Tyrosine Phosphorylation Motifs Research Articles

Role of Abl and Src family kinases in actin-cytoskeletal rearrangements induced by the Helicobacter pylori CagA protein

The clinical outcome of infections with Helicobacter pylori is determined by a complex interplay of host–pathogen interactions, and persistent infection with this pathogen is the major cause of developing chronic gastritis, peptic ulcers and gastric cancer. Highly virulent strains encode a so-called type IV secretion system which translocates the CagA effector protein into gastric epithelial target cells. Injected CagA becomes tyrosine-phosphorylated on EPIYA sequence motifs by Src and Abl family kinase members. CagA then binds to and activates/inactivates various signalling proteins in a phosphorylation-dependent and phosphorylation-independent manner. In this way injected CagA can act as a master key that evolved during evolution the ability to highjack multiple downstream signalling cascades. Here we review our knowledge on the tyrosine phosphorylation motifs in CagA, the recent advances in the interaction of CagA with Src and Abl tyrosine kinases and their role in signalling events leading to changes of the phosphorylation status of actin-binding proteins cortactin, ezrin and vinculin followed by actin-cytoskeletal rearrangements, cell scattering and elongation. Detailed investigation of these pathways will help to yield novel insights and to elucidate the mechanisms of H. pylori-induced pathogenesis.

Phylogenetic analysis, based on EPIYA repeats in the cagA gene of Indian Helicobacter pylori, and the implications of sequence variation in tyrosine phosphorylation motifs on determining the clinical outcome

The population of India harbors one of the world’s most highly diverse gene pools, owing to the influx of successive waves of immigrants over regular periods in time. Several phylogenetic studies involving mitochondrial DNA and Y chromosomal variation have demonstrated Europeans to have been the first settlers in India. Nevertheless, certain controversy exists, due to the support given to the thesis that colonization was by the Austro-Asiatic group, prior to the Europeans. Thus, the aim was to investigate pre-historic colonization of India by anatomically modern humans, using conserved stretches of five amino acid (EPIYA) sequences in the cagA gene of Helicobacter pylori. Simultaneously, the existence of a pathogenic relationship of tyrosine phosphorylation motifs (TPMs), in 32 H. pylori strains isolated from subjects with several forms of gastric diseases, was also explored. High resolution sequence analysis of the above described genes was performed. The nucleotide sequences obtained were translated into amino acids using MEGA (version 4.0) software for EPIYA. An MJ-Network was constructed for obtaining TPM haplotypes by using NETWORK (version 4.5) software. The findings of the study suggest that Indian H. pylori strains share a common ancestry with Europeans. No specific association of haplotypes with the outcome of disease was revealed through additional network analysis of TPMs.

Identification of Novel Phosphorylation Motifs Through an Integrative Computational and Experimental Analysis of the Human Phosphoproteome.

Protein phosphorylation occurs in certain sequence/structural contexts that are still incompletely understood. The amino acids surrounding the phosphorylated residues are important in determining the binding of the kinase to the protein sequence. Upon phosphorylation these sequences also determine the binding of certain domains that specifically bind to phosphorylated sequences. Thus far, such 'motifs' have been identified through alignment of a limited number of well identified kinase substrates. RESULTS: Experimentally determined phosphorylation sites from Human Protein Reference Database were used to identify 1,167 novel serine/threonine or tyrosine phosphorylation motifs using a computational approach. We were able to statistically validate a number of these novel motifs based on their enrichment in known phosphopeptides datasets over phosphoserine/threonine/tyrosine peptides in the human proteome. There were 299 novel serine/threonine or tyrosine phosphorylation motifs that were found to be statistically significant. Several of the novel motifs that we identified computationally have subsequently appeared in large datasets of experimentally determined phosphorylation sites since we initiated our analysis. Using a peptide microarray platform, we have experimentally evaluated the ability of casein kinase I to phosphorylate a subset of the novel motifs discovered in this study. Our results demonstrate that it is feasible to identify novel phosphorylation motifs through large phosphorylation datasets. Our study also establishes peptide microarrays as a novel platform for high throughput kinase assays and for the validation of consensus motifs. Finally, this extended catalog of phosphorylation motifs should assist in a systematic study of phosphorylation networks in signal transduction pathways.

Porcine insulin receptor substrate 4 (IRS4) gene: cloning, polymorphism and association study

Using PCR and inverse PCR techniques we obtained a 4,498 bp nucleotide sequence FN424076 encompassing the complete coding sequence of the porcine insulin receptor substrate 4 (IRS4) gene and its proximal promoter. The 1,269 amino acid porcine protein deduced from the nucleotide sequence shares 92% identity with the human IRS4 and possesses the same domains and the same number of tyrosine phosphorylation motifs as the human protein. We detected substitution FN424076:g.96C<G in the promoter region that segregates in Meishan and a synonymous substitution FN424076:g.1829T<C in the coding sequence with allele C present only in Meishan. Linkage mapping placed the IRS4 gene at position 82 cM on the current USDA-USMARC linkage map of porcine chromosome X. Association analyses were performed on 555 animals of 12th-15th generation of the Meishan × Large White cross and showed that both SNPs were highly significantly associated with backfat depth (P=0.0005) and that the SNP FN424076:g1829T<C was also associated with loin depth (P=0.017). The Meishan alleles increased back fat depth and decreased loin depth. IRS4 can be considered a positional candidate gene for at least some of the QTL located at the centromeric region of porcine chromosome X.

A molecule in teleost fish, related with human MHC-encoded G6F, has a cytoplasmic tail with ITAM and marks the surface of thrombocytes and in some fishes also of erythrocytes

In teleost fish, a novel gene G6F-like was identified, encoding a type I transmembrane molecule with four extracellular Ig-like domains and a cytoplasmic tail with putative tyrosine phosphorylation motifs including YxN and an immunoreceptor tyrosine-based activation motif (ITAM). G6F-like maps to a teleost genomic region where stretches corresponding to human chromosomes 6p (with the MHC), 12p (with CD4 and LAG-3), and 19q are tightly linked. This genomic organization resembles the ancestral "Ur-MHC" proposed for the jawed vertebrate ancestor. The deduced G6F-like molecule shows sequence similarity with members of the CD4/LAG-3 family and with the human major histocompatibility complex-encoded thrombocyte marker G6F. Despite some differences in molecular organization, teleost G6F-like and tetrapod G6F seem orthologous as they map to similar genomic location, share typical motifs in transmembrane and cytoplasmic regions, and are both expressed by thrombocytes/platelets. In the crucian carps goldfish (Carassius auratus auratus) and ginbuna (Carassius auratus langsdorfii), G6F-like was found expressed not only by thrombocytes but also by erythrocytes, supporting that erythroid and thromboid cells in teleost fish form a hematopoietic lineage like they do in mammals. The ITAM-bearing of G6F-like suggests that the molecule plays an important role in cell activation, and G6F-like expression by erythrocytes suggests that these cells have functional overlap potential with thrombocytes.

The SH2 domain protein Shep1 regulates the in vivo signaling function of the scaffolding protein Cas

The members of the p130Cas (Cas) family are important scaffolding proteins that orchestrate cell adhesion, migration and invasiveness downstream of integrin adhesion receptors and receptor tyrosine kinases by recruiting enzymes and structural molecules. Shep1, BCAR3/AND-34 and NSP1 define a recently identified family of SH2 domain-containing proteins that constitutively bind Cas proteins through a Cdc25-type nucleotide exchange factor-like domain. To gain insight into the functional interplay between Shep1 and Cas in vivo, we have inactivated the Shep1 gene in the mouse through Cre-mediated deletion of the exon encoding the SH2 domain. Analysis of Cas tyrosine phosphorylation in the brains of newborn mice, where Shep1 is highly expressed, revealed a strong decrease in Cas substrate domain phosphorylation in knockout compared to wild-type brains. Src family kinases bind to Cas via their SH3 and SH2 domains, which contributes to their activation, and phosphorylate multiple tyrosines in the Cas substrate domain. These tyrosine-phosphorylated motifs represent docking sites for the Crk adaptor, linking Cas to the downstream Rac1 and Rap1 GTPases to regulate cell adhesion and actin cytoskeleton organization. Accordingly, we detected lower Cas–Crk association and lower phosphorylation of the Src activation loop in Shep1 knockout brains compared to wild-type. Conversely, Shep1 transfection in COS cells increases Cas tyrosine phosphorylation. The SH2 domain is likely critical for the effects of Shep1 on Cas and Src signaling because the knockout mice express Shep1 fragments that lack the amino-terminal region including the SH2 domain, presumably due to aberrant translation from internal ATG codons. These fragments retain the ability to increase Cas levels in transfected cells, similar to full-length Shep1. However, they do not affect Cas phosphorylation on their own or in the presence of co-transfected full-length Shep1. They also do not show dominant negative effects on the activity of full-length Shep1 in vivo because the heterozygous mice, which express the fragments, have a normal life span. This is in contrast to the homozygous knockout mice, most of which die soon after birth. These data demonstrate that Shep1 plays a critical role in the in vivo regulation of Src activity and Cas downstream signaling through Crk, and suggest that the SH2 domain of Shep1 is critical for these effects.

Helicobacter pylori CagA Phosphorylation Status Determines the gp130-activated SHP2/ERK and JAK/STAT Signal Transduction Pathways in Gastric Epithelial Cells

The Helicobacter pylori protein CagA may undergo tyrosine phosphorylation following its entry into human gastric epithelial cells with downstream effects on signal transduction. Disruption of the gp130 receptor that modulates the balance of the SHP2/ERK and JAK/STAT pathways enhanced peptic ulceration and gastric cancer in gp130 knock-out mice. In this study, we evaluated the effect of translocated CagA in relation to its tyrosine phosphorylation status on the gp130-mediated signal switch between the SHP2/ERK and JAK/STAT3 pathways. We showed that in the presence of CagA, SHP2 was recruited to gp130. Phosphorylated CagA showed enhanced SHP2 binding activity and ERK1/2 phosphorylation, whereas unphosphorylated CagA showed preferential STAT3 activation. These findings indicate that the phosphorylation status of CagA affects the signal switch between the SHP2/ERK and JAK/STAT3 pathways through gp130, providing a novel mechanism to explain H. pylori signaling.

Application of PCR amplicon sequencing using a single primer pair in PCR amplification to assess variations in Helicobacter pylori CagA EPIYA tyrosine phosphorylation motifs

BackgroundThe presence of various EPIYA tyrosine phosphorylation motifs in the CagA protein of Helicobacter pylori has been suggested to contribute to pathogenesis in adults. In this study, a unique PCR assay and sequencing strategy was developed to establish the number and variation of cagA EPIYA motifs.FindingsMDA-DNA derived from gastric biopsy specimens from eleven subjects with gastritis was used with M13- and T7-sequence-tagged primers for amplification of the cagA EPIYA motif region. Automated capillary electrophoresis using a high resolution kit and amplicon sequencing confirmed variations in the cagA EPIYA motif region. In nine cases, sequencing revealed the presence of AB, ABC, or ABCC (Western type) cagA EPIYA motif, respectively. In two cases, double cagA EPIYA motifs were detected (ABC/ABCC or ABC/AB), indicating the presence of two H. pylori strains in the same biopsy.ConclusionAutomated capillary electrophoresis and Amplicon sequencing using a single, M13- and T7-sequence-tagged primer pair in PCR amplification enabled a rapid molecular typing of cagA EPIYA motifs. Moreover, the techniques described allowed for a rapid detection of mixed H. pylori strains present in the same biopsy specimen.

Helicobacter pylori CagA protein polymorphisms and their lack of association with pathogenesis.

To investigate Helicobacter pylori (H. pylori) CagA diversity and to evaluate the association between protein polymorphisms and the occurrence of gastric pathologies. One hundred and twenty-two clinical isolates of H. pylori cultured from gastric biopsies obtained from Colombian patients with dyspepsia were included as study material. DNA extracted from isolates was used to determine cagA status, amplifying the C-terminal cagA gene region by polymerase chain reaction. One hundred and six strains with a single amplicon were sequenced and results were used to characterize the 3' variable region of the cagA gene. To establish the number and type of tyrosine phosphorylation motifs Glutamine acid-Proline-Isoleucine-Tyrosine-Alanine (EPIYA) bioinformatic analysis using Amino Acid Sequence Analyzer-Amino Acid Sequence Analyzer software was conducted. Analysis of the association between the number of EPIYA motifs and the gastric pathology was performed using chi(2) test and analysis of the presence of EPIYA-C motifs in relation to the pathology was made by logistic regression odds ratios. Comparisons among EPIYA types found and those reported in GenBank were performed using a proportion test in Statistix Analytical Software version 8.0. After amplification of the 3' of the cagA gene, 106 from 122 isolates presented a single amplicon and 16 showed multiple amplicons. As expected, diversity in the size of the cagA unique fragments among isolates was observed. The 106 strains that presented a single amplicon after 3' cagA amplification came from patients with gastritis (19 patients), atrophic gastritis (21), intestinal metaplasia (26), duodenal ulcer (22) and gastric cancer. DNA sequence analysis showed that the differences in size of 3' cagA unique fragments was attributable to the number of EPIYA motifs: 1.9% had two EPIYA motifs, 62.3% had three, 33.0% had four and 2.8% had five motifs. The majority of tested clinical strains (62.3%) were found to harbor the ABC combination of EPIYA motifs and a significant statistical difference was observed between the frequencies of ABCC tyrosine phosphorylation motifs and Western strains sequences deposited in GenBank. The present report describes a lack of association between H. pylori CagA-protein polymorphisms and pathogenesis. ABCC high frequency variations compared with Western-strains sequences deposited in GenBank require more investigation.

CD33-Negative Acute Myeloid Leukemia (AML) Has Similar Characteristics to CD33-Positive Disease.

Abstract 4129Several immunophenotypic subgroups within AML are associated with specific disease characteristics, e.g., CD19-positive AML is associated with t(8;21)(q22;q22) and CD56 expression is associated with adverse outcome. CD33 is a sialic acid-binding immunoglobulin-like lectin (Siglec) located on chromosome 19q13.3. It is highly expressed on early multilineage hematopoietic progenitors but absent from the pluripotent hematopoietic stem cells. It contains two tyrosine phosphorylation motifs whose phosphorylation depends on Src and is the target of gemtuzumab ozogamicin. We asked if lack of CD33 expression (<10%) on the surface of AML blasts was associated with different disease characteristics. We identified 15 such newly diagnosed AML cases and compared their characteristics with 45 (1:3 ratio) cases matched by age and French-American-British subtypes (only cases with M0, M1 and M2 were included). The mean white blood cell count at diagnosis (18.3 ×109/L, 41.7×109/L; p=0.149) and percent of secondary AML [either antecedent hematologic disorder (13%, 18%; p=1) or therapy-related (7%, 16%; p=0.6657)] among the CD33-negative group was similar to the CD33-positive cohort. The percent of unknown (21%, 19%), intermediate (64%, 51%) and unfavorable (14%, 30%) karyotype (p=0.5484) and the percent of cases with FLT3 (20%, 18%; p=0.6058) and NPM1 (7%, 22%; p=0.1562) was also similar among the CD33-negative and the CD33-positive cohorts. The patients were treated with available protocols. There was no significant difference between CD33-negative and CD33-positive treatment assignment. A total of 50% of the CD33-negative cases achieved complete remission compared to 48% of the CD33-positive cases. The median overall survival was 17.1 [95% confidence interval (CI) 10.3, 22.5] versus 10.6 (95% CI 7.1, 16.9) months (p=0.7488) for the CD33-negative compared with the CD33-positive cases. Finally, disease-free survival was also similar between the two groups; 11.0 (95% CI 6.2, 22.6) months for the CD33-negative cohort versus 9.5 (95% CI 4.5, 118.2) months for the CD33-positive group (p=0.966). In summary, CD33 expression does not alter disease characteristics and does not appear to affect outcome in AML unless treatment with gemtuzumab ozogamicin is considered. Disclosures:No relevant conflicts of interest to declare.

Cell Surface Biliverdin Reductase Mediates Biliverdin-induced Anti-inflammatory Effects via Phosphatidylinositol 3-Kinase and Akt

Biliverdin reductase A (BVR) catalyzes the reduction of biliverdin (BV) to bilirubin (BR) in all cells. Others and we have shown that biliverdin is a potent anti-inflammatory molecule, however, the mechanism by which BV exerts its protective effects is unclear. We describe and elucidate a novel finding demonstrating that BVR is expressed on the external plasma membrane of macrophages (and other cells) where it quickly converts BV to BR. The enzymatic conversion of BV to BR on the surface by BVR initiates a signaling cascade through tyrosine phosphorylation of BVR on the cytoplasmic tail. Phosphorylated BVR in turn binds to the p85alpha subunit of phosphatidylinositol 3-kinase and activates downstream signaling to Akt. Using bacterial endotoxin (lipopolysaccharide) to initiate an inflammatory response in macrophages, we find a rapid increase in BVR surface expression. One of the mechanisms by which BV mediates its protective effects in response to lipopolysaccharide is through enhanced production of interleukin-10 (IL-10) the prototypical anti-inflammatory cytokine. IL-10 regulation is dependent in part on the activation of Akt. The effects of BV on IL-10 expression are lost with blockade of Akt. Inhibition of surface BVR with RNA interference attenuates BV-induced Akt signaling and IL-10 expression and in vivo negates the cytoprotective effects of BV in models of shock and acute hepatitis. Collectively, our findings elucidate a potentially important new molecular mechanism by which BV, through the enzymatic activity and phosphorylation of surface BVR (BVR)(surf) modulates the inflammatory response.

CagA and VacA Polymorphisms Do Not Correlate with Severity of Histopathological Lesions inHelicobacter pylori-Infected Greek Children

The presence of various numbers of EPIYA tyrosine phosphorylation motifs in the CagA protein of Helicobacter pylori has been suggested to contribute to pathogenesis in adults. In this prospective study, we characterized H. pylori isolates from symptomatic children, with reference to the diversity of functional EPIYA motifs in the CagA protein and vacA isotypes, and assessed the potential correlation with the histopathological manifestations of the infection. We analyzed 105 H. pylori isolates from 98 children and determined the diversity of EPIYA motifs in CagA by amplification and sequencing of the 3' variable region of the cagA gene as well as vacA isotypes for the signal, middle, and intermediate regions. CagA phosphorylation and levels of secreted IL-8 were determined following in vitro infection of AGS gastric epithelial cells. Histopathological evaluation of H. pylori colonization, activity, and severity of the associated gastritis was performed according to the updated Sydney criteria. EPIYA A (GLKN[ST]EPIYAKVNKKK), EPIYA B (Q[V/A]ASPEPIY[A/T]QVAKKVNAKI), and EPIYA C (RS[V/A]SPEPIYATIDDLG) motifs were detected in the ABC (46.6%) and ABCC (17.1%) combinations. No isolates harboring more than two EPIYA C motifs in CagA were found. The presence of isogenic strains with variable numbers of CagA EPIYA C motifs within the same patient was detected in seven cases. Occurrence of increasing numbers of EPIYA C motifs correlated strongly with presence of a high-vacuolation (s1 or s2/i1/m1) phenotype and age. A weak positive correlation was observed between vacuolating vacA genotypes and presence of nodular gastritis. However, CagA- and VacA-dependent pathogenicities were not found to contribute to severity of histopathology manifestations in H. pylori-infected children.

CEACAM1 : modulateur central du métabolisme, de la progression tumorale,de l’angiogenèse et de l’immunité

CEACAM1 (carcinoembryonic antigen cell adhesion molecule 1), a member of the immunoglobulin (Ig) superfamily, is a heavily glycosylated protein. This glycoprotein exhibits an intracytoplasmic region that can be either long (71-73 amino acids) with two inhibitory tyrosine-phosphorylated motifs and several phosphorylated serine residues, or short (10 amino acid). CEACAM1 is a multifunctional protein that plays a role in intercellular adhesion, as an inhibitor of tumor development, as a bacterial adhesin, and as a receptor for the mouse hepatitis virus. Moreover, CEACAM1 is an active regulator of cell signaling, modulating the insulin or EGF receptor pathways in epithelial cells or the Zap-70 pathway in hematopoietic cells. The recent development of genetically modified mouse models altering the Ceacam1 gene corroborates most of these data, but also highlights CEACAM1's functional complexity. Thus, in addition to the functions identified previously, CEACAM1 is an important regulator of lipid metabolism, of tumor progression as a regulator of the Wnt signaling pathway, of normal and tumor neo-angiogenesis and of immunity.

Genome sequence analysis of Helicobacter pylori strains associated with gastric ulceration and gastric cancer

BackgroundPersistent colonization of the human stomach by Helicobacter pylori is associated with asymptomatic gastric inflammation (gastritis) and an increased risk of duodenal ulceration, gastric ulceration, and non-cardia gastric cancer. In previous studies, the genome sequences of H. pylori strains from patients with gastritis or duodenal ulcer disease have been analyzed. In this study, we analyzed the genome sequences of an H. pylori strain (98-10) isolated from a patient with gastric cancer and an H. pylori strain (B128) isolated from a patient with gastric ulcer disease.ResultsBased on multilocus sequence typing, strain 98-10 was most closely related to H. pylori strains of East Asian origin and strain B128 was most closely related to strains of European origin. Strain 98-10 contained multiple features characteristic of East Asian strains, including a type s1c vacA allele and a cagA allele encoding an EPIYA-D tyrosine phosphorylation motif. A core genome of 1237 genes was present in all five strains for which genome sequences were available. Among the 1237 core genes, a subset of alleles was highly divergent in the East Asian strain 98-10, encoding proteins that exhibited <90% amino acid sequence identity compared to corresponding proteins in the other four strains. Unique strain-specific genes were identified in each of the newly sequenced strains, and a set of strain-specific genes was shared among H. pylori strains associated with gastric cancer or premalignant gastric lesions.ConclusionThese data provide insight into the diversity that exists among H. pylori strains from diverse clinical and geographic origins. Highly divergent alleles and strain-specific genes identified in this study may represent useful biomarkers for analyzing geographic partitioning of H. pylori and for identifying strains capable of inducing malignant or premalignant gastric lesions.

SH2 domain binding to phosphopeptide ligands: potential for drug targeting

SH2 domains are modular components of a wide range of functionally diverse proteins involved in mammalian signal transduction including enzymes, adaptors, regulators and transcription factors. Members of the SH2 domain family recognize a wide variety of short tyrosine phosphorylated peptide motifs. Biochemical and structural studies have revealed key aspects of these interactions that account for their ability to discriminate between different sequence motifs. While the mechanism of phosphotyrosine (pTyr) recognition is remarkably conserved among the SH2 domains, differences in recognition of phosphopeptide residues N and especially C-terminal to the pTyr have been identified that contribute to selectivity. The basis for SH2- phosphopeptide recognition is discussed in light of the available structural and biochemical data with a focus on recent information regarding SH2 domains within a new class found within the signal transducer and activator of transcription (STAT) protein family.

Identification of the repeated number of C and D regions of tyrosine phosphorylation motifs in Helicobacter pylori cagA using multiplex PCR

Various tyrosine phosphorylation motif regions of H. pylori cagA exist. The number of these regions was found to have some influence on cell signaling, which was found to be more pronounced when in D (ESS) region than in C (WSS) region. A molecular biological method with multiplex PCR was developed to distinguish C and D regions, and to identify the repetition number of tyrosine phosphorylation of the cagA gene. Multiplex PCR using novel primer sets was performed on 73 strains of H. pylori isolated from Korean patients with upper gastrointestinal diseases. The Western cagA was identified in only 3 strains (4.1%) whereas East Asia cagA was identified in 69 strains (94.5%). These results were reconfirmed through a sequencing analysis. The method developed in this study would be useful for monitoring the repeated number of C and D regions of tyrosine phosphorylation motifs in H. pylori cagA.

Differences in Helicobacter pylori CagA tyrosine phosphorylation motif patterns between western and East Asian strains, and influences on interleukin-8 secretion

Helicobacter pylori strains from East Asia have an 'East Asian' type of CagA that is more active and predominantly comprises a single type. Strains from other countries have a 'western' type of CagA, which is less active and comprises many different types generated by intragenomic recombination. Co-culture of AGS gastric epithelial cells with isolates of western strains that displayed microevolution in CagA showed that isolates with additional copies of the C motif induced significantly more interleukin (IL)-8 secretion. Co-culture of AGS cells with western and East Asian strains, each expressing CagA with a single copy of the C or D motif, showed that East Asian strains induced significantly more IL-8 secretion. Analysis of the different CagA types from data deposited in GenBank and from the literature showed that western CagA is significantly more likely to undergo duplication of tyrosine phosphorylation motif C than East Asian CagA is of the corresponding D motif. Taken together, the data suggest that the already highly active East Asian CagA with one D motif has no requirement to increase its virulence, whereas the less active western CagA displays flexibility in its capacity to increase its number of tyrosine phosphorylation motifs to become more virulent.

Haemophilus ducreyi LspA Proteins Are Tyrosine Phosphorylated by Macrophage-Encoded Protein Tyrosine Kinases

The LspA proteins (LspA1 and LspA2) of Haemophilus ducreyi are necessary for this pathogen to inhibit the phagocytic activity of macrophage cell lines, an event that can be correlated with a reduction in the level of active Src family protein tyrosine kinases (PTKs) in these eukaryotic cells. During studies investigating this inhibitory mechanism, it was discovered that the LspA proteins themselves were tyrosine phosphorylated after wild-type H. ducreyi cells were incubated with macrophages. LspA proteins in cell-free concentrated H. ducreyi culture supernatant fluid could also be tyrosine phosphorylated by macrophages. This ability to tyrosine phosphorylate the LspA proteins was not limited to immune cell lineages but could be accomplished by both HeLa and COS-7 cells. Kinase inhibitor studies with macrophages demonstrated that the Src family PTKs were required for this tyrosine phosphorylation activity. In silico methods and site-directed mutagenesis were used to identify EPIYG and EPVYA motifs in LspA1 that contained tyrosines that were targets for phosphorylation. A total of four tyrosines could be phosphorylated in LspA1, with LspA2 containing eight predicted tyrosine phosphorylation motifs. Purified LspA1 fusion proteins containing either the EPIYG or EPVYA motifs were shown to be phosphorylated by purified Src PTK in vitro. Macrophage lysates could also tyrosine phosphorylate the LspA proteins and an LspA1 fusion protein via a mechanism that was dependent on the presence of both divalent cations and ATP. Several motifs known to interact with or otherwise affect eukaryotic kinases were identified in the LspA proteins.

Differences in Virulence Markers betweenHelicobacter pyloriStrains from Iraq and Those from Iran: Potential Importance of Regional Differences inH. pylori-Associated Disease

Helicobacter pylori causes peptic ulceration and gastric adenocarcinoma; the latter is common in Iran but not in Iraq. We hypothesized that more virulent H. pylori strains may be found in Iran than in Iraq and so compared established and newly described virulence factors in strains from these countries. We studied 59 unselected dyspeptic patients from Iran and 49 from Iraq. cagA was found in similar proportions of strains from both countries (76% in Iran versus 71% in Iraq) and was significantly associated with peptic ulcer disease in Iraq (P <or= 0.01) but not in Iran. cagA alleles encoding four or more tyrosine phosphorylation motifs were found in 12% of the Iranian strains but none of the Iraqi strains (P = 0.02). There were no significant differences in the vacA signal-, middle-, or intermediate-region types between Iranian and Iraqi strains. Among the strains from Iran, vacA genotypes showed no specific peptic ulcer associations, but among the strains from Iraq, vacA i1 strains were associated with gastric ulcer (P <or= 0.02), mimicking their previously demonstrated association with gastric cancer in Iran. dupA was found in similar proportions of Iranian and Iraqi strains (38% and 32%, respectively) and was associated with peptic ulceration in Iraqi patients (P <or= 0.01) but not Iranian patients. H. pylori strains from Iraq and Iran possess virulence factors similar to those in Western countries. The presence of cagA with more phosphorylation motifs in Iranian strains may contribute to the higher incidence of gastric cancer. However, the association between strain virulence markers and disease in Iraq but not Iran suggests that other host and environmental factors may be more important in the disease-prone Iranian population.

QS292. Signal Transduction in Macrophages Through Carcinoembryonic Antigen (CEA) and Its Receptor hnRNP M

Introduction: Secretion of carcinoembryonic antigen (CEA) is an indicator of poor prognosis for colorectal cancer. Circulating CEA is removed by receptor mediated endocytosis by Kupffer cells and this results in an inflammatory response characterized by a cytokine cascade (IL-1, IL-6, IL-10 and TNF-α) that increases the metastatic potential of the cancer cells. The transcription factor c-jun has been shown to be a binding partner with the CEA receptor hnRNP M. There is also activation of the AP-1 transcription factor in Kupffer cells exposed to CEA. We have used differentiated THP-1 macrophages as a model for CEA induced macrophage activation and have demonstrated hnRNP M on the surface of these terminally differentiated cells. Exposure to CEA results in production of cytokines and these influence the ability of CEA producing cancers to implant and grow in the liver. Methods: THP-1 a human monocyte cell line was differentiated overnight to produce a macrophage phenotype with phorbol myristate acetate (PMA) (200ng/ml). Differentiated THP-1 cells were treated with CEA (2μg?ml) and the cells harvested at a number of time points between 0 and 4 hours. Cells were extracted following treatment at these various times, with RIPA buffer containing a cocktail of protease and phosphatase inhibitors. These extracts were run on one or 2-D SDS-PAGE and transferred to membranes for Western blotting. Membranes were probed with antibodies against c-jun, phospho c-jun, JNK 1,2 and 3, p38, phospho-tyrosine and phospho serine/threonine. Results: We showed a significant down regulation of c-jun expression on treatment of the differentiated cells with CEA while an increase in c-jun phosphorylation was observed. JNK-1, and 2 expressions also increase with CEA treatment that is consistent with phosphorylation of c-jun. JNK-3 levels however, decrease following CEA binding. There was also a down regulation of the JNK pathway protein p38. These data point to role for the JNK/c-jun/AP-1 system in signaling by hnRNP-M4. Because hnRNP M4 has a tyrosine phosphorylation motif in its N-terminal domain we have examined the effect of CEA binding on phosphorylation at this locus. Use of western blotting showed increased tyrosine phosphorylation of both isoforms of hnRNP M (M3 and M4). Using 2D electrophoresis we have shown changes in the isoelectric point of the receptor isoforms that would be consistent with phosphorylation. Use of phospho-serine/threonine antibodies showed phosphorylation of 3 proteins distinct from hnRNP M in differentiated THP-1 cells. CEA treatment causes a decline in one band and an increase in phosphorylation of the other two. We are presently attempting to identify these proteins. Conclusions: There is involvement of the c-jun pathway in the initiation of macrophages by CEA to produce cytokines and involves up regulation of JNK-1 and 2. Activation of macrophages by CEA probably involves the transcription factor AP-1. Both isoforms of the receptor appear to be phosphorylated on the consensus tyrosine located in the N-terminal domain. These pathways may be potential targets for intervention to prevent implantation of CEA producing colorectal cancer cells in the liver.