The src-family protein-tyrosine kinase p59hck is located on the secretory granules in human neutrophils and translocates towards the phagosome during cell activation.
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ABSTRACT:
SUBMITTER: Mohn H
PROVIDER: S-EPMC1135781 | biostudies-other | 1995 Jul
REPOSITORIES: biostudies-other
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Project description:Neutrophils play a critical role in innate immunity and also influence adaptive immune responses. This occurs in good part through their production of inflammatory and immunomodulatory cytokines, in conjunction with their prolonged survival at inflamed foci. While a picture of the signaling machinery underlying these neutrophil responses is now emerging, much remains to be uncovered. In this study, we report that neutrophils constitutively express various Src family isoforms (STKs), as well as Syk, and that inhibition of these protein tyrosine kinases selectively hinders inflammatory cytokine generation by acting posttranscriptionally. Accordingly, STK or Syk inhibition decreases the phosphorylation of signaling intermediates (e.g., eIF-4E, S6K, and MNK1) involved in translational control. By contrast, delayed apoptosis appears to be independent of either STKs or Syk. Our data therefore significantly extend our understanding of which neutrophil responses are governed by STKs and Syk and pinpoint some signaling intermediates that are likely involved. In view of the foremost role of neutrophils in several chronic inflammatory conditions, our findings identify potential molecular targets that could be exploited for future therapeutic intervention.
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Project description:Protein kinases are enzymes that catalyze the covalent transfer of the γ-phosphate of an adenosine triphosphate (ATP) molecule onto a tyrosine, serine, threonine, or histidine residue in the substrate and thus send a chemical signal to networks of downstream proteins. They are important cellular signaling enzymes that regulate cell growth, proliferation, metabolism, differentiation, and migration. Unregulated protein kinase activity is often associated with a wide range of diseases, therefore making protein kinases major therapeutic targets. A prototypical system of central interest to understand the regulation of kinase activity is provided by tyrosine kinase c-Src, which belongs to the family of Src-related non-receptor tyrosine kinases (SFKs). Although the broad picture of autoinhibition via the regulatory domains and via the phosphorylation of the C-terminal tail is well characterized from a structural point of view, a detailed mechanistic understanding at the atomic-level is lacking. Advanced computational methods based on all-atom molecular dynamics (MD) simulations are employed to advance our understanding of tyrosine kinase activation. The computational studies suggest that the isolated kinase domain (KD) is energetically most favorable in the inactive conformation when the activation loop (A-loop) of the KD is not phosphorylated. The KD makes transient visits to a catalytically competent active-like conformation. The process of bimolecular trans-autophosphorylation of the A-loop eventually locks the KD in the active state. Activating point mutations may act by slightly increasing the population of the active-like conformation, enhancing the availability of the A-loop to be phosphorylated. The Src-homology 2 (SH2) and Src-homology 3 (SH3) regulatory domains, depending upon their configuration, either promote the inactive or the active state of the kinase domain. In addition to the roles played by the SH3, SH2, and KD, the Src-homology 4-Unique domain (SH4-U) region also serves as a key moderator of substrate specificity and kinase function. Thus, a fundamental understanding of the conformational propensity of the SH4-U region and how this affects the association to the membrane surface are likely to lead to the discovery of new intermediate states and alternate strategies for inhibition of kinase activity for drug discovery. The existence of a multitude of KD conformations poses a great challenge aimed at the design of specific inhibitors. One promising computational strategy to explore the conformational flexibility of the KD is to construct Markov state models from aggregated MD data.
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Project description:BackgroundBreast cancer is the most common female cancer and a major cause of morbidity and mortality. Progress in breast cancer therapeutics has been attained with the introduction of targeted therapies for specific sub-sets. However, other subsets lack targeted interventions and thus there is persisting need for identification and characterization of molecular targets in order to advance breast cancer therapeutics.AimTo analyze the role of lesions in neurotrophic receptor tyrosine kinase (NTRK) genes in breast cancers.MethodsAnalysis of publicly available genomic breast cancer datasets was performed for identification and characterization of cases with fusions and other molecular abnormalities involving NTRK1, NTRK2 and NTRK3 genes.ResultsNTRK fusions are present in a small number of breast cancers at the extensive GENIE project data set which contains more than 10000 breast cancers. These cases are not identified as secretory in the database, suggesting that the histologic characterization is not always evident. In the breast cancer The Cancer Genome Atlas (TCGA) cohort the more common molecular lesion in NTRK genes is amplification of NTRK1 observed in 7.9% of breast cancers.ConclusionNeurotrophin receptors molecular lesions other than fusions are observed more often than fusions. However, currently available NTRK inhibitors are effective mainly for fusion lesions. Amplifications of NTRK1, being more frequent in breast cancers, could be a viable therapeutic target if inhibitors efficacious for them become available.
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Project description:The Anion Cl-/HCO3- Exchangers AE1, AE2, and AE3 are membrane pH regulatory ion transporters ubiquitously expressed in vertebrate tissues. Besides relieving intracellular alkaline and CO2 loads, the AEs have an important function during development and cell death and play a central role in such cellular properties as cell shape, metabolism, and contractility. The activity of AE(s) are regulated by neurohormones. However, little is known as to the intracellular signal transduction pathways that underlie this modulation. We show here that, in cardiomyocytes that express both AE1 and AE3, the purinergic agonist, ATP, triggers activation of anion exchange. The AE activation is observed in cells in which AE3 expression was blocked but not in cells microinjected with neutralizing anti-AE1 antibodies. ATP induces tyrosine phosphorylation of AE1, activation of the tyrosine kinase Fyn, and association of both Fyn and FAK with AE1. Inhibition of Src family kinases in vivo by genistein, herbimycin A, or ST638 prevents purinergic activation of AE1. Microinjection of either anti-Cst.1 antibody or recombinant CSK, both of which prevent activation of Src family kinase, significantly decreases ATP-induced activation of AE. Microinjection of an anti-FAK antibody as well as expression in cardiomyocytes of Phe397 FAK dominant negative mutant, also prevents purinergic activation of AE. Therefore, tyrosine kinases play a key role in acute regulation of intracellular pH and thus in cell function including excitation-contraction coupling of the myocardium.
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Project description:We investigated the basis for previous observations that fluorescent-labeled neuropeptide Y (NPY) is usually released within 200 ms after fusion, whereas labeled tissue plasminogen activator (tPA) is often discharged over many seconds. We found that tPA and NPY are endogenously expressed in small and different subpopulations of bovine chromaffin cells in culture. We measured the mobility of these proteins (tagged with fluorophore) within the lumen of individual secretory granules in living chromaffin cells, and related their mobilities to postfusion release kinetics. A method was developed that is not limited by standard optical resolution, in which a bright flash of strongly decaying evanescent field (∼64 nm exponential decay constant) produced by total internal reflection (TIR) selectively bleaches cerulean-labeled protein proximal to the glass coverslip within individual granules. Fluorescence recovery occurred as unbleached protein from distal regions within the 300 nm granule diffused into the bleached proximal regions. The fractional bleaching of tPA-cerulean (tPA-cer) was greater when subsequently probed with TIR excitation than with epifluorescence, indicating that tPA-cer mobility was low. The almost equal NPY-cer bleaching when probed with TIR and epifluorescence indicated that NPY-cer equilibrated within the 300 ms bleach pulse, and therefore had a greater mobility than tPA-cer. TIR-fluorescence recovery after photobleaching revealed a significant recovery of tPA-cer (but not NPY-cer) fluorescence within several hundred milliseconds after bleaching. Numerical simulations, which take into account bleach duration, granule diameter, and the limited number of fluorophores in a granule, are consistent with tPA-cer being 100% mobile, with a diffusion coefficient of 2 × 10(-10) cm(2)/s (∼1/3000 of that for a protein of similar size in aqueous solution). However, the low diffusive mobility of tPA cannot alone explain its slow postfusion release. In the accompanying study, we suggest that, additionally, tPA itself stabilizes the fusion pore with dimensions that restrict its own exit.
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Project description:Src, originally identified as an oncogene, is a membrane-anchored tyrosine kinase and the Src family kinase (SFK) prototype. SFKs regulate the signalling induced by a wide range of cell surface receptors leading to epithelial cell growth and adhesion. In the intestine, the SFK members Src, Fyn and Yes regulate epithelial cell proliferation and migration during tissue regeneration and transformation, thus implicating conserved and specific functions. In patients with colon cancer, SFK activity is a marker of poor clinical prognosis and a potent driver of metastasis formation. These tumorigenic activities are linked to SFK capacity to promote the dissemination and tumour-initiating capacities of epithelial tumour cells. However, it is unclear how SFKs promote colon tumour formation and metastatic progression because SFK-encoding genes are unfrequently mutated in human cancer. Here, we review recent findings on SFK signalling during intestinal homeostasis, regeneration and tumorigenesis. We also describe the key nongenetic mechanisms underlying SFK tumour activities in colorectal cancer, and discuss how these mechanisms could be exploited in therapeutic strategies to target SFK signalling in metastatic colon cancer.
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Project description:The translocated actin recruiting phosphoprotein (Tarp) is injected into the cytosol shortly after Chlamydia trachomatis attachment to a target cell and subsequently phosphorylated by an unidentified tyrosine kinase. A role for Tarp phosphorylation in bacterial entry is unknown. In this study, recombinant C. trachomatis Tarp was employed to identify the host cell kinase(s) required for phosphorylation. Each tyrosine rich repeat of L2 Tarp harbors a sequence similar to a Src and Abl kinase consensus target. Furthermore, purified p60-src, Yes, Fyn, and Abl kinases were able to phosphorylate Tarp. Mutagenesis of potential tyrosines within a single tyrosine rich repeat peptide indicated that both Src and Abl kinases phosphorylate the same residues suggesting that C. trachomatis Tarp may serve as a substrate for multiple host cell kinases. Surprisingly, chemical inhibition of Src and Abl kinases prevented Tarp phosphorylation in culture and had no measurable effect on bacterial entry into host cells.
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Project description:Fatal familial insomnia (FFI), genetic Creutzfeldt-Jakob disease (gCJD), and Gerstmann-Sträussler-Scheinker (GSS) syndrome are neurodegenerative disorders linked to prion protein (PrP) mutations. The pathogenic mechanisms are not known, but increasing evidence points to mutant PrP misfolding and retention in the secretory pathway. We previously found that the D178N/M129 mutation associated with FFI accumulates in the Golgi of neuronal cells, impairing post-Golgi trafficking. In this study we further characterized the trafficking defect induced by the FFI mutation and tested the 178N/V129 variant linked to gCJD and a nine-octapeptide repeat insertion associated with GSS. We used transfected HeLa cells, embryonic fibroblasts and primary neurons from transgenic mice, and fibroblasts from carriers of the FFI mutation. In all these cell types, the mutant PrPs showed abnormal intracellular localizations, accumulating in the endoplasmic reticulum (ER) and Golgi. To test the efficiency of the membrane trafficking system, we monitored the intracellular transport of the temperature-sensitive vesicular stomatite virus glycoprotein (VSV-G), a well-established cargo reporter, and of endogenous procollagen I (PC-I). We observed marked alterations in secretory trafficking, with VSV-G accumulating mainly in the Golgi complex and PC-I in the ER and Golgi. A redacted version of mutant PrP with reduced propensity to misfold did not impair VSV-G trafficking, nor did artificial ER or Golgi retention of wild-type PrP; this indicates that both misfolding and intracellular retention were required to induce the transport defect. Pharmacological activation of Src family kinase (SFK) improved intracellular transport, suggesting that mutant PrP impairs secretory trafficking through corruption of SFK-mediated signaling.
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Project description:FilGAP is a Rac-specific GTPase-activating protein (GAP) that suppresses lamellae formation. In this study, we have identified RBM10 (RNA Binding Motif domain protein 10) as a FilGAP-interacting protein. Although RBM10 is mostly localized in the nuclei in human melanoma A7 cells, forced expression of Src family tyrosine kinase Fyn induced translocation of RBM10 from nucleus into cell peripheries where RBM10 and FilGAP are co-localized. The translocation of RBM10 from nucleus appears to require catalytic activity of Fyn since kinase-negative Fyn mutant failed to induce translocation of RBM10 in A7 cells. When human breast carcinoma MDA-MB-231 cells are spreading on collagen-coated coverslips, endogenous FilGAP and RBM10 were localized at the cell periphery with tyrosine-phosphorylated proteins. RBM10 appears to be responsible for targeting FilGAP at the cell periphery because depletion of RBM10 by siRNA abrogated peripheral localization of FilGAP during cell spreading. Association of RBM10 with FilGAP may stimulate RacGAP activity of FilGAP. First, forced expression of RBM10 suppressed FilGAP-mediated cell spreading on collagen. Conversely, depletion of endogenous RBM10 by siRNA abolished FilGAP-mediated suppression of cell spreading on collagen. Second, FilGAP suppressed formation of membrane ruffles induced by Fyn and instead produced spiky cell protrusions at the cell periphery. This protrusive structure was also induced by depletion of Rac, suggesting that the formation of protrusions may be due to suppression of Rac by FilGAP. We found that depletion of RBM10 markedly reduced the formation of protrusions in cells transfected with Fyn and FilGAP. Finally, depletion of RBM10 blocked FilGAP-mediated suppression of ruffle formation induced by EGF. Taken together, these results suggest that Src family tyrosine kinase signaling may regulate FilGAP through association with RBM10.
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