Cellular signaling by tyrosine phosphorylation in keloid and normal human dermal fibroblasts

Keloids represent a dysregulated response to cutaneous wounding that results in disfiguring scars. Unique to humans, keloids are characterized by an accumulation of extracellular matrix components. The underlying molecular mechanisms of keloid pathogenesis, however, remain largely uncharacterized. Similarly, cellular signaling mechanisms, which may indicate inherent differences in the way keloid fibroblasts and normal human dermal fibroblasts interact with extracellular matrix or other cells, have not been investigated. As part of a fundamental assessment of cellular response to injury in keloid fibroblasts, phosphorylation studies were performed using three different keloid (n = 3) and normal human dermal (n = 3) fibroblast cell lines. These studies were undertaken to elucidate whether keloid and normal human dermal fibroblasts exhibit different tyrosine kinase activity. Initially, distinct tyrosine phosphorylation patterns of keloid and normal human dermal fibroblasts were demonstrated. Next, the phosphorylation patterns were correlated with known molecules that may be important to keloid pathogenesis. On the basis of molecular weight, it was hypothesized that the highly phosphorylated bands seen in keloid fibroblasts represented epidermal growth factor receptor (EGFR); discoidin domain receptor 1 (DDR1); and Shc, an adaptor protein known to bind many tyrosine kinases, including EGFR and DDR1. Individual immunoblotting using EGFR, DDR1, and Shc antibodies revealed greater expression in keloid fibroblasts compared with normal human dermal fibroblasts. These data substantiate for the first time the finding of greater phosphorylation by the above-mentioned molecules, which may be important in keloid pathogenesis.     

Regulation of airway goblet cell mucin secretion by tyrosine phosphorylation signaling pathways

Mucus hyperproduction in pulmonary obstructive diseases results from increased goblet cell numbers and possibly increased cellular mucin synthesis, occurring in response to inflammatory mediators acting via receptor tyrosine kinases (RYK) and tyrosine phosphorylation (Y-Pi) signaling pathways. Yet, increased mucin synthesis does not lead necessarily to increased secretion, as mucins are stored in secretory granules and secreted in response to extracellular signals, commonly assumed to be mediated by G protein-coupled receptors (GPCRs). We asked whether activation 1) of Y-Pi signaling pathways, in principal, and 2) of the novel PKC isoform, nPKCdelta, by Y-Pi, specifically, might lead to regulated mucin secretion. nPKCdelta in SPOC1 cells was tyrosine phosphorylated by exposure to purinergic agonist (ATPgammaS) or PMA, actions that were blocked by the Src kinase inhibitor, PP1. Mucin secretion, however, was not affected by PP1. Hence, activation of nPKCdelta by Y-Pi is unlikely to participate in GPCR-related mucin secretion. Mucin secretion from both SPOC1 and normal human bronchial epithelial (NHBE) cells was stimulated by generalized protein Y-Pi induced by the tyrosine phosphatase inhibitor, pervanadate (PV). PV-induced SPOC1 cell mucin secretion was not affected by inhibition of Src kinases (genistein or PP1), or of PI3 kinase (LY-294002). MAP kinase pathway inhibitors, RAF1 kinase inhibitor-I and U0126 (MEK), inhibited SPOC1 cell PV-induced secretion by approximately 50%. Significantly, the phospholipase C (PLC) inhibitor, U-73122, essentially abolished PV- and ATPgammaS-induced mucin secretion from both SPOC1 and NHBE cells. Hence, PLC signaling may play a key role in regulated mucin secretion, whether the event is initiated by mediators interacting with GPCRs or RYKs.     

Structural basis of syndecan-4 phosphorylation as a molecular switch to regulate signaling

The syndecan transmembrane proteoglycans are involved in the organization of the actin cytoskeleton and have important roles as cell surface receptors during cell-matrix interactions. We have shown that the syndecan-4 cytoplasmic domain (4L) forms oligomeric complexes that bind to and stimulate PKCalpha activity in the presence of PtdIns(4,5)P2, emphasizing the importance of multimerization in the regulation of PKCalpha activation. Oligomerization of the cytoplasmic domain of syndecan-4 is regulated either positively by PtdIns(4,5)P2 or negatively by phosphorylation of serine 183. Phosphorylation results in reduced PKCalpha activity by inhibiting PtdIns(4,5)P2-dependent oligomerization of the syndecan-4 cytoplasmic domain. Data from NMR and gel-filtration chromatography show that the phosphorylated cytoplasmic domain (p-4L) exists as a dimer, similar to 4L, but not as higher-order oligomers. NMR analysis showed that the overall conformation of p-4L is a compact intertwined dimer with an unusually symmetric clamp shape, and its molecular surface is mostly positively charged. The two parallel strands form a cavity in the center of the dimeric twist. An especially marked effect of phosphorylation of the syndecan-4 cytoplasmic domain is a dramatic conformational change near the C2 region that ablates an interaction site with the PDZ domain of syntenin. Wound healing studies further suggest that syndecan-4 phosphorylation might influence cell migration behavior. We conclude that the phosphorylation (Ser183) of syndecan-4 can play a critical role as a molecular switch to regulate its functions through conformational change.     

Inhibition of capacitation-associated tyrosine phosphorylation signaling in rat sperm by epididymal protein Crisp-1

Ejaculated sperm are unable to fertilize an egg until they undergo capacitation. Capacitation results in the acquisition of hyperactivated motility, changes in the properties of the plasma membrane, including changes in proteins and glycoproteins, and acquisition of the ability to undergo the acrosome reaction. In all mammalian species examined, capacitation requires removal of cholesterol from the plasma membrane and the presence of extracellular Ca2+ and HCO3-. We designed experiments to elucidate the conditions required for in vitro capacitation of rat spermatozoa and the effects of Crisp-1, an epididymal secretory protein, on capacitation. Protein tyrosine phosphorylation, a hallmark of capacitation in sperm of other species, occurs during 5 h of in vitro incubation, and this phosphorylation is dependent upon HCO3-, Ca2+, and the removal of cholesterol from the membrane. Crisp-1, which is added to the sperm surface in the epididymis in vivo, is lost during capacitation, and addition of exogenous Crisp-1 to the incubation medium inhibits tyrosine phosphorylation in a dose-dependent manner, thus inhibiting capacitation and ultimately the acrosome reaction. Inhibition of capacitation by Crisp-1 occurs upstream of the production of cAMP by the sperm.     

Characterizing tyrosine phosphorylation signaling in lung cancer using SH2 profiling

Background

Tyrosine kinases drive the proliferation and survival of many human cancers. Thus profiling the global state of tyrosine phosphorylation of a tumor is likely to provide a wealth of information that can be used to classify tumors for prognosis and prediction. However, the comprehensive analysis of tyrosine phosphorylation of large numbers of human cancer specimens is technically challenging using current methods.

Methodology/Principal Findings

We used a phosphoproteomic method termed SH2 profiling to characterize the global state of phosphotyrosine (pTyr) signaling in human lung cancer cell lines. This method quantifies the phosphorylated binding sites for SH2 domains, which are used by cells to respond to changes in pTyr during signaling. Cells could be grouped based on SH2 binding patterns, with some clusters correlated with EGF receptor (EGFR) or K-RAS mutation status. Binding of specific SH2 domains, most prominently RAS pathway activators Grb2 and ShcA, correlated with EGFR mutation and sensitivity to the EGFR inhibitor erlotinib. SH2 binding patterns also reflected MET activation and could identify cells driven by multiple kinases. The pTyr responses of cells treated with kinase inhibitors provided evidence of distinct mechanisms of inhibition.

Conclusions/Significance

This study illustrates the potential of modular protein domains and their proteomic binding profiles as powerful molecular diagnostic tools for tumor classification and biomarker identification.     

Protein tyrosine phosphorylation and calcium signaling in thyroid FRTL-5 cells

We examined the importance of tyrosine kinase(s) on the ATP-evoked Ca²⁺ entry and DNA synthesis of thyroid FRTL-5 cells. ATP rapidly and transiently tyrosine phosphorylated a 72-kDa protein(s). This phosphorylation was abolished by pertussis toxin and by the tyrosine kinase inhibitor genistein, and was dependent on Ca²⁺ entry. Pretreatment of the cells with genistein did not affect the release of sequestered Ca²⁺, but the capacitative Ca²⁺ or Ba²⁺ entry evoked by ATP or thapsigargin was attenuated. Pretreatment of the cells with orthovanadate enhanced the increase in intracellular free Ca²⁺ ([Ca²⁺]_i), whereas the Ba²⁺ entry was not increased. Phorbol 12-myristate 13-acetate (PMA) phosphorylated the same protein(s) as did ATP. Genistein inhibited the ATP-evoked phosphorylation of MAP kinase and attenuated both the ATP- and the PMA-evoked DNA synthesis. However, genistein did not inhibit the ATP-evoked expression of c-fos. Furthermore, genistein enhanced the ATP-evoked release of arachidonic acid. Thus, ATP activates a tyrosine kinase via a Ca²⁺-dependent mechanism. A genistein-sensitive mechanism participates, in part, in the ATP-evoked activation of DNA synthesis. Genistein inhibits only modestly capacitative Ca²⁺ entry in FRTL-5 cells. J. Cell. Physiol. 175:211–219, 1998. © 1998 Wiley-Liss, Inc.     

Calcium signaling induced by adhesion mediates protein tyrosine phosphorylation and is independent of pHi

Our goal was to evaluate early signaling events that occur as epithelial cells make initial contact with a substrate and to correlate them with phosphorylation. The corneal epithelium was chosen to study signaling events that occur with adhesion because it represents a simple system in which the tissue adheres to a basal lamina, is avascular, and is bathed by a tear film in which changes in the local environment are hypothesized to alter signaling. To perform these experiments we developed a novel adhesion assay to capture the changes in intracellular Ca(2+) and pH that occur as a cell makes its initial contact with a substrate. The first transient cytosolic Ca(2+) peak was detected only as the cell made contact with the substrate and was demonstrated using fluorimetric assays combined with live cell imaging. We demonstrated that this transient Ca(2+) peak always preceded a cytoplasmic alkalization. When the intracellular environment was modified, the initial response was altered. Pretreatment with 1,2-bis(o-aminophenoxy)ethane-N,N, N'N'-tetraacetic acid (BAPTA), an intracellular chelator, inhibited Ca(2+) mobilization, whereas benzamil altered the duration of the oscillations. Thapsigargin caused an initial Ca(2+) release followed by a long attenuated response. An inositol triphosphate analog induced a large initial response, whereas heparin inhibited Ca(2+) oscillations. Inhibitors of tyrosine phosphorylation did not alter the initial mobilization of cytosolic Ca(2) but clearance of cytosolic Ca(2+) was inhibited. Exposing corneal epithelial cells to BAPTA, benzamil, or thapsigargin also attenuated the phosphorylation of the focal adhesion protein paxillin. However, although heparin inhibited Ca(2+) oscillations, it did not alter phosphorylation of paxillin. These studies demonstrate that the initial contact that a cell makes with a substrate modulates the intracellular environment, and that changes in Ca(2+) mobilization can alter later signaling events such as the phosphorylation of specific adhesion proteins. These findings may have implications for wound repair and development.     

Ultraviolet radiation rapidly induces tyrosine phosphorylation and calcium signaling in lymphocytes.

UV radiation is known to induce lymphocyte nonresponsiveness both in vitro and in vivo. We have found that UV radiation rapidly induced tyrosine phosphorylation and calcium signaling in normal human peripheral blood lymphocytes. In the leukemic T cell line Jurkat and the Burkitt's lymphoma cell line Ramos, UV rapidly induced tyrosine phosphorylation in a wavelength-dependent manner, giving strong signals after UVB and UVC, but not UVA, irradiation. Similarly, in Jurkat cells UV-induced calcium signals were dependent on the dose of UVB or UVC irradiation over a range of 150-1200 J/m2, but only a small signal was observed for UVA at a dose of 1200 J/m2. The UV-induced calcium signals were blocked by the tyrosine kinase inhibitor herbimycin A, indicating that they were dependent on tyrosine phosphorylation. Phospholipase C (PLC) gamma 1 was tyrosine phosphorylated in response to UV irradiation but to a lesser extent than observed after CD3 cross-linking. However, PLC gamma 1-associated proteins demonstrated to bind to the PLC gamma 1 SH2 domain were tyrosine phosphorylated strongly after UV irradiation. A similar dose response was observed for the inhibition by herbimycin A of UV-induced calcium signals and UV-induced tyrosine phosphorylation of PLC gamma 1 and associated proteins. We propose that in contrast to CD3/Ti stimulation, UV aberrantly triggers lymphocyte signal transduction pathways by a mechanism that bypasses normal receptor control.     

Identification of proteins undergoing tyrosine phosphorylation during mouse sperm capacitation

Mammalian sperm are not able to fertilize immediately upon ejaculation; they become fertilization-competent after undergoing changes in the female reproductive tract collectively termed capacitation. Although it has been established that capacitation is associated with an increase in tyrosine phosphorylation, little is known about the role of this event in sperm function. In this work we used a combination of two dimensional gel electrophoresis and mass spectrometry to identify proteins that undergo tyrosine phosphorylation during capacitation. Some of the identified proteins are the mouse orthologues of human sperm proteins known to undergo tyrosine phosphorylation. Among them we identified VDAC, tubulin, PDH E1 beta chain, glutathione S-transferase, NADH dehydrogenase (ubiquinone) Fe-S protein 6, acrosin binding protein precursor (sp32), proteasome subunit alpha type 6b and cytochrome b-c1 complex. In addition to previously described proteins, we identified two testis-specific aldolases as substrates for tyrosine phosphorylation. Genomic and EST analyses suggest that these aldolases are retroposons expressed exclusively in the testis, as has been reported elsewhere. Because of the importance of glycolysis for sperm function, we hypothesize that tyrosine phosphorylation of these proteins can play a role in the regulation of glycolysis during capacitation. However, neither the Km nor the Vmax of aldolase changed as a function of capacitation when its enzymatic activity was assayed in vitro, suggesting other levels of regulation for aldolase function.     

Identification of the insulin-responsive tyrosine phosphorylation sites on IRSp53

The 53-kDa insulin receptor substrate protein (IRSp53) is part of a regulatory network that organises the actin cytoskeleton in response to stimulation by small GTPases, promoting formation of actin-rich cell protrusions such as filopodia and lamellipodia. It had been established earlier that IRSp53 is tyrosine phosphorylated in response to stimulation of the insulin and insulin-related growth factor receptors, but the consequences of tyrosine phosphorylation for IRSp53 function are unknown. Here, we have used a variety of IRSp53 truncation and point mutants to identify insulin-responsive tyrosine phosphorylation sites on IRSp53. We have found that the C-terminal half of IRSp53 (residues 251-521) undergoes tyrosine phosphorylation in response to insulin stimulation of the insulin beta receptor or epidermal growth factor stimulation via the epidermal growth factor receptor, and that the key residue for insulin receptor-mediated phosphorylation is tyrosine 310, located in a region between the N-terminal IRSp53/MIM homology domain (IMD, residue 1-250) and the central SH3 domain (residues 374-438) that is predicted to be natively unstructured. Mutation of tyrosine 310 to phenylalanine or glutamic acid abrogates the phosphorylation in response to insulin stimulation, but not in response to stimulation of the epidermal growth factor receptor. The N-terminal IMD, which mediates dimerisation of IRSp53, is required for efficient tyrosine phosphorylation downstream of either the insulin or epidermal growth factor receptor stimulation, yet does not appear to include a tyrosine-phosphorylated site itself. Thus, we have identified tyrosine 310 as a primary site of tyrosine phosphorylation in response to insulin signalling and we have shown that although IRSp53 is tyrosine phosphorylated in response to epidermal growth factor receptor signalling, tyrosine 310 is not crucial. Furthermore, the tyrosine phosphorylation status does not appear to affect the cell morphology and production of filopod-like structures upon expression of IRSp53.     

Identification of the angiogenesis signaling domain in pleiotrophin defines a mechanism of the angiogenic switch

Neoplasms progress through genetic and epigenetic mutations that deregulate pathways in the malignant cell that stimulate more aggressive growth of the malignant cell itself and/or remodel the tumor microenvironment to support the developing tumor mass. The appearance of new blood vessels in malignant tumors is known as the "angiogenic switch." The angiogenic switch triggers a stage of rapid tumor growth supported by extensive tumor angiogenesis and a more aggressive tumor phenotype and its onset is a poor prognostic indicator for host survival. Identification of the factors that stimulate the angiogenic switch thus is of high importance. Pleiotrophin (PTN the protein, Ptn the gene) is an angiogenic factor and the Ptn gene has been found to be constitutively expressed in many human tumors of different cell types. These studies use a nude mouse model to test if Ptn constitutively expressed in premalignant cells is sufficient to trigger an angiogenic switch in vivo. We introduced an ectopic Ptn gene into "premalignant" SW-13 cells and analyzed the phenotype of SW-13 Ptn cell tumor implants in the flanks of nude mice. SW-13 Ptn cell subcutaneous tumor implants grew very rapidly and had a striking increase in the density of new blood vessels compared to the SW-13 cell tumor implants, suggesting that constitutive PTN signaling in the premalignant SW-13 cell implants in the nude mouse recapitulates fully the angiogenic switch. It was found also that ectopic expression of the C-terminal domain of PTN in SW-13 cell implants was equally effective in initiating an angiogenic switch as the full-length PTN whereas implants of SW-13 cells in nude mice that express the N-terminal domain of PTN grew rapidly but failed to develop tumor angiogenesis. The data suggest the possibility that mutations that activate Ptn in premalignant cells are sufficient to stimulate an angiogenic switch in vivo and, since these mutations are frequently found in human malignancies, that constitutive PTN signaling may be an important contributor to progression of human tumors. The data also suggest that the C-terminal and the N-terminal domains of PTN equally initiate switches in premalignant cells to cells of a more aggressive tumor phenotype but the separate domains of PTN signal different mechanisms and perhaps signal through activation of a separate receptor-like protein.     

PDGF beta-receptor stimulates tyrosine phosphorylation of GAP and association of GAP with a signaling complex

Platelet-derived growth factor (PDGF) stimulated the tyrosine phosphorylation of the GTPase activating protein (GAP) in 3T3 cells and in CHO cells expressing wild-type PDGF receptors, but not in several CHO cell lines expressing mutant receptors defective in transmitting mitogenic signals. Following PDGF treatment of cells, GAP physically associated with the PDGF receptor and with Raf-1, phospholipase c-gamma, and PI-3 kinase, suggesting that PDGF induced the formation of complexes of signaling molecules. The association of GAP with the PDGF receptor and the phosphorylation of GAP with the PDGF receptor and the phosphorylation of GAP were reconstituted in vitro using purified protein and in insect cells expressing murine PDGF receptor and human GAP. However, in cells transformed by activated c-Ha-ras, which are defective in certain responses to PDGF, GAP failed to associate with the PDGF receptor or increase its phosphotyrosine content in response to PDGF. The association of GAP with ligand-activated PDGF receptors may directly link PDGF and ras signaling pathways.     

Proteasome inhibitors regulate tyrosine phosphorylation of IRS-1 and insulin signaling in adipocytes

Insulin rapidly stimulates the tyrosine kinase activity of its receptor, resulting in the phosphorylation of insulin receptor substrates (IRS), which in turn associates and activates PI 3-kinase, leading to an increase in glucose uptake. Phosphorylation of IRS proteins and activation of downstream kinases by insulin are transient and the mechanisms for the subsequent downregulation of their activity are largely unknown. We report here that the insulin-induced IRS-1 tyrosine phosphorylation and PI 3-kinase association to IRS-1 were strongly sustained by the proteasome inhibitors, MG132 and lactacystin. In contrast, no effect was detected on the insulin receptor and IRS-2 tyrosine phosphorylation. Interestingly, lactacystin also preserved PKB activation and insulin-induced glucose uptake. In contrast, calpeptin, a calpain inhibitor, was ineffective. Tyrosine phosphatase assays were also performed, showing that lactacystin was not functioning directly as a tyrosine phosphatase inhibitor "in vitro." In conclusion, proteasome inhibitors can regulate the tyrosine phosphorylation of IRS-1 and the downstream insulin signaling pathway, leading to glucose transport.     

Identification of four phosphorylation sites in the N-terminal region of tyrosine hydroxylase

As reported previously [Vulliet et al. (1985) FEBS Lett. 182 335-339], tyrosine hydroxylase purified from rat pheochromocytoma is phosphorylated at an identical site (site A) by cyclic AMP-dependent protein kinase, the calmodulin-dependent multiprotein kinase and protein kinase C, while the calmodulin-dependent multiprotein kinase also phosphorylates another unique site (site C). Preparations of tyrosine hydroxylase purified from this source are also contaminated with traces of a fourth protein kinase which phosphorylates another unique site (site E). We have isolated tryptic peptides containing each of these sites and determined their amino acid sequences. By comparison of these data with the known cDNA sequence for rat tyrosine hydroxylase, we have been able to identify these sites as Ser-8 (site E), Ser-19 (site C), and Ser-40 (site A). In some preparations of tyrosine hydroxlyase, cyclic AMP-dependent protein kinase also phosphorylated a secondary site which was identified as ser-153. All of these phosphorylation sites are in the amino-terminal region, where there is no significant homology with the closely related enzyme, phenylalanine hydroxylase. Our data also establish that the initiator methionine is removed by post-translational processing to leave pro-2 as the amino-terminus of the mature protein. The significance of these results for the mechanism of action of extracellular signals on catecholamine biosynthesis is discussed.     

EGF induces tyrosine phosphorylation of phospholipase C-II: a potential mechanism for EGF receptor signaling

Binding of EGF to cells expressing human EGF receptor stimulated rapid tyrosine phosphorylation of phospholipase C-II (PLC-II), as revealed by immunoblotting analysis with phosphotyrosine-specific antibodies. Tyrosine phosphorylation of PLC-II was stimulated by low physiological concentrations of EGF (1 nM), was quantitative, and was already maximal after a 30 sec incubation with 50 nM EGF at 37 degrees C. Interestingly, antibodies specific for PLC-II were able to coimmunoprecipitate the EGF receptor and antibodies against EGF receptor also coimmunoprecipitated PLC-II. According to this analysis, approximately 1% of EGF receptor molecules were associated with PLC-II molecules. The protein tyrosine kinase inhibitor tyrphostin RG50864, which blocks EGF-dependent cell proliferation, blocked EGF-induced tyrosine phosphorylation of PLC-II, its association with EGF receptor, and EGF-induced Ca2+ release. Hence, EGF-induced tyrosine phosphorylation of PLC-II may be a regulatory event linking the tyrosine kinase activity of EGF receptor to the PIP2 hydrolysis signaling pathway.     

Trask phosphorylation defines the reverse mode of a phosphotyrosine signaling switch that underlies cell anchorage state

Phosphotyrosine signaling in anchored epithelial cells constitutes a spacially ordained signaling program that largely functions to promote integrin-linked focal adhesion complexes, serving to secure cell anchorage to matrix and as a bidirectional signaling hub that coordinates the physical state of the cell and its environment with cellular functions including proliferation and survival. Cells release their adhesions during processes such as mitosis, migration or tumorigenesis, but the fate of signaling through tyrosine phosphorylation in unanchored cells remains poorly understood. In an examination of epithelial cells in the unanchored state, we find abundant phosphotyrosine signaling, largely recommitted to an anti-adhesive function mediated through the Src family phosphorylation of their transmembrane substrate Trask/CDCP1/gp140. Src-Trask phosphorylation inhibits integrin clustering and focal adhesion assembly and signaling, defining an active phosphotyrosine signaling program underlying the unanchored state. Src-Trask signaling and Src-focal adhesion signaling inactivate each other, constituting two opposing modes of phosphotyrosine signaling that define a switch underline cell anchorage state. Src kinases are prominent drivers of both signaling modes, identifying their position at the helm of adhesion signaling capable of specifying anchorage state through substrate selection. These experimental studies along with concurring phylogenetic evidence suggest that phosphorylation on tyrosine is a signaling function fundamentally linked with the regulation of integrins.Key words: Trask, CDCP1, gp140, tyrosine phosphorylation, integrin, Src     

Peroxynitrite activates kinases of the src family and upregulates tyrosine phosphorylation signaling

The hypothesis that peroxynitrite may act as a signaling molecule able to upregulate protein tyrosine phosphorylation is discussed. This article focuses on the mechanisms for activating kinases of the src family, an important class of nonreceptor tyrosine kinases implicated in the regulation of cell communication, proliferation, migration, differentiation, and survival. Recent in vitro findings show that in erythrocytes, synaptosomes, and cerebellar primary culture cells peroxynitrite is able to inhibit phosphatases and to activate different members of the src kinase family through different mechanisms involving cysteine-dependent and -independent processes. The ability of nitrotyrosine-containing peptides with SH2 binding affinity to activate src kinases is also discussed.     

A new tyrosine phosphorylation site in PLC gamma 1: the role of tyrosine 775 in immune receptor signaling

Phospholipase Cgamma (PLCgamma) is a ubiquitous gatekeeper of calcium mobilization and diacylglycerol-mediated events induced by the activation of Ag and growth factor receptors. The activity of PLCgamma is regulated through its controlled membrane translocation and tyrosine (Y) phosphorylation. Four activation-induced tyrosine phosphorylation sites have been previously described (Y472, Y771, Y783, and Y1254), but their specific roles in Ag receptor-induced PLCgamma1 activation are not fully elucidated. Unexpectedly, we found that the phosphorylation of a PLCgamma1 construct with all four sites mutated to phenylalanine was comparable with that observed with wild-type PLCgamma1, suggesting the existence of an unidentified site(s). Sequence alignment with known phosphorylation sites in PLCgamma2 indicated homology of PLCgamma1 tyrosine residue 775 (Y775) with PLCgamma2 Y753, a characterized phosphorylation site. Tyrosine 775 was characterized as a phosphorylation site using phospho-specific anti-Y775 antiserum, and by mutational analysis. Phosphorylation of Y775 did not depend on the other tyrosines, and point mutation of PLCgamma1 Y775, or the previously described Y783, substantially reduced AgR-induced calcium, NF-AT, and AP-1 activation. Mutation of Y472, Y771, and Y1254 had no effect on overall PLCgamma1 phosphorylation or activation. Although the concomitant mutation of Y775 and Y783 abolished downstream PLCgamma1 signaling, these two tyrosines were sufficient to reconstitute the wild-type response in the absence of functional Y472, Y771, and Y1254. These data establish Y775 as a critical phosphorylation site for PLCgamma1 activation and confirm the functional importance of Y783.     

Mechanism of tyrosine hydroxylase activation by phosphorylation

It was found that the fluorescence of 1,N⁶-ethenoadenosine triphosphate (ε-ATP) bound to myosin subfragment-1 (S-1) is resistant to quenching by acrylamide, while free ε-ATP is effectively quenched. Thus in the presence of acrylamide the bound ε-ATP is still highly fluorescent, while free ε-ATP is much less fluorescent. The Stern-Volmer constants of bound and free ε-ATP are 6.83 and 57.86 M^?1, respectively. Therefore it is easy to distinguish spectro-scopically the nucleotide-ligated S-1 from nucleotide-free S-1. Moreover acrylamide does not alter the S-1-Mg²⁺-ε-ATPase behavior.