Time Course Of Tyrosine Phosphorylation Research Articles

A proteomic approach to the investigation of early events involved in the activation of vascular smooth muscle cells

Vascular smooth muscle cells (VSMC) are mature cells that maintain great plasticity. This distinctive quality is the basis of the migration and proliferation of VSMC in cardiovascular diseases. We have investigated, via a proteomic approach, the molecular changes that promote VSMC switching from a quiescent to an activated-proliferating phenotype. In particular, we focus on the modulation in tyrosine phosphorylation that occurs in cell activation by serum or by single growth factors, such as insulin-like growth factor 1 (IGF-1) or platelet-derived growth factor (PDGF-BB). A comparison of profiles from two-dimensional polyacrylamide gel electrophoresis analysis of quiescent and activated-proliferating VSMC has revealed a number of differences in protein expression. Several differentially expressed proteins have been identified by mass spectrometry, and their changes during the time course of tyrosine phosphorylation have been documented from time zero up to 48 h after stimulus. The tyrosine-phosphorylation level generally decreases within a few minutes of stimulation, followed by a rapid dramatic recovery of some chaperones and redox enzymes, but no significant recovery for glucose metabolism enzymes. With respect to cytoskeleton components, no remarkable fluctuations have been detected at the earliest time points, except for those relating to alpha-actin, which displays an impressive decrease. A comparison of the early stages of cell stimulation after serum or after single growth factor administration has revealed important differences in the phosphorylation of chaperones, thereby suggesting their crucial role in VSMC activation.

Evidence for the involvement of proline-directed serine/threonine phosphorylation in sperm capacitation

To become fertilization competent, mammalian sperm undergo changes in the female reproductive tract termed capacitation. Capacitation correlates with an increase in tyrosine phosphorylation; however, less is known about the role of serine/threonine phosphorylation in this process. Proline-directed phosphorylation is one of the major regulatory phosphorylation events in many cellular processes such as cell proliferation and differentiation. Using mitotic phosphoprotein monoclonal-2 (MPM-2) antibody in this study, we observed that several mouse sperm proteins in the range of 70-250 kDa underwent increased serine/threonine-proline phosphorylation during capacitation. In contrast to the time course of tyrosine phosphorylation, proline-directed phosphorylation could be observed at shorter time points of sperm incubation, and it was found to be independent of NaHCO(3) and adenosine 3'5'-cyclic monophosphate (cAMP). Similar to the regulation of the increase in tyrosine phosphorylation, cholesterol acceptors such as bovine serum albumin (BSA) or 2-hydroxypropyl-beta-cyclodextrin (2-OH-propyl-beta-CD) were essential for the regulation of proline-directed phosphorylation in mouse sperm. Furthermore, it was also found to be BSA dependent in human sperm. Among proline-directed kinases, extracellular signal-regulated kinase 1/2 (ERK1/2) is present in mammalian sperm; nevertheless, U0126 and PD098059, two inhibitors of the ERK pathway, did not block this phosphorylation in mouse sperm. In conclusion, capacitation is associated with an increase in proline-directed phosphorylation linked to cholesterol efflux in the sperm.

Novelty of the Pyruvate Metabolic Enzyme Dihydrolipoamide Dehydrogenasein Spermatozoa: CORRELATION OF ITS LOCALIZATION, TYROSINE PHOSPHORYLATION,AND ACTIVITY DURING SPERMCAPACITATION

Spermatozoa are cells distinctly different from other somatic cells of the body, capacitation being one of the unique phenomena manifested by this gamete. We have shown earlier that dihydrolipoamide dehydrogenase, a post-pyruvate metabolic enzyme, undergoes capacitation-dependent tyrosine phosphorylation, and the functioning of the enzyme is required for hyperactivation (enhanced motility) and acrosome reaction of hamster spermatozoa (Mitra, K., and Shivaji, S. (2004) Biol. Reprod. 70, 887-899). In this report we have investigated the localization of this mitochondrial enzyme in spermatozoa revealing non-canonical extra-mitochondrial localization of the enzyme in mammalian spermatozoa. In hamster spermatozoa, dihydrolipoamide dehydrogenase along with its host complex, the pyruvate dehydrogenase complex, are localized in the acrosome and in the principal piece of the sperm flagella. The localization of dihydrolipoamide dehydrogenase, however, appears to be in the mitochondria in the spermatocytes, but in spermatids it appears to show a juxtanuclear localization (like Golgi). The capacitation-dependent time course of tyrosine phosphorylation of dihydrolipoamide dehydrogenase appears to be different in the principal piece of the flagella and the acrosome in hamster spermatozoa. Activity assays of this bi-directional enzyme suggest a strong correlation between the tyrosine phosphorylation and the bi-directional enzyme activity. This is the first report of a direct correlation of the localization, tyrosine phosphorylation, and activity of the important metabolic enzyme, dihydrolipoamide dehydrogenase, implicating dual involvement and regulation of the enzyme during sperm capacitation.

The functional role of the JAK-STAT pathway in post-infarction remodeling.

Recently, the Janus kinase/signal transducer and activator of transcription (JAK-STAT) signaling pathway was found to be prominently associated with activation of the autocrine loop of the heart tissue-localized renin angiotensin system (RAS). We investigated if the JAK-STAT pathway is activated in the post-myocardial infarction (MI) non-ischemic myocardium (NIM), destined to undergo remodeling and whether blockade of the pathway in vivo can modify early post-MI remodeling. We investigated the time course of tyrosine phosphorylation of JAK-STAT and gp130 proteins in the NIM of post-MI rat heart as well as the binding activity of STAT proteins to the St-domain of the angiotensinogen gene promoter. We further compared the effects of in vivo blockade of RAS by the AT(1) receptor (AT(1)R) blocker losartan with the in vivo blockade of JAK-STAT pathway by the specific JAK2 blocker tyrphostin AG490 on certain aspects of early post-MI remodeling. We showed that JAK2, STATs 1, 3, 5a and 6 and gp130 proteins are tyrosine phosphorylated as early as 5-30 min post-MI and that STATs 1, 3, and 5a remain activated up to 7 days post-MI. Gel mobility shift assay showed a strong binding activity of STAT proteins to the St-domain of angiotensinogen gene promoter in 1-day post-MI NIM. The binding was significantly reduced in rat hearts previously treated with losartan or tyrphostin AG490. Supershift experiments identified STATs 3 and 5a as specifically interacting with the St-domain. Both AT(1)R and JAK2 blockade resulted in significant amelioration of the increase of protein phosphatase 1 activity and decrease in basal level of p16-phospholamban that may underlie early diastolic dysfunction, as well as partial amelioration of early downregulation of Kv4.2 gene expression that may underlie increased arrhythmogenicity of 3-day post-MI heart. On the other hand, while blockade of AT(1)R significantly ameliorated apoptotic changes in 1-day post-MI border zone, blockade of JAK2 increased apoptosis. The study provides compelling evidence in favor of the linkage of the JAK-STAT pathway with the angiotensin II autocrine loop and uncovers a mechanism by which selective activation of a set of STAT proteins underlies mobilization of the gene activation program intrinsic to post-MI remodeling. It also suggests that drugs that inhibit JAK-STAT phosphorylation may provide a new approach to modify post-MI remodeling. This needs to be confirmed in long term in vivo studies in the post-MI heart.

Association of IgG Fc receptor II with tyrosine kinases in the human basophilic leukemia cell line KU812F

Background: We previously reported that crosslinking of IgG Fc receptor II (FcγRII) induces intracellular calcium mobilization, but not histamine release in human basophils. To clarify functional activities of FcγRII on human basophils, we analyzed the FcγRII-mediated signaling events in the human basophilic leukemia cell line KU812F. Methods: Flow cytometric methods were used to investigate the effect on intracellular calcium mobilization of cross-linking of FcγRII. KU812F cells were pre-incubated with anti-FcγRII monoclonal antibody (IV.3). After the addition of various concentrations of the tyrosine kinase inhibitor genistein or buffer alone, cells were stimulated with goat antimouse IgG F(ab') 2 (GAM) and analyzed with the flow cytometer. Next, in order to test the signaling events after cross-linking of FcγRII, we examined tyrosine kinase activity. The time-course of tyrosine phosphorylation after cross-linking of FcγRII and the effect of genistein on this tyrosine phosphorylation were tested by immunoblotting. Immunoprecipitation was also performed to identify the type of tyrosine kinase associated with signal transduction of FcγRII. Results: The tyrosine kinase inhibitor genistein inhibited intracellular calcium mobilization caused by cross-linking of FcγRII in a dose-dependent manner. Rapid tyrosine phosphorylation after FcγRII cross-linking was shown by immunoblot analysis and this phosphorylation was inhibited by genistein. Furthermore, tyrosine phosphorylation of Lyn and Syk was observed upon cross-linking of FcγRII. Conclusions: Tyrosine phosphorylation is necessary for the signaling pathway through FcγRII and tyrosine phosphorylation of Lyn and Syk, at least, is actively involved in this signal transduction.

Conversion of Threonine 757 to Valine Enhances Stat5a Transactivation Potential

The growth hormone family of cytokines transduces intracellular signals through the Jak2-Stat5 pathway to activate the transcription of target genes. Amino acids within the C termini of Stats constitute the transactivation domain but also regulate the time course of tyrosine phosphorylation and extent of DNA binding. We mutated Thr(757) in the C-terminal of Stat5a (Thr-Stat5) to Val (Val-Stat5) and Asp (Asp-Stat5) and examined the effect on nuclear translocation, DNA binding, and prolactin-induced transcriptional activation of a Stat5-responsive luciferase reporter gene. Val-Stat5 produced a 5-fold higher increase in transcriptional activity relative to Thr-Stat5; Asp-Stat5 produced a similar response to Thr-Stat5. The increased transactivation was ligand induced and was not due to differences in basal expression of Val-Stat5 or to a constitutively activated Stat5 protein. Similar rates of loss of DNA binding ability and phosphorylation of Val- and Thr-Stat5 were observed following a single pulse of prolactin, indicating that the dephosphorylation pathways were unaltered. The serine-threonine kinase inhibitor H7 inhibited the transactivation potential of Thr-, Val-, and Asp-Stat5 to a similar extent, eliminating phosphorylation of Thr(757) as a regulatory mechanism. The results suggest that Thr(757) modulates the transactivation potential of Stat5 by a mechanism(s) that is dependent on the formation of Stat5 dimers and/or their nuclear translocation.

Developmental Regulation of Tyrosine Phosphorylation of the Nicotinic Acetylcholine Receptor inTorpedoElectrocyte

Tyrosine phosphorylation is thought to play a critical role in the clustering of acetylcholine receptors (AChR) at the developing neuromuscular junction. Yet,in vitroapproaches have led to conflicting conclusions regarding the function of tyrosine phosphorylation of AChR β subunit in AChR clustering. In this work, we followedin situthe time course of tyrosine phosphorylation of AChR in developingTorpedoelectrocyte. We observed that tyrosine phosphorylation of the AChR β and δ subunits occurs at a late stage of embryonic development after the accumulation of AChRs and rapsyn in the membrane and the onset of innervation. Interestingly, in the mature postsynaptic membrane, we observed two populations of AChR differing both in their phosphotyrosine content and distribution. Our data are consistent with the notion that tyrosine phosphorylation of the AChR is related to downstream events in the pathway regulating AChR accumulation rather than to initial clustering events.

Regulation of the Association of p120cbl with Grb2 in Jurkat T Cells

The c-cbl protooncogene product (p120(cbl)) is a known substrate of multiple tyrosine kinases. It is found in complexes with critical signal transduction molecules, including the linker protein Grb2. Here, we demonstrate using an immobilized Grb2-binding peptide that the Grb2-p120(cbl) complex dissociates in vivo following engagement of the T-cell antigen receptor in Jurkat T-cells. The early kinetics of this dissociation correlate with the known time course of tyrosine phosphorylation of p120(cbl) and other substrates. This dissociation persists in vivo even when p120(cbl) becomes dephosphorylated to basal levels. However, this decreased association is not observed in protein overlay assays on nitrocellulose membranes in which a Grb2 fusion protein is used to detect p120(cbl) from stimulated or unstimulated cells. These data suggest that the tyrosine phosphorylation of p120(cbl) does not completely account for the regulation of its association with Grb2. Additionally, we used truncation mutations of p120(cbl) to map the p120(cbl)-Grb2 interaction to amino acids 481-528 of p120(cbl); this interaction is stronger in longer constructs that include additional proline-rich motifs. The in vivo regulation of the Grb2-p120(cbl) complex further supports the idea of a significant role for p120(cbl) in receptor-mediated signaling pathways.

Shc is the predominant signaling molecule coupling insulin receptors to activation of guanine nucleotide releasing factor and p21ras-GTP formation.

Insulin stimulates tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and She in Rat1 fibroblasts overexpressing wild type insulin receptors. We investigated the relative role of IRS-1 and She in insulin activation of guanine nucleotide releasing factor (GNRF) and p21ras-GTP formation. The time course of insulin-stimulated tyrosine phosphorylation of IRS-1 was rapid, whereas Shc phosphorylation was relatively slow. Growth factor receptor bound protein-2 (Grb2) associated with IRS-1 rapidly and gradually dissociated after 5 min, whereas Grb2 association with Shc was slower and reached a maximum at 10 min after insulin stimulation. Thus, the kinetics of Grb2 association with IRS-1 and She corresponded closely to the time course of tyrosine phosphorylation of IRS-1 and Shc, respectively. Importantly, 3-13-fold more Grb2 was associated with Shc than with IRS-1. In addition, the kinetics of insulin-stimulated GNRF activity and p21ras-GTP formation corresponded more closely to the time course of Shc phosphorylation than to the kinetics of IRS-1 phosphorylation. Furthermore, immunoprecipitation of Shc proteins from cell lysates of insulin-stimulated cells removed 67% of the GNRF activity, whereas precipitation of IRS-1 had a negligible effect on GNRF activity. Thus, although both IRS-1 and Shc associate with Grb2, the current results indicate that Shc plays a more important role than IRS-1 in insulin stimulation of GNRF activity and subsequent p21ras-GTP formation.

Stimulation of human neutrophils with formyl-methionyl-leucyl-phenylalanine induces tyrosine phosphorylation and activation of two distinct mitogen-activated protein-kinases.

Stimulation of human polymorphonuclear neutrophils with the chemotactic peptide FMLP induces an increase in the phosphorylation of several proteins on tyrosine residues. Immunoblotting of whole cell lysates with antiphosphotyrosine antibodies demonstrated this increase to be predominant in two proteins with apparent m.w. of 40,000 and 42,000, respectively. We identify these two proteins as members of the mitogen-activated-protein (MAP) kinase family. These results are based on comigration of the tyrosine phosphorylated proteins with two proteins of same apparent m.w., recognized by a series of antibodies raised against sequences contained within members of this family. This was further confirmed by immunoprecipitation with either an antiphosphotyrosine antibody or a MAP kinase-specific antibody. To assess whether neutrophil stimulation with FMLP increased MAP kinase activity, the cells were stimulated with various doses of FMLP and the phosphotransferase activity in cell lysates was measured against two identified substrates of MAP kinase: myelin basic protein and T-669, the epidermal growth factor receptor Thr 669 synthetic peptide modeled after the major site of phosphorylation of the receptor. A dose- and time-dependent increase in MAP kinase activity was detected in the lysates from FMLP-stimulated PMN, which closely correlated with the dose and time course of tyrosine phosphorylation of the 40- and 42-kDa proteins. Additionally, the MAP kinases in crude lysates were enriched by DEAE-cellulose chromatography and further resolved on a MonoQ column by fast performance liquid chromatography. The respective eluates contained the two proteins, each recognized by anti-MAP kinase antibodies and, after stimulation, each was phosphorylated on tyrosine residues and expressed T-669 phosphotransferase activity. Treatment with genistein, a tyrosine kinase inhibitor, reduced the tyrosine phosphorylation of the 40- and 42-kDa proteins in a dose-dependent manner, decreased the activation of the MAP kinases, and inhibited the production of superoxide anion by FMLP-stimulated neutrophils. Therefore, this study demonstrates that human polymorphonuclear neutrophils contain two distinct members of the MAP kinase family, antigenically related to sea star p44mpk and rat p43erk1 and that both MAP kinase isoforms are implicated in the cascade of protein phosphorylation induced by neutrophil stimulation with FMLP. Furthermore, these data suggest that tyrosine phosphorylation and activation of these two enzymes may play a regulatory role in the signal transduction pathway leading to the respiratory burst induced by FMLP.

Tyrosine phosphorylation in cells treated with transforming growth factor‐β

Cells stimulated with epidermal growth factor (EGF) or any one of a diverse group of other mitogenic agents display an increased tyrosine phosphorylation of a pair of 42,000 Mr proteins. Transforming Growth Factor-beta (TGF-beta) is able to potentiate the mitogenic effects of Epidermal Growth Factor on some fibroblastic cells (such as the NRK-49F cell line) and, in addition, permits the anchorage-independent growth of these cells. In this study we asked whether these growth-regulatory actions of Transforming Growth Factor-beta are associated with changes in tyrosine phosphorylation of cellular proteins, in particular the 42,000 Mr proteins. We found no effect of Transforming Growth Factor-beta on the extent or time-course of tyrosine phosphorylation, either by itself or in combination with Epidermal Growth Factor. Since the tyrosine phosphorylation of the 42,000 Mr proteins is stimulated both by receptors with tyrosine kinase activity and by diacylglycerol analogs (but not by Transforming Growth Factor-beta), we suggest that the activity of the receptor for Transforming Growth Factor-beta is linked neither to tyrosine phosphorylation nor to phosphatidyl inositol turnover.