Phosphorylation Sites In Domain Research Articles

Inhibition of MARCKS phosphorylation attenuates of dendritic cell migration in a murine model of acute asthma

BackgroundMARCKS (myristoylated alanine-rich C kinase substrates) serves as a substrate for protein kinase C, residing in the plasma membrane while acts as an actin filament crosslinking protein. This investigation aims to elucidate phosphorylated MARCKS (p-MARCKS) levels and activity in allergic asthma patients and explore the therapeutic potential of peptide inhibitors targeting p-MARCKS in an acute mouse model of allergic asthma. MethodsImmunohistochemistry and histology staining were employed on lung tissue slides to evaluate p-MARCKS expression and allergic asthma symptoms. Airway resistance was measured using invasive whole-body plethysmography. Flow cytometry detected lung dendritic cell migration, and migration/maturation assays were conducted on isolated murine bone marrow-derived dendritic cells (BM-DCs). ResultsElevated p-MARCKS expression was observed in both human asthmatic tissues and animal models immunized with ovalbumin or Alternaria alternata. Remarkably, asthmatic individuals showed elevated high p-MARCKS expression in lung tissues. Intraperitoneal injection of the peptide MPS, targeting the MARCKS phosphorylation site domain, before allergen challenged, effectively suppressed MARCKS phosphorylation in murine lung tissues. MPS inhibited both in vivo and in vitro migration and maturation of dendritic cells (BM-DCs) and reduced Th2-related lymphocyte activation in bronchoalveolar lavage fluid (BALF). MPS pretreatment additionally suppressed all symptoms associated with allergic airway asthma, including a reduction in inflammatory cell influx, airway mucous cell metaplasia, and airway hyperreactivity. ConclusionThese findings suggest that phosphorylated MARCKS occurs in asthmatic lung tissue, and the inhibition of MARCKS phosphorylation by the MPS peptide reduces dendritic cell migration and Th2-related lymphocytes in the lungs in a murine model of acute asthma.

Abstract P6-10-15: Targeting MARCKS in inflammatory breast cancer increased paclitaxel sensitivity

Abstract Background. Because of its high metastatic potential, inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer. We previously demonstrated that Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) protein overexpression was associated with shorter metastasis-free survival (MFS) in IBC patients, but not in non-IBC (nIBC) patients. However, the mechanism of action of MARCKS and its particular association to poorer outcome in IBC compared to nIBC are poorly understood. Methods. We evaluated in vitro the inhibitory effect of MPS (MARCKS phosphorylation site domain), a peptide targeting MARCKS phosphorylation site domain (PSD) in single and in combination with paclitaxel treatment, on cell proliferation and cell motility in two cell lines of different phenotype (SUM149 for IBC and MDA-MB-231 for nIBC), as well as its distinct molecular mechanisms of action. We also assessed the clinical relevance of the protein expression of MARCKS and phosphatase and tensin homolog (PTEN) in a large series of IBC vs. nIBC patients. Results. In vitro, the treatment with MPS peptide impaired cell proliferation, migration, and invasion in SUM149 compared to MDA-MB-231 cells. More important, MARCKS inhibition increased paclitaxel sensitivity when using combination therapy in SUM149 cells compared to MDA-MB-231 cells. Interestingly, we could partially explain this specific inhibitory effect in IBC cells using western blot: MARCKS inhibition in single and in combination induced up and downregulation of the PTEN/AKT signaling pathway respectively in IBC compared to nIBC cells. Importantly, a negative correlation of MARCKS and PTEN was only found in the clinical IBC samples (180 patients) compared to nIBC samples (355 patients). More importantly, the group of patients with negative MARCKS and positive PTEN protein expression was associated to better 5-year MFS only in IBC patients. Conclusion. These results indicate two major findings: first, the important prognostic value of the negative correlation of MARCKS and PTEN expression in IBC patients, and second the specific role of MARCKS in regulating different downstream pathways and increasing the paclitaxel response in combination treatment in IBC compared to non-IBC. They suggest a potential IBC-specific targetable biomarker, the inhibition of which might impair disease aggressiveness and perhaps enhance patients’ survival. Citation Format: Maroua Manai, Ines Bini, Pascal Finetti, Haifa Bichiou, Carolina Reduzzi, Naziha Ben Hamida, Marc Lopez, Khaled Rahal, Karima Mrad, Mohamed Manai, Massimo Cristofanilli, Hamouda Boussen, Raoudha Doghri, Maher Kharrat, François Bertucci. Targeting MARCKS in inflammatory breast cancer increased paclitaxel sensitivity [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-15.

In silico identification and experimental validation of cellular uptake by a new cell penetrating peptide P1 derived from MARCKS

Viral vectors for vaccine delivery are challenged by recently reported safety issues like immunogenicity and risk for cancer development, and thus there is a growing need for the development of non-viral vectors. Cell penetrating peptides (CPPs) are non-viral vectors that can enter plasma membranes efficiently and deliver a broad range of cargoes. Our bioinformatic prediction and wet-lab validation data suggested that peptide P1 derived from MARCKS protein phosphorylation site domain is a new potential CPP candidate. We found that peptide P1 can efficiently internalize into various cell lines in a concentration-dependent manner. Receptor-mediated endocytosis pathway is the major mechanism of P1 penetration, although P1 also directly penetrates the plasma membrane. We also found that peptide P1 has low cytotoxicity in cultured cell lines as well as mouse red blood cells. Furthermore, peptide P1 not only can enter into cultured cells itself, but it also can interact with plasmid DNA and mediate the functional delivery of plasmid DNA into cultured cells, even in hard-to-transfect cells. Combined, these findings indicate that P1 may be a promising vector for efficient intracellular delivery of bioactive cargos.

Tackling MARCKS-PIP3 circuit attenuates fibroblast activation and fibrosis progression.

Targeting activated fibroblasts, including myofibroblast differentiation, has emerged as a key therapeutic strategy in patients with idiopathic pulmonary fibrosis (IPF). However, there is no available therapy capable of selectively eradicating myofibroblasts or limiting their genesis. Through an integrative analysis of the regulator genes that are responsible for the activation of IPF fibroblasts, we noticed the phosphatidylinositol 4,5-bisphosphate (PIP2)-binding protein, myristoylated alanine-rich C-kinase substrate (MARCKS), as a potential target molecule for IPF. Herein, we have employed a 25-mer novel peptide, MARCKS phosphorylation site domain sequence (MPS), to determine if MARCKS inhibition reduces pulmonary fibrosis through the inactivation of PI3K/protein kinase B (AKT) signaling in fibroblast cells. We first observed that higher levels of MARCKS phosphorylation and the myofibroblast marker α-smooth muscle actin (α-SMA) were notably overexpressed in all tested IPF lung tissues and fibroblast cells. Treatment with the MPS peptide suppressed levels of MARCKS phosphorylation in primary IPF fibroblasts. A kinetic assay confirmed that this peptide binds to phospholipids, particularly PIP2, with a dissociation constant of 17.64 nM. As expected, a decrease of phosphatidylinositol (3,4,5)-trisphosphate pools and AKT activity occurred in MPS-treated IPF fibroblast cells. MPS peptide was demonstrated to impair cell proliferation, invasion, and migration in multiple IPF fibroblast cells in vitro as well as to reduce pulmonary fibrosis in bleomycin-treated mice in vivo. Surprisingly, we found that MPS peptide decreases α-SMA expression and synergistically interacts with nintedanib treatment in IPF fibroblasts. Our data suggest MARCKS as a druggable target in pulmonary fibrosis and also provide a promising antifibrotic agent that may lead to effective IPF treatments.-Yang, D. C., Li, J.-M., Xu, J., Oldham, J., Phan, S. H., Last, J. A., Wu, R., Chen, C.-H. Tackling MARCKS-PIP3 circuit attenuates fibroblast activation and fibrosis progression.

Targeting MARCKS phosphorylation site domain shows therapeutic potential for allergic asthma

Abstract Prevalence of asthma is increased all over the world, especially in Taiwan, study analyzed data of 997,729 enrolees from the National Health Insurance register from 2000 to 2007, the prevalence of asthma increase from 2.9% to 11.9%. Various signaling pathways and mechanisms have been identified and targeted for therapeutic application. Among them, one treatment, based on the use of MANS peptide, which is corresponding to the myristoylated N-terminal 24-amino acid portion of the MARCKS (myristoylated alanine-rich C kinase substrates) protein, to compete MARCKS’ binding to membrane has produced useful results. There is a lack of information regarding to the level/activity of phosphorylated MARCKS (p-MARCKS) in allergic asthma. It is also unclear if this activity can be directly suppressed for the allergic asthma treatment application. In this study, we have found elevated p-MARCKS in most of asthmatic tissues from human as well as from the animal model. This elevation can be directly suppressed by a peptide, MPS, targeting MARCKS phosphorylation site domain, prior to allergen sensitization. In addition, MPS pre-treatment also suppressed all of the symptoms associated with allergic airway asthma, which includes a reduction of inflammatory cells influx and TH2 cytokine presence in the lumen, and also a reduction of airway mucous cell metaplasia and airway hyperreactivity. In addition to inhibition of TH2 responses, lung dendritic cells migrate to mediastinal lymph nodes also decreased by MPS treatment. These results suggest that MARCKS phosphorylation site domain is a potential therapeutic target for the treatment of allergic asthma.

Comparative Analysis of CDPK Family in Maize, Arabidopsis, Rice, and Sorghum Revealed Potential Targets for Drought Tolerance Improvement.

Calcium dependent protein kinases (CDPKs) play significant role in regulation of plant growth and development in response to various stresses including drought. A set of 32 CDPK genes identified in maize were further used for searching of orthologs in the model plant Arabidopsis (72) and major food crops such as rice (78) and sorghum (91). We comprehensively studied the phylogenetic relationship, annotations, gene duplications, gene structure, divergence time, 3-D protein structures and tissue-specific drought induced expression of CDPK genes in all four species. Variation in intron frequency in the studied species was one of the reasons for the functional diversity of CDPK genes to various stress responses. Protein kinase and protein kinase C phosphorylation site domains were the most conserved motifs identified in all species. Four groups were identified from the sequence-based phylogenetic analysis, in which maize CDPKs were clustered in group III. Expression data showed that the CDPK genes were highly expressed in leaf of maize, rice, and sorghum whereas in Arabidopsis the maximum expression was observed in root. The expression assay showed 5, 6, 11, and 9 were the commonly and differentially expressed drought-related orthologous genes in maize, Arabidopsis, rice, and sorghum, respectively. 3-D protein structure were predicted for the nine genes (Arabidopsis: 2, maize: 2, rice: 3, and sorghum: 2) showing differential expression in at least three species. The predicted 3-D structures were further evaluated and validated by Ramachandran plot, ANOLEA, ProSA, and Verify-3D. The superimposed 3-D structure of drought-related orthologous proteins retained similar folding pattern owing to their conserved nature. Functional annotation revealed the involvement of CDPK genes in various pathways such as osmotic homeostasis, cell protection, and root growth. The interactions of CDPK genes in various pathways play crucial role in imparting drought tolerance through different ABA and MAPK signaling cascades. These selected candidate genes could be targeted in development of drought tolerant genotypes in maize, rice, and sorghum through appropriate breeding approaches. Our comparative experiments of CDPK genes could also be extended in the drought stress breeding programmes of the related species.

Upregulation of MARCKS in kidney cancer and its potential as a therapeutic target.

Targeted therapeutics, such as those abrogating hypoxia inducible factor (HIF)/vascular endothelial growth factor signaling, are initially effective against kidney cancer (or renal cell carcinoma, RCC); however, drug resistance frequently occurs via subsequent activation of alternative pathways. Through genome-scale integrated analysis of the HIF-α network, we identified the major protein kinase C substrate MARCKS (myristoylated alanine-rich C kinase substrate) as a potential target molecule for kidney cancer. In a screen of nephrectomy samples from 56 patients with RCC, we found that MARCKS expression and its phosphorylation are increased and positively correlate with tumor grade. Genetic and pharmacologic suppression of MARCKS in high-grade RCC cell lines in vitro led to a decrease in cell proliferation and migration. We further demonstrated that higher MARCKS expression promotes growth and angiogenesis in vivo in an RCC xenograft tumor. MARCKS acted upstream of the AKT/mTOR pathway, activating HIF-target genes, notably vascular endothelial growth factor-A. Following knockdown of MARCKS in RCC cells, the IC50 of the multikinase inhibitor regorafenib was reduced. Surprisingly, attenuation of MARCKS using the MPS (MARCKS phosphorylation site domain) peptide synergistically interacted with regorafenib treatment and decreased survival of kidney cancer cells through inactivation of AKT and mTOR. Our data suggest a major contribution of MARCKS to kidney cancer growth and provide an alternative therapeutic strategy of improving the efficacy of multikinase inhibitors.

Marcks Peptide Inhibitor Displays Synergistic Cytotoxicity with Bortezomib in Drug Resistant Multiple Myeloma Cells but Enhances Autophagic Effect

Myristoylated alanine-rich C kinase substrate (MARCKS) is a protein which has recently been connected to the progression of multiple cancer types including multiple myeloma (MM). MARCKS can be phosphorylated and then activated by several PKCs in its phosphorylation site domain (PSD). All known biological functions of MARCKS including its oncogenic effects are conducted by its phosphorylated form. Although in an earlier study we have shown that MARCKS knocking down could lead to cell cycle arrest and apoptosis in drug resistant MM cell lines, whether it is possible to antagonize drug resistance of MM cells by inhibitors of MARCKS phosphorylation is still unclear.Here we investigated the anti-myeloma activity of MARCKS inhibitors in MM cells by applying MARCKS PSD peptide inhibitor (MPS), a validated 25-amino-acid peptide targeting the MARCKS PSD. Two drug resistant MM cell lines MM1R and 8226R5 as well as normal peripheral blood mononuclear cells (PBMCs) derived from three healthy volunteers were exposed to MPS for 48 hours. Compared with its mutated (inactive) form, treatment of this inhibitor led to a dose dependent cytotoxic effect on drug resistant MM cells, while it did not affect cell viability of normal PBMCs. Furthermore, MPS treatment significantly sensitized MM1R and 8226R5 cells to bortezomib exposure after 48 hrs. Co-treatment of these two agents exhibited synergistic effect on both of the above resistant MM cell lines (on 8226R5, the combination index (CI) = 0.79; on MM1R, CI=0.88).We next investigated the mechanisms by which MPS may induce cell death and overcome bortezomib resistance. Following MPS treatment, accumulation of p27Kip1as well as dampened phosphorylation of Akt1 were identified both in MM1R and 8226R5 MM cell lines, indicating that MPS treatment may lead to cell cycle arrest and growth suppression. Interestingly, the level of LC3BII, a marker of autophagy, was also significantly upregulated, which suggested an enhancement of autophagic activity after MARCKS inhibition. To confirm the correlation between MARCKS inhibition and autophagy induction, we knocked down MARCKS by transfecting shRNA lentiviral vectors into above drug resistant MM cells. Western blots showed a significantly upregulated LC3BII in both cell lines with MARCKS silencing, compared with cells transfected by scrambled shRNA. Accumulation of lysosomes as well as autophagosomes was also observed separately via cellular staining with acridine orange and florescence-labeled LC3BII antibody.Upregulation of autophagy has been tightly related to the induction of drug resistance in cancer cells. Thus, drug resistant MM cells with impaired MARCKS activity may become more dependent on the pro-survival outcome of autophagy when exposed to anti-tumor drugs like bortezomib. To examine this hypothesis, both MARCKS-silenced MM1R and 8226R5 cells as well as negative control cells expressing scramble shRNA were co-treated with bortezomib and autophagy inhibitor chloroquine (CQ) for 48hrs. MTT assays showed that the combination of CQ and bortezomib resulted in more cell death in MARCKS-silenced drug resistant MM cells than negative control cells (~40% and ~30% more cell death in MM1R (p<0.01) and 8226R5 (p<0.05) cell lines, respectively). In MARCKS-silenced cells, synergistic effects of CQ and bortezomib were also more significant than in negative control cells. Hence depletion of MARCKS activity makes MM cells more vulnerable to bortezomib when cellular autophagic activity is intercepted. We are currently examining the in vivo activity of MPS using xenograft mice models. Taken together, our results indicate that MPS peptide is effective in killing myeloma cells by inhibition of phosphorylation of MARCKS but also enhances autophagic response. This previously unappreciated correlation between MARCKS suppression and autophagy enhancement suggests that the combination of bortezomib, MARCKS inhibitor, as well as autophagy antagonists like chloroquine may contribute to the development of novel MM therapies. DisclosuresNo relevant conflicts of interest to declare.

Novel 2-Carbonylbenzo[b]thiophene 1,1-Dioxide Derivatives as Potent Inhibitors of STAT3 Signaling Pathway.

Signal transducer and activator of transcription 3 (STAT3) is considered to be an attractive therapeutic target for cancer therapy. In this study, a series of 2-carbonylbenzo[b]thiophene 1,1-dioxide derivatives (CBT) were designed to inhibit the STAT3 SH2 domain phosphorylation site Try 705. We demonstrated that incorporation of basic flexible groups through amide bond linkage to benzo[b]thiophene 1,1-dioxide (BTP) achieved compounds with higher antiproliferative potency than BTP itself. The most potent compound 6o, as indicated from luciferase reporter gene assay, inhibited the STAT3 pathway by decreasing the phosphorylation level of STAT3 Tyr705, while the phosphorylation level of other upstream tyrosine kinases in this pathway was not significantly inhibited. Compound 6o was also shown to trigger ROS generation and accumulation, thus consequently attributed partially to the observed cell apoptosis. This study provided important structural information for the development of inhibitors targeting the STAT3 pathway.

Targeting myristoylated alanine-rich C kinase substrate phosphorylation site domain in lung cancer. Mechanisms and therapeutic implications.

Phosphorylation of myristoylated alanine-rich C kinase substrate (phospho-MARCKS) at the phosphorylation site domain (PSD) is crucial for mucus granule secretion and cell motility, but little is known concerning its function in lung cancer. We aimed to determine if MARCKS PSD activity can serve as a therapeutic target and to elucidate the molecular basis of this potential. The clinical relevance of phospho-MARCKS was first confirmed. Next, we used genetic approaches to verify the functionality and molecular mechanism of phospho-MARCKS. Finally, cancer cells were pharmacologically inhibited for MARCKS activity and subjected to functional bioassays. We demonstrated that higher phospho-MARCKS levels were correlated with shorter overall survival of lung cancer patients. Using shRNA silencing and ectopic expression of wild-type and PSD-mutated (S159/163A) MARCKS, we showed that elevated phospho-MARCKS promoted cancer growth and erlotinib resistance. Further studies demonstrated an interaction of phosphoinositide 3-kinase with MARCKS, but not with phospho-MARCKS. Interestingly, phospho-MARCKS acted in parallel with increased phosphatidylinositol (3,4,5)-triphosphate pools and AKT activation in cells. Through treatment with a 25-mer peptide targeting the MARCKS PSD motif (MPS peptide), we were able to suppress tumor growth and metastasis in vivo, and reduced levels of phospho-MARCKS, phosphatidylinositol (3,4,5)-triphosphate, and AKT activity. This peptide also enhanced the sensitivity of lung cancer cells to erlotinib treatment, especially those with sustained activation of phosphoinositide 3-kinase/AKT signaling. These results suggest a key role for MARCKS PSD in cancer disease and provide a unique strategy for inhibiting the activity of MARCKS PSD as a treatment for lung cancer.

Abstract 2596: Suppression of lung cancer malignancy by peptides targeting MARCKS phosphorylation site domain: A comparative study

Abstract We have recently reported elevated MARCKS phosphorylation (p-MARCKS) in lung cancer tissues but not in their adjacent normal non-cancer tissue sections. We have extended this study to a tumor tissue array from a cohort of 110 human patients with lung cancer. Immunohistochemistry results have shown a significant association of elevated p-MARCKS with advanced-stage, lymph node metastasis and malignant phenotypes of lung cancer, confirming the importance of p-MARCKS in lung cancer progression. To see if this newly discover biomarker can be a target for therapeutic treatment for the suppression of lung cancer malignancy, we have employed two peptides for this potential; one is the MANS peptide developed by our collaborator many years ago to target myristoylated N-terminal amino acid region for the control of airway mucus granule secretion, the other peptide is MPS that directly targets the MARCKS phosphorylation site domain. For MANS peptide, our recent publication has shown it is effective in blocking lung cancer cell migration and invasiveness in vitro as well as in vivo metastasis. A similar observation was seen for MPS peptide treatment. However, in contrast to MANS peptide, MPS treatment can directly block PIP3-dependent AKT signaling and repress lung tumor growth in vivo in addition to the suppression of metastasis, while MANS can only reduce cancer metastasis. These results support the initial notion on the involvement of p-MARCKS as a biomarker in lung cancer and the potential to target the phosphorylation site domain for controlling cancer progression. Our data have also suggested a direct targeting MARCKS phosphorylation site domain by MPS peptide is more effective in the suppression of lung cancer malignancy. Citation Format: Ching-Hsien Chen, Sarah Statt, Phillip Thai, Jasmine G. Lee, Kenneth B. Adler, Pan-Chyr Yang, Reen Wu. Suppression of lung cancer malignancy by peptides targeting MARCKS phosphorylation site domain: A comparative study. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2596. doi:10.1158/1538-7445.AM2014-2596

Role of MARCKS in regulated secretion from mast cells and airway goblet cells

MARCKS (myristoylated alanine-rich C kinase substrate) is postulated to regulate the passage of secretory granules through cortical actin in the early phase of exocytosis. There are, however, three proposed mechanisms of action, all of which were derived from studies using synthetic peptides representing either the central phosphorylation site domain or the upstream, NH2-terminal domain: it tethers actin to the plasma membrane and/or to secretory granules, and/or it sequesters PIP2. Using MARCKS-null mice, we probed for a loss of function secretory phenotype in mast cells harvested from embryonic livers and maturated in vivo [embryonic hepatic-derived mast cells (eHMCs)]. Both wild-type (WT) and MARCKS-null eHMCs exhibited full exocytic responses upon FcϵRI receptor activation with DNP-BSA (2,4-dinitrophenyl-BSA), whether they were in suspension or adherent. The secretory responses of MARCKS-null eHMCs were consistently higher than those of WT cells, but the differences had sporadic statistical significance. The MARCKS-null cells exhibited faster secretory kinetics, however, achieving the plateau phase of the response with a t½ ∼2.5-fold faster. Hence, MARCKS appears to be a nonessential regulatory protein in mast cell exocytosis but exerts a negative modulation. Surprisingly, the MARCKS NH2-terminal peptide, MANS, which has been reported to inhibit mucin secretion from airway goblet cells (Li Y, Martin LD, Spizz G, Adler KB. J Biol Chem 276: 40982-40990, 2001), inhibited hexosaminidase secretion from WT and MARCKS-null eHMCs, leading us to reexamine its effects on mucin secretion. Results from studies using peptide inhibitors with human bronchial epithelial cells and with binding assays using purified mucins suggested that MANS inhibited the mucin binding assay, rather than the secretory response.

The mobility of two kinase domains in the Escherichia coli chemoreceptor array varies with signalling state

Motile bacteria sense their physical and chemical environment through highly cooperative, ordered arrays of chemoreceptors. These signalling complexes phosphorylate a response regulator which in turn governs flagellar motor reversals, driving cells towards favourable environments. The structural changes that translate chemoeffector binding into the appropriate kinase output are not known. Here, we apply high-resolution electron cryotomography to visualize mutant chemoreceptor signalling arrays in well-defined kinase activity states. The arrays were well ordered in all signalling states, with no discernible differences in receptor conformation at 2-3 nm resolution. Differences were observed, however, in a keel-like density that we identify here as CheA kinase domains P1 and P2, the phosphorylation site domain and the binding domain for response regulator target proteins. Mutant receptor arrays with high kinase activities all exhibited small keels and high proteolysis susceptibility, indicative of mobile P1 and P2 domains. In contrast, arrays in kinase-off signalling states exhibited a range of keel sizes. These findings confirm that chemoreceptor arrays do not undergo large structural changes during signalling, and suggest instead that kinase activity is modulated at least in part by changes in the mobility of key domains.

SH3 Domain Tyrosine Phosphorylation – Sites, Role and Evolution

BackgroundSH3 domains are eukaryotic protein domains that participate in a plethora of cellular processes including signal transduction, proliferation, and cellular movement. Several studies indicate that tyrosine phosphorylation could play a significant role in the regulation of SH3 domains.ResultsTo explore the incidence of the tyrosine phosphorylation within SH3 domains we queried the PhosphoSite Plus database of phosphorylation sites. Over 100 tyrosine phosphorylations occurring on 20 different SH3 domain positions were identified. The tyrosine corresponding to c–Src Tyr-90 was by far the most frequently identified SH3 domain phosphorylation site. A comparison of sequences around this tyrosine led to delineation of a preferred sequence motif ALYD(Y/F). This motif is present in about 15% of human SH3 domains and is structurally well conserved. We further observed that tyrosine phosphorylation is more abundant than serine or threonine phosphorylation within SH3 domains and other adaptor domains, such as SH2 or WW domains. Tyrosine phosphorylation could represent an important regulatory mechanism of adaptor domains.ConclusionsWhile tyrosine phosphorylation typically promotes signaling protein interactions via SH2 or PTB domains, its role in SH3 domains is the opposite - it blocks or prevents interactions. The regulatory function of tyrosine phosphorylation is most likely achieved by the phosphate moiety and its charge interfering with binding of polyproline helices of SH3 domain interacting partners.

Consensus in silico computational modelling of the p22phox subunit of the NADPH oxidase

The p22phox protein is an essential subunit of the cytochrome b558 of the NADPH oxidase (Nox) complex which by generating reactive oxygen species (ROS) plays important role in regulating cellular function. p22phox stabilises the Nox enzyme, assists in catalytic core maturation and in the meantime provides an anchoring site for cytosolic regulatory subunits to bind. However, the protein structure of the p22phox is still uncertain. In this study we use an in silico computational bioinformatic approach to produce a consensus 3-dimensional model of the p22phox. Based on published protein sequence data of human p22phox and by using transmembrane specific protein prediction algorithms, we found that p22phox consists of two domains: an N-terminal transmembrane domain (124 a.a.) and a C-terminal cytoplasmic domain (71 a.a.). In its predicted most stable form, p22phox contains three transmembrane helices leading to an extracellular N-terminus and an extensive (39 a.a.) extracellular loop between helices 2 and 3. Furthermore, we locate the cytosolic domain phosphorylation site at threonine147 which literature shows is capable of priming the p22phox, in order to accept its binding partners. Our results are consistent with the biological characterisation of p22phox derived from experiments using specific antibody or genetic manipulation. Our 3-D protein model provides insights into the biological function of p22phox and cytochrome b558, and can be used as tool to investigate the regulatory mechanism of Nox isoforms.

Molecular Characterisation of Long-Acting Insulin Analogues in Comparison with Human Insulin, IGF-1 and Insulin X10

Aims/HypothesisThere is controversy with respect to molecular characteristics of insulin analogues. We report a series of experiments forming a comprehensive characterisation of the long acting insulin analogues, glargine and detemir, in comparison with human insulin, IGF-1, and the super-mitogenic insulin, X10.MethodsWe measured binding of ligands to membrane-bound and solubilised receptors, receptor activation and mitogenicity in a number of cell types.ResultsDetemir and glargine each displayed a balanced affinity for insulin receptor (IR) isoforms A and B. This was also true for X10, whereas IGF-1 had a higher affinity for IR-A than IR-B. X10 and glargine both exhibited a higher relative IGF-1R than IR binding affinity, whereas detemir displayed an IGF-1R:IR binding ratio of ≤1. Ligands with high relative IGF-1R affinity also had high affinity for IR/IGF-1R hybrid receptors. In general, the relative binding affinities of the analogues were reflected in their ability to phosphorylate the IR and IGF-1R. Detailed analysis revealed that X10, in contrast to the other ligands, seemed to evoke a preferential phosphorylation of juxtamembrane and kinase domain phosphorylation sites of the IR. Sustained phosphorylation was only observed from the IR after stimulation with X10, and after stimulation with IGF-1 from the IGF-1R. Both X10 and glargine showed an increased mitogenic potency compared to human insulin in cells expressing many IGF-1Rs, whereas only X10 showed increased mitogenicity in cells expressing many IRs.ConclusionsDetailed analysis of receptor binding, activation and in vitro mitogenicity indicated no molecular safety concern with detemir.

Abstract 3933: ERα phosphorylation patterns at hinge domain Ser294 and Ser305 differentiate ligand-dependent from ligand-independent receptor activation

Abstract Domain-specific phosphorylation of estrogen receptor alpha (ERα) is critical for its transcription factor activity and may determine its predictive and therapeutic biomarker value. Recent attention has turned to the poorly understood ERα hinge domain in light of preliminary evidence that phosphorylation at serine 305 (pSer305) associates with poor clinical outcome and endocrine resistance. Using multiple reaction monitoring (MRM) mass spectrometry (MS), we have studied a number of ERα overexpressing human breast cancer cells lines (MCF-7, BT474, T47D) and found that robust ERα phosphorylation occurs on a neighboring hinge domain site at Ser294, but only upon ligand (estradiol, tamoxifen) stimulation of the receptor since growth factor cell stimulation (EGF, insulin, heregulin-α, forskolin) fails to induce pSer294. As well, pSer294 formation appears to be essential for full ligand induction of ERα gene transcription in experimental models. Selective inhibition of cyclin-dependent kinases (CDKs) by roscovitine or SNS-032 in the breast cancer cell lines blocks ligand induced pSer294 formation, indicating that this hinge domain phosphorylation is mediated by CDKs; likewise, cell-free studies with recombinant ERα and specific cyclin-CDK complexes reveal that Ser294 phosphorylation is most effectively induced by the transcription-regulating and cell cycle-independent kinases, CDK7 and CDK9. In contrast to this ligand specific induction of pSer294, western analysis of immunopurified ERα from MCF-7 probed with a newly available pSer305 monoclonal demonstrates that pSer305 formation occurs in response to growth factor stimulation (EGF, insulin, forskolin) but not in response to cell stimulation by ligand alone. Interestingly, co-treatment with ligand and growth factor reduces pSer294 formation relative to ligand stimulation alone while co-treatment reduces pSer305 formation relative to growth factor stimulation alone, suggesting that these two hinge phosphorylation sites not only respond reciprocally to specify different aspects of ERα function but are also antagonistically responsive to crosstalk. Unlike the better studied N-terminal domain phosphorylation sites at Ser118 and Ser167, which are phosphorylated jointly and additively upon combined ligand-dependent and ligand-independent ERα activation, the two hinge domain sites at Ser294 and Ser305 appear to respond in a reciprocal fashion differentiating ligand-dependent from ligand-independent ERα activation. Pending current development of a pSer294 monoclonal, future immunohistochemical interrogation of the ERα hinge domain phosphorylation status at Ser294 and Ser305 is expected to improve the biomarker value of this receptor by better predicting ERα overexpressing breast cancers most likely to respond to endocrine agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3933. doi:1538-7445.AM2012-3933

Cold temperature induces mucin hypersecretion from normal human bronchial epithelial cells in vitro through a transient receptor potential melastatin 8 (TRPM8)–mediated mechanism

Cold air stimulus is a major environmental factor that exacerbates chronic inflammatory airway diseases, such as chronic obstructive pulmonary disease (COPD) and asthma. At the molecular level, cold is detected by transient receptor potential melastatin 8 (TRPM8). To date, TRPM8 expression has not been characterized in the airway epithelium of patients with COPD. The role of TRPM8 channels in a series of airway responses induced by cold stimuli and the molecular and biochemical pathways of TRPM8 in regulating cold-induced responses are largely unknown. We sought to explore the role of TRPM8 in cold air-provoked mucus hypersecretion and the potential signaling pathway involved in this process. The expression of TRPM8 in the bronchial epithelium was examined by means of immunohistochemistry, RT-PCR, and Western blotting. TRPM8 receptor function and hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) were characterized by means of Ca(2+) imaging and spatiotemporal dynamics of phospholipase C (PLC) δ1-pleckstrin homology-green fluorescent protein, respectively. The expression of MUC5AC mRNA and MUC5AC mucin protein was measured by using real-time PCR and ELISA, respectively. Four serine residues in the myristoylated alanine-rich C kinase substrate (MARCKS)-phosphorylation site domain were mutated to identify the function of MARCKS in TRPM8-mediated airway mucus hypersecretion. TRPM8 protein and mRNA expression were significantly increased in patients with COPD compared with expression seen in healthy subjects. Cold produced robust increases in intracellular Ca(2+) levels and promoted translocation of PLCδ1-pleckstrin homology-green fluorescent protein. Cold increased expression of MUC5AC mRNA and intracellular and secreted MUC5AC protein in a nonsustained way. Phosphorylation site domain-mutant MARCKS cDNA hindered MUC5AC secretion induced by cold. These results indicate that the TRPM8 receptor is involved in cold-induced mucus hypersecretion through the Ca(2+)-PLC-PIP2-MARCKS signaling pathway.

MAP Kinase analyser: A tool for plant kinase and substrate analysis

MAPK (Mitogen Activated Protein Kinase) is a Ser/Thr kinase, which plays a crucial role in plant growth and development, transferring the extra cellular stimuli into intracellular response etc. Manual identification of these MAPK in the plant genome is tedious and time taking process. There are number of online servers which predict the P-site (phosphorylation site), find the motifs and domain but there is no specific tool which can identify all them together. In order to identify the P-Site, phosphorylation site consensus sequences and domain of the MAPK in plant genome, we developed a tool, MAP Kinase analyzer. MAP kinase analyzer take protein sequence as input in the fasta format and the output of tool includes: 1) The prediction of the phosphorylation site viz., Serine (S), Threonine (T), and Tyrosine (Y), Contex, Position, Score and phosphorylating kinase as well as the graphical output; 2) Phosphorylation site consensus sequence pattern for different kinases and 3) Domain information about the MAPK's. The MAP kinase analyser tool and supplementary files can be downloaded from http://www.bioinfogbpuat/mapk_OWN_1/.

Distinct roles for Crk adaptor isoforms in actin reorganization induced by extracellular signals

Crk family adaptors, consisting of Src homology 2 (SH2) and SH3 protein-binding domains, mediate assembly of protein complexes in signaling. CrkI, an alternately spliced form of Crk, lacks the regulatory phosphorylation site and C-terminal SH3 domain present in CrkII and CrkL. We used gene silencing combined with mutational analysis to probe the role of Crk adaptors in platelet-derived growth-factor receptor beta (PDGFbetaR) signaling. We demonstrate that Crk adaptors are required for formation of focal adhesions, and for PDGF-stimulated remodeling of the actin cytoskeleton and cell migration. Crk-dependent signaling is crucial during the early stages of PDGFbetaR activation, whereas its termination by Abl family tyrosine kinases is important for turnover of focal adhesions and progression of dorsal-membrane ruffles. CrkII and CrkL preferentially activate the small GTPase Rac1, whereas variants lacking a functional C-terminal SH3 domain, including CrkI, preferentially activate Rap1. Thus, differences in the activity of Crk isoforms, including their effectors and their ability to be downregulated by phosphorylation, are important for coordinating dynamic changes in the actin cytoskeleton in response to extracellular signals.