Magoh) complex is a component of the exon junction complex (EJC) required for mRNA metabolism involving nonsense-mediated mRNA decay (NMD). RBM8A is a phosphorylated protein that plays some roles in NMD. However, the detailed status and mechanism of the phosphorylation of RBM8A is not completely understood. Therefore, in this study, we analyzed in detail RBM8A phosphorylation in human cells. Accordingly, analysis of the phosphorylation status of RBM8A protein in whole-cell lysates by using Phos-tag gels revealed that the majority of endogenous RBM8A was phosphorylated throughout the cell-cycle progression. Nuclear and cytoplasmic RBM8A and RBM8A in the EJC were also found to be mostly phosphorylated. We also screened the phosphorylated serine by mutational analysis using Phos-tag gels to reveal modifications of serine residues 166 and 168. A single substitution at position 168 that concomitantly abolished the phosphorylation of serine 166 suggested the priority of kinase reaction between these sites. Furthermore, analysis of the role of the binding protein Magoh in RBM8A phosphorylation revealed its inhibitory effect invitro and invivo. Thus, we conclude that almost all synthesized RBM8A proteins are rapidly phosphorylated in cells and that phosphorylation occurs before the complex formation with Magoh.

Tips on improving the efficiency of electrotransfer of target proteins from Phos-tag SDS-PAGE gel

The sensitivity of Western blotting analysis after Phos-tag SDS-PAGE is occasionally inferior to that after normal (Phos-tag-free) SDS-PAGE under similar experimental conditions, possibly as a result of inefficient electrotransfer from the Phos-tag gel to the blotting membrane. We therefore present tips on improving the efficiency of electrotransfer of proteins in semidry and wet-tank blotting. When model samples containing several standard phosphoproteins were subjected to semidry blotting, their electrotransfer efficiencies after Phos-tag SDS-PAGE were markedly inferior to those of their dephosphorylated counterparts in the same gel. This was ameliorated by immersing the electrophoresed Phos-tag gel in a transfer buffer containing 1 mM EDTA for 30 min before electroblotting. Similarly, phosphoproteomes in crude cell extracts were inefficiently transferred by semidry blotting, but the efficiencies of their electrotransfer were improved by pretreatment with EDTA. In contrast, the efficiencies of wet-tank blotting of the same samples were not dependent on the degree of phosphorylation, and the efficiencies of electrotransfer of all proteins from Phos-tag gels were similar to those from normal gels. In some cases involving the use of a Phos-tag gel, addition of 0.1% w/v of SDS to the transfer buffer significantly improved the electrotransfer.