Phosphorylated Amino Acid Research Articles

Phosphorylation of the Consensus Sites of Protein Kinase A on α1D L-type Calcium Channel

The novel alpha(1D) L-type Ca(2+) channel is expressed in supraventricular tissue and has been implicated in the pacemaker activity of the heart and in atrial fibrillation. We recently demonstrated that PKA activation led to increased alpha(1D) Ca(2+) channel activity in tsA201 cells by phosphorylation of the channel protein. Here we sought to identify the phosphorylated PKA consensus sites on the alpha(1) subunit of the alpha(1D) Ca(2+) channel by generating GST fusion proteins of the intracellular loops, N terminus, proximal and distal C termini of the alpha(1) subunit of alpha(1D) Ca(2+) channel. An in vitro PKA kinase assay was performed for the GST fusion proteins, and their phosphorylation was assessed by Western blotting using either anti-PKA substrate or anti-phosphoserine antibodies. Western blotting showed that the N terminus and C terminus were phosphorylated. Serines 1743 and 1816, two PKA consensus sites, were phosphorylated by PKA and identified by mass spectrometry. Site directed mutagenesis and patch clamp studies revealed that serines 1743 and 1816 were major functional PKA consensus sites. Altogether, biochemical and functional data revealed that serines 1743 and 1816 are major functional PKA consensus sites on the alpha(1) subunit of alpha(1D) Ca(2+) channel. These novel findings provide new insights into the autonomic regulation of the alpha(1D) Ca(2+) channel in the heart.

Proteomic Analysis of HCV Cirrhosis and HCV-Induced HCC: Identifying Biomarkers for Monitoring HCV-Cirrhotic Patients Awaiting Liver Transplantation

Progression from chronic hepatitis C virus (HCV) infection to cirrhosis and hepatocellular carcinoma (HCC) results in protein changes in the peripheral blood. We evaluated global protein expression in plasma samples of HCV-cirrhotic and HCV-cirrhotic-HCC patients. Plasma samples from 25 HCV-cirrhotic-HCC and 10 HCV-cirrhotic patients were quantitatively evaluated for protein expression. Tryptic peptides were analyzed using Thermo linear ion-trap mass spectrometer (LTQ) coupled with a Surveyor HPLC system (Thermo). SEQUEST and X!Tandem database search algorithms were used for peptide sequence identification. Protein relative quantification was performed using the area under the curve from the select ion chromatogram. A significant fold change between groups was based on controlling the false discovery rate (FDR) at less than 5%. We identified and quantified 2320 proteins from the analysis of the different protein pattern between HCV-cirrhosis and HCV-HCC samples. Gene ontology terms classified the more important biologic process related to these proteins as signal transduction, regulation of transcription DNA-dependent, protein amino acid phosphorylation, cell adhesion, transport, and immune response. Seven proteins showed significant expression changes with a FDR less than 5% between cirrhosis and tumor groups. Moreover, 18 proteins showed significant expression changes (FDR <5%) when plasma samples from HCV-cirrhosis were compared with early HCV-HCC. Differential protein expression was observed between samples from HCV patients with cirrhosis with and without HCC. Also, differences were observed between early and advanced HCV-HCC samples. This study provides important information for discovery of potential biomarkers for early HCC diagnosis in HCV cirrhotic patients.

Controlling Peptide Folding with Repulsive Interactions between Phosphorylated Amino Acids and Tryptophan

Controlling Peptide Folding with Repulsive Interactions between Phosphorylated Amino Acids and Tryptophan

Characterization of biomimetc material, Mussel adhesive protein, using computer tools and servers.

In this paper six different mussel adhesion proteins (MAPs) retrieved from SwissProt database are analyzed using computer tools and servers. Primary analysis showed that all the MAPs are hydrophobic in nature due to the high content of non-polar amino acid residues. The sequence analysis showed the absence of sulphide bridges and non polar amino acid tryptophan in MAPs. Signal peptide is a common feature in MAPs. The aliphatic index computed by Expasy's Protparm infers that most of the MAPs are stable at a wide range of temperature. Secondary structure analysis showed that MAPs could results in a better interaction with water. The secondary structure analysis showed MAPs contain more βhelices and turns. The SOSUI server predicts one transmembarne region in Mytilus galprovincialis. The predicted transmembrane region was visualized and analyzed using helical wheel plots generated by EMBOSS PepWheel tool. The absence of disulphide bonds in MAPS was confirmed by SYC_REC tools and from 3 Dimensional structures created by Rasmol tool. The cystein position identified by Rasmol tool might be correct as the evaluated parameters (Rampage, CE and ProQ) are within the accepted limits for the model 3D structure. The signal peptide identified by SignalP server showed that they are secretary pathway in localization except in Mytilus edulus, where it is cytoplasmic. Based on the signal peptides reliability class the MAPs are classified into three groups. The phosphorylation sites of serine, threonoine and tyrosine are predicted by NetPhos server. Interestingly tyrosine is the most phosphorylated amino acid in the MAPs; hence it is inferred that MAPs can be purified by using antibodies.

高エネルギー電子移動解離が可能なタンデム質量分析装置の開発

A JEOL JMS-HX110 double-focusing mass spectrometer was combined with the collision cell and spherical electric sector of a HITACHI M80-B instrument to perform tandem mass spectrometry experiments. A quadrupole lens doublet was mounted between the mass spectrometer and the collision cell to improve ion transmission into the collision cell. The detection system was also improved to expand the mass range of product ions. These improvements provided reliable spectra of high-energy electron transfer dissociation (HE-ETD) of peptides using an alkali metal target. The present results demonstrated that HE-ETD by a single collision with an alkali metal target could determine the amino acid sequence and phosphorylation site of peptides.

An integrated comparative phosphoproteomic and bioinformatic approach reveals a novel class of MPM-2 motifs upregulated in EGFRvIII-expressing glioblastoma cells

Glioblastoma (GBM, WHO grade IV) is an aggressively proliferative and invasive brain tumor that carries a poor clinical prognosis with a median survival of 9 to 12 months. In a prior phosphoproteomic study performed in the U87MG glioblastoma cell line, we identified tyrosine phosphorylation events that are regulated as a result of titrating EGFRvIII, a constitutively active mutant of the epidermal growth factor receptor (EGFR) associated with poor prognosis in GBM patients. In the present study, we have used the phosphoserine/phosphothreonine-specific antibody MPM-2 (mitotic protein monoclonal #2) to quantify serine/threonine phosphorylation events in the same cell lines. By employing a bioinformatic tool to identify amino acid sequence motifs regulated in response to increasing oncogene levels, a set of previously undescribed MPM-2 epitope sequence motifs orthogonal to the canonical "pS/pT-P" motif was identified. These motifs contain acidic amino acids in combinations of the -5, -2, +1, +3, and +5 positions relative to the phosphorylated amino acid. Phosphopeptides containing these motifs are upregulated in cells expressing EGFRvIII, raising the possibility of a general role for a previously unrecognized acidophilic kinase (e.g. casein kinase II (CK2)) in cell proliferation downstream of EGFR signaling.

Profiling of Phosphorylated Proteins in Human Fetal Liver

Constitutively phosphorylated proteins in Human fetal liver (HFL) aged 16-24 wk of gestation were studied using a 2-DE step followed by western blotting detection and MS identification. We found 166 phosphorylated protein spots with quantitative information and identified 101 gene products. Of theses identified proteins, 57 contain phosphoserine, 49 contain phosphothreonine, 51 contain phosphotyrosine, and 64 were newly identified phosphorylated proteins. The possible phosphorylation sites were further predicted using Netphos, ScanProsite and Scansite programs and most proteins were predicted the same site by at least 2 programs. Integrating the functional categories, protein abundance and the degree of phosphorylation of these proteins, we found proteins related to carbohydrate, lipid and amino acid metabolism were highly expressed with also the high degree of all serine, threonine and tyrosine phosphorylation; proteins associated with hematopoiesis were relatively highly expressed but with a relatively low degree of phosphorylation at serine, threonine and tyrosine; the proteins for signal transduction; biosynthesis of secondary metabolites and those whose function were unknown were lowly expressed, but with the zhigh degree of phosphorylation and interestingly, serine was the main phosphorylated amino acid of signal transducers; threonine in enzymes of biosynthesis of secondary metabolites; and tyrosine in proteins with unknown function.

195 TRANSCRIPTOME PROFILING OF BOVINE ENDOMETRIUM BASED ON THE PREGNANCY OUTCOME AFTER TRANSFER OF IN VIVO-DERIVED EMBRYOS

Transcriptome profiling of pre-transfer cycle endometrium in relation to its ability to sustain the upcoming pregnancy may pave the way to develop or identify molecular markers that can be utilized to detect and select receptive endometrium before embryo transfer. Here, we aim to show differential expression of genes between endometrium biopsies derived from recipients during pre-transfer cycle based on the pregnancy successes after embryo transfer. For this, endometrium biopsies were taken from 56 Simmental cyclic heifers of the same age at day 7 and 14 of the estrous cycle. On the next cycle, in vivo-produced day 7 blastocysts were transferred to all animals at day 7 of the estrous cycle. Pregnancy diagnosis was done at 28, 42, and 56 days of gestation. Thirty-two cows were returned to heat after 21 days, 7 were pregnant until day 42 but no pregnancy after that, and 15 resulted in calf delivery. Subsequently, the endometrium biopsies sampled during the pre-transfer period were categorized based on the pregnancy outcome. Those endometrial biopsies taken at days 7 and 14 during the pre-transferred period from those that resulted in successful calf delivery were named as d7CD and d14CD, respectively, and those endometrial samples collected at days 7 and 14 during pre-transferred period from those groups that resulted in no pregnancy were named as d7NP and d14NP, respectively. Total RNA was extracted from 3 pools of each experimental group in 3 replicates using RNeasy mini kit (Qiagen, Hilden, Germany). A total of 12 biotin-labeled cRNA samples were hybridized on 12 bovine Affymetrix arrays (Affymetrix, Santa Clara, CA, USA) consisting of 24128 probe sets. The microarray data normalization and background correction was performed using guanine cytosine robust multi-array analysis, and the data analysis was performed using LIMMA written on R package, which maintained the bioconductor. The results showed that 1130 transcripts were differentially expressed between d7CD and d7NP, of which 626 and 504 were up- and down-regulated, respectively, in d7CD. Genes that involve in regulation of transcription (PPARA, NR2F1, MYB, MYB, and CHF2) and the collagen families (COL1A1 and COL1A2) were enriched in d7CD. A total of 234 transcripts were differentially expressed between d14CD and d14NP, of which 94 and 140 were up- and down-regulated, respectively, in d14CD compared to d14NP. Transcripts involved in protein amino acid phosphorylation (MA2K6, GATM, AK3L1, and MAPK10) were found to be enriched in d14CD compared to d14NP. In conclusion, pre-transfer endometrium biopsies showed significant differences in transcriptome profile depending on the pregnancy outcome after transfer of in vivo-derived blastocysts and enable to identify transcripts related to pregnancy establishment.

Requirements for Protein Phosphorylation and the Kinase Activity of Polo-like Kinase 1 (Plk1) for the Kinetochore Function of Mitotic Arrest Deficiency Protein 1 (Mad1)

Mitotic arrest deficiency protein 1 (Mad1) is associated with microtubule-unattached kinetochores in mitotic cells and is a component of the spindle assembly checkpoint (SAC). Here, we have studied the phosphorylation of Mad1 and mapped using liquid chromatography-tandem mass spectrometry several phosphorylated amino acids in this protein. One phosphorylated residue, Thr680, was characterized to be important for the kinetochore localization of Mad1 and its SAC function. We also found that in mitotic cells Mad1 co-immunoprecipitated with Plk1. Depletion of cellular Plk1 using small interfering RNAs and inhibition of the kinase activity of Plk1 using a kinase-dead mutant or a small molecule inhibitor attenuated Mad1 phosphorylation and its association with kinetochores. Collectively, these findings indicate mechanistic roles contributed by protein phosphorylation and Plk1 to the SAC activity of Mad1.

An in silico strategy identified the target gene candidates regulated by dehydration responsive element binding proteins (DREBs) in Arabidopsis genome

Identification of downstream target genes of stress-relating transcription factors (TFs) is desirable in understanding cellular responses to various environmental stimuli. However, this has long been a difficult work for both experimental and computational practices. In this research, we presented a novel computational strategy which combined the analysis of the transcription factor binding site (TFBS) contexts and machine learning approach. Using this strategy, we conducted a genome-wide investigation into novel direct target genes of dehydration responsive element binding proteins (DREBs), the members of AP2-EREBPs transcription factor super family which is reported to be responsive to various abiotic stresses in Arabidopsis. The genome-wide searching yielded in total 474 target gene candidates. With reference to the microarray data for abiotic stresses-inducible gene expression profile, 268 target gene candidates out of the total 474 genes predicted, were induced during the 24-h exposure to abiotic stresses. This takes about 57% of total predicted targets. Furthermore, GO annotations revealed that these target genes are likely involved in protein amino acid phosphorylation, protein binding and Endomembrane sorting system. The results suggested that the predicted target gene candidates were adequate to meet the essential biological principle of stress-resistance in plants.

Mechanisms and regulation of vitamin C uptake: studies of the hSVCT systems in human liver epithelial cells

Humans use two sodium-ascorbate cotransporters (hSVCT1 and hSVCT2) for transporting the dietary essential micronutrient ascorbic acid, the reduced and active form of vitamin C. Although the human liver plays a pivotal role in regulating and maintaining vitamin C homeostasis, vitamin C transport physiology and regulation of the hSVCT systems in this organ have not been well defined. Thus, this research used a human hepatic cell line (HepG2), confirming certain results with primary human hepatocytes and determined the initial rate of ascorbic acid uptake to be Na(+) gradient, pH dependent, and saturable as a function of concentration over low and high micromolar ranges. Additionally, hSVCT2 protein and mRNA are expressed at higher levels in HepG2 cells and native human liver, and the cloned hSVCT2 promoter has more activity in HepG2 cells. Results using short interfering RNA suggest that in HepG2 cells, decreasing hSVCT2 message levels reduces the overall ascorbic acid uptake process more than decreasing hSVCT1 message levels. Activation of PKC intracellular regulatory pathways caused a downregulation in ascorbic acid uptake not mediated by a single predicted PKC-specific amino acid phosphorylation site in hSVCT1 or hSVCT2. However, PKC activation causes internalization of hSVCT1 but not hSVCT2. Examination of other intracellular regulatory pathways on ascorbic acid uptake determined that regulation also potentially occurs by PKA, PTK, and Ca(2+)/calmodulin, but not by nitric oxide-dependent pathways. These studies are the first to determine the overall ascorbic acid uptake process and relative expression, regulation, and contribution of the hSVCT systems in human liver epithelial cells.

Emerging roles for protein histidine phosphorylation in cellular signal transduction: lessons from the islet β‐cell

Protein phosphorylation represents one of the key regulatory events in physiological insulin secretion from the islet β-cell. In this context, several classes of protein kinases (e.g. calcium-, cyclic nucleotide- and phospholipid-dependent protein kinases and tyrosine kinases) have been characterized in the β-cell. The majority of phosphorylated amino acids identified include phosphoserine, phosphothreonine and phosphotyrosine. Protein histidine phosphorylation has been implicated in the prokaryotic and eukaryotic cellular signal transduction. Most notably, phoshohistidine accounts for 6% of total protein phosphorylation in eukaryotes, which makes it nearly 100-fold more abundant than phosphotyrosine, but less abundant than phosphoserine and phosphothreonine. However, very little is known about the number of proteins with phosphohistidines, since they are highly labile and are rapidly lost during phosphoamino acid identification under standard experimental conditions. The overall objectives of this review are to: (i) summarize the existing evidence indicating the subcellular distribution and characterization of various histidine kinases in the islet β-cell, (ii) describe evidence for functional regulation of these kinases by agonists of insulin secretion, (iii) present a working model to implicate novel regulatory roles for histidine kinases in the receptor-independent activation, by glucose, of G-proteins endogenous to the β-cell, (iv) summarize evidence supporting the localization of protein histidine phosphatases in the islet β-cell and (v) highlight experimental evidence suggesting potential defects in the histidine kinase signalling cascade in islets derived from the Goto-Kakizaki (GK) rat, a model for type 2 diabetes. Potential avenues for future research to further decipher regulatory roles for protein histidine phosphorylation in physiological insulin secretion are also discussed.

Characteristic negative ion fragmentations of deprotonated peptides containing post‐translational modifications: mono‐phosphorylated Ser, Thr and Tyr. A joint experimental and theoretical study

Peptides and proteins may contain post-translationally modified phosphorylated amino acid residues, in particular phosphorylated serine (pSer), threonine (pThr) and tyrosine (pTyr). Following earlier work by Lehmann et al., the [M-H]- anions of peptides containing pSer and pThr functionality show loss of the elements of H3PO4. This process, illustrated for Ser (and using a model system), is CH3CONH-C(CH2OPO3H2)CONHCH(3) --> [CH3CONHC(==CH2)CONHCH3 (-OPO3H2)] (a) --> [CH3CONHC(==CH2)CONHCH3-H]- + H3PO4, a process endothermic by 83 kJ mol(-1) at the MP2/6-31++G(d,p)//HF/6-31++G(d,p) level of theory. In addition, intermediate (a) may decompose to yield CH3CONHC(==CH2)CONHCH3 + H2PO4 - in a process exothermic by 3 kJ mol(-1). The barrier to the transition state for these two processes is 49 kJ mol(-1). Characteristic cleavages of pSer and pThr are more energetically favourable than the negative ion backbone cleavages of peptides described previously. In contrast, loss of HPO3 from [M-H]- is characteristic of pTyr. The cleavage [NH2CH(CH2-C6H4-OPO3H-)CO2H] --> [NH2C(CH2-C6H4-O-)CO2H (HPO3)] (b) --> NH2CH(CH2-C6H4-O-)CO2H + HPO3 is endothermic by 318 kJ mol(-1) at the HF/6-31+G(d)//AM1 level of theory. In addition, intermediate (b) also yields NH2CH(CH2-C6H4-OH)CO2H + PO3 - (reaction endothermic by 137 kJ mol(-1)). The two negative ion cleavages of pTyr have a barrier to the transition state of 198 kJ mol(-1) (at the HF/6-31+G(d)//AM1 level of theory) comparable with those already reported for negative ion backbone cleavages.

Giardia lamblia: Characterization of ecto-phosphatase activities

Ecto-phosphatase activities of Giardia lamblia were characterized in intact cells, which are able to hydrolyze the artificial substrate p-nitrophenylphosphate ( p-NPP) to p-nitrophenol ( p-NP) at a rate of 8.4 ± 0.8 nmol p-NP/h/10 7 cells. The ecto-phosphatase activities were inhibited at high pH as well as by classical inhibitors of acid phosphatases, such as sodium fluoride and sodium molybdate and by inorganic phosphate, the final product of the reaction. Experiments using a classical inhibitor of phosphotyrosine phosphatase, sodium orthovanadate, also showed that the ecto-phosphatase activity was inhibited in a dose-dependent manner. Different phosphorylated amino acids were used as substrates for the G. lamblia ecto-phosphatase activities the highest rate of phosphate release was achieved using phosphotyrosine. Not only p-NPP hydrolysis but also phosphotyrosine hydrolysis was inhibited by sodium orthovanadate. Phosphotyrosine but not phospho-serine or phospho-threonine inhibited the p-nitrophenylphosphatase activity. We also observed a positive correlation between the ecto-phosphatase activity and the capacity to encystation of G. lamblia trophozoites.

Evaluating the coupling efficiency of phosphorylated amino acids for SPOT synthesis

A high demand of interest concerning binding assays to study the consequences of posttranscriptional phosphorylation may be addressed by peptide array-based methods. A crucial factor for de novo chemical approaches to generate such arrays is the possibility to rationally permutate phosphorylation events along a huge number of sequences. The simple principle behind this advantage is the stepwise synthesis of peptides, which allows the incorporation of either phosphorylated or nonphosphorylated derivates at serine, threonine, and tyrosine positions. In spite of several reported applications of phosphopeptide arrays, there is, to our best knowledge, no reported analysis of the efficiency of the involved techniques. Here, we analyze different coupling conditions to introduce phosphoamino acids in standard SPOT synthesis. Our results clearly indicate that EEDQ is the preferable activator and can also be used in fully automated SPOT synthesis.

Exploring the intramolecular phosphorylation sites in human Chk2

A comparative biochemical analysis was performed using recombinant human protein kinase Chk2 (checkpoint kinase 2) expressed in bacteria and insect cells. Dephosphorylated, inactive, recombinant human Chk2 could be reactivated in a concentration-dependent manner. Despite distinct time-dependent autophosphorylation kinetics by monitoring the phosphorylation of amino acid residues T68, S19, S33/35, T432, in Chk2 wildtype and Chk2 mutants (T68A, T68D and Q69E) they gave identical specific activities. However, upon gel filtration of Chk2 wildtype and the mutants, only Chk2 wildtype and the T68D mutant led to the formation of a ‘pure’ dimer; dephosphorylated wildtype Chk2 eluted as a monomer. Transfection of HEK293 cells with Chk2 wildtype and Chk2 mutants in the absence or presence of DNA damage showed significant T68 phosphorylation already in the absence of DNA damaging reagents. Upon DNA damage, phosphorylation of additional Chk2 sites was observed (S19, S33/35). A comparison of ATM +/+ and ATM −/− cells with respect to phosphorylation of residues T68, S19, S33/35 in the absence and presence of DNA damage showed in all cases phosphorylation of T68, although signal intensity was increased ca. three-fold after DNA damage. Mass spectrometric analyses of human recombinant Chk2 isolated from bacteria and insect cells showed distinct differences. The number of phosphorylated residues in human recombinant Chk2 isolated from bacteria was 16, whereas in the case of the recombinant human Chk2 from insect cells it was 8. Except for phosphorylated amino acid T378 which was not found in the Chk2 isolated from bacteria, all other phosphorylated residues identified in human Chk2 from insect cells were present also in Chk2 from bacteria.

Vasopressin-stimulated Increase in Phosphorylation at Ser269 Potentiates Plasma Membrane Retention of Aquaporin-2

Vasopressin controls water excretion through regulation of aquaporin-2 (AQP2) trafficking in renal collecting duct cells. Using mass spectrometry, we previously demonstrated four phosphorylated serines (Ser256, Ser261, Ser264, and Ser269) in the carboxyl-terminal tail of rat AQP2. Here, we used phospho-specific antibodies and protein mass spectrometry to investigate the roles of vasopressin and cyclic AMP in the regulation of phosphorylation at Ser269 and addressed the role of this site in AQP2 trafficking. The V2 receptor-specific vasopressin analog dDAVP increased Ser(P)269-AQP2 abundance more than 10-fold, but at a rate much slower than the corresponding increase in Ser256 phosphorylation. Vasopressin-mediated changes in phosphorylation at both sites were mimicked by cAMP addition and inhibited by protein kinase A (PKA) antagonists. In vitro kinase assays, however, demonstrated that PKA phosphorylates Ser256, but not Ser269. Phosphorylation of AQP2 at Ser269 did not occur when Ser256 was replaced by an unphosphorylatable amino acid, as seen in both S256L-AQP2 mutant mice and in Madin-Darby canine kidney cells expressing an S256A mutant, suggesting that Ser269 phosphorylation depends upon prior phosphorylation at Ser256. Immunogold electron microscopy localized Ser(P)269-AQP2 solely in the apical plasma membrane of rat collecting duct cells, in contrast to the other three phospho-forms (found in both apical plasma membrane and intracellular vesicles). Madin-Darby canine kidney cells expressing an S269D "phosphomimic" AQP2 mutant showed constitutive localization at the plasma membrane. The data support a model in which vasopressin-mediated phosphorylation of AQP2 at Ser269:(a) depends on prior PKA-mediated phosphorylation of Ser256 and (b) enhances apical plasma membrane retention of AQP2.

Ubiquitin Chain Editing Revealed by Polyubiquitin Linkage-Specific Antibodies

Posttranslational modification of proteins with polyubiquitin occurs in diverse signaling pathways and is tightly regulated to ensure cellular homeostasis. Studies employing ubiquitin mutants suggest that the fate of polyubiquitinated proteins is determined by which lysine within ubiquitin is linked to the C terminus of an adjacent ubiquitin. We have developed linkage-specific antibodies that recognize polyubiquitin chains joined through lysine 63 (K63) or 48 (K48). A cocrystal structure of an anti-K63 linkage Fab bound to K63-linked diubiquitin provides insight into the molecular basis for specificity. We use these antibodies to demonstrate that RIP1, which is essential for tumor necrosis factor-induced NF-kappaB activation, and IRAK1, which participates in signaling by interleukin-1beta and Toll-like receptors, both undergo polyubiquitin editing in stimulated cells. Both kinase adaptors initially acquire K63-linked polyubiquitin, while at later times K48-linked polyubiquitin targets them for proteasomal degradation. Polyubiquitin editing may therefore be a general mechanism for attenuating innate immune signaling.

The Role of Phosphorylated Residues in Peptide–Peptide Noncovalent Complexes Formation

Electrospray mass spectrometry (ESI-MS) has become the tool of choice for the study of noncovalent complexes. Our previous work has highlighted the role of phosphorylated amino acid residues in the formation of noncovalent complexes through electrostatic interaction with arginine residues' guanidinium groups. In this study, we employ tandem mass spectrometry to investigate the gas-phase stability and dissociation pathways of these noncovalent complexes. The only difference in the three phosphopeptides tested is the nature of the phosphorylated amino acid residue. In addition the absence of acidic residues and an amidated carboxyl terminus insured that the only negative charge came from the phosphate, which allowed for the comparison of the noncovalent bond between arginine residues and each of the different phosphorylated residues. Dissociation curves were generated by plotting noncovalent complex ion intensities as a function of the nominal energy given to the noncovalent complex ion before entering the collision cell. These results showed that noncovalent complexes formed with phosphorylated tyrosine were the most stable, followed by serine and threonine, which had similar stability.

P190A RhoGAP Is a Glycogen Synthase Kinase-3-β Substrate Required for Polarized Cell Migration

The Rho GTPases are critical regulators of the actin cytoskeleton and are required for cell adhesion, migration, and polarity. Among the key Rho regulatory proteins in the context of cell migration are the p190 RhoGAPs (p190A and p190B), which function to modulate Rho signaling in response to integrin engagement. The p190 RhoGAPs undergo complex regulation, including phosphorylation by several identified kinases, interactions with phospholipids, and association with a variety of cellular proteins. Here, we have identified an additional regulatory mechanism unique to p190A RhoGAP that involves priming-dependent phosphorylation by glycogen synthase-3-beta (GSK-3beta), a kinase previously implicated in establishing cell polarity. We found that p190A-deficient fibroblasts exhibit a defect in directional cell migration reflecting a requirement for GSK-3beta-mediated phosphorylation of amino acids in the C-terminal "tail" of p190A. This phosphorylation leads to inhibition of p190A RhoGAP activity in vitro and in vivo. These studies identify p190A as a novel GSK-3beta substrate and reveal a mechanism by which GSK-3beta contributes to cellular polarization in directionally migrating cells via effects on Rho GTPase activity.