Cholesterol substitution increases the structural heterogeneity of caveolae

Caveolin-1 binds cholesterol and caveola formation involves caveolin-1 oligomerization and cholesterol association. The role of cholesterol in caveolae has so far been addressed by methods that compromise membrane integrity and abolish caveolar invaginations. To study the importance of sterol specificity for the structure and function of caveolae, we replaced cholesterol in mammalian cells with its immediate precursor desmosterol by inhibiting 24-dehydrocholesterol reductase. Desmosterol could substitute for cholesterol in maintaining cell growth, membrane integrity, and preserving caveolar invaginations. However, in desmosterol cells the affinity of caveolin-1 for sterol and the stability of caveolin oligomers were decreased. Moreover, caveolar invaginations became more heterogeneous in dimensions and in the number of caveolin-1 molecules per caveola. Despite the altered caveolar structure, caveolar ligand uptake was only moderately inhibited. We found that in desmosterol cells, Src kinase phosphorylated Cav1 at Tyr(14) more avidly than in cholesterol cells. Taken the role of Cav1 Tyr(14) phosphorylation in caveolar endocytosis, this may help to preserve caveolar uptake in desmosterol cells. We conclude that a sterol C24 double bond interferes with caveolin-sterol interaction and perturbs caveolar morphology but facilitates Cav1 Src phosphorylation and allows caveolar endocytosis. More generally, substitution of cholesterol by a structurally closely related sterol provides a method to selectively modify membrane protein-sterol affinity, structure and function of cholesterol-dependent domains without compromising membrane integrity.     

Co-Regulation of Cell Polarization and Migration by Caveolar Proteins PTRF/Cavin-1 and Caveolin-1

Caveolin-1 and caveolae are differentially polarized in migrating cells in various models, and caveolin-1 expression has been shown to quantitatively modulate cell migration. PTRF/cavin-1 is a cytoplasmic protein now established to be also necessary for caveola formation. Here we tested the effect of PTRF expression on cell migration. Using fluorescence imaging, quantitative proteomics, and cell migration assays we show that PTRF/cavin-1 modulates cellular polarization, and the subcellular localization of Rac1 and caveolin-1 in migrating cells as well as PKCα caveola recruitment. PTRF/cavin-1 quantitatively reduced cell migration, and induced mesenchymal epithelial reversion. Similar to caveolin-1, the polarization of PTRF/cavin-1 was dependent on the migration mode. By selectively manipulating PTRF/cavin-1 and caveolin-1 expression (and therefore caveola formation) in multiple cell systems, we unveil caveola-independent functions for both proteins in cell migration.     

Epithelial growth factor-induced phosphorylation of caveolin 1 at tyrosine 14 stimulates caveolae formation in epithelial cells

Caveolae are flask-shaped endocytic structures composed primarily of caveolin-1 (Cav1) and caveolin-2 (Cav2) proteins. Interestingly, a cytoplasmic accumulation of Cav1 protein does not always result in a large number of assembled caveolae organelles, suggesting a regulatory mechanism that controls caveolae assembly. In this study we report that stimulation of epithelial cells with epithelial growth factor (EGF) results in a profound increase in the number of caveolar structures at the plasma membrane. Human pancreatic tumor cells (PANC-1) and normal rat kidney cells (NRK), as a control, were treated with 30 ng/ml EGF for 0, 5, and 20 min before fixation and viewing by electron microscopy. Cells fixed without EGF treatment exhibited modest numbers of plasma membrane-associated caveolae. Cells treated with EGF for 5 or 20 min showed an 8-10-fold increase in caveolar structures, some forming long, pronounced caveolar "towers" at the cell-cell borders. It is known that Cav1 is Src-phosphorylated on tyrosine 14 in response to EGF treatment, although the significance of this modification is unknown. We postulated that phosphorylation could provide the stimulus for caveolae assembly. To this end, we transfected cells with mutant forms of Cav1 that could not be phosphorylated (Cav1Y14F) and tested if this altered protein reduced the number of EGF-induced caveolae. We observed that EGF-stimulated PANC-1 cells expressing the mutant Cav1Y14F protein exhibited a 90-95% reduction in caveolae number compared with cells expressing wild type Cav1. This study provides novel insights into how cells regulate caveolae formation and implicates EGF-based signaling cascades in the phosphorylation of Cav1 as a stimulus for caveolae assembly.     

Molecular Characterization of Caveolin-induced Membrane Curvature

The generation of caveolae involves insertion of the cholesterol-binding integral membrane protein caveolin-1 (Cav1) into the membrane, however, the precise molecular mechanisms are as yet unknown. We have speculated that insertion of the caveolin scaffolding domain (CSD), a conserved amphipathic region implicated in interactions with signaling proteins, is crucial for caveola formation. We now define the core membrane-juxtaposed region of Cav1 and show that the oligomerization domain and CSD are protected by tight association with the membrane in both mature mammalian caveolae and a model prokaryotic system for caveola biogenesis. Cryoelectron tomography reveals the core membrane-juxtaposed domain to be sufficient to maintain oligomerization as defined by polyhedral distortion of the caveolar membrane. Through mutagenesis we demonstrate the importance of the membrane association of the oligomerization domain/CSD for defined caveola biogenesis and furthermore, highlight the functional significance of the intramembrane domain and the CSD for defined caveolin-induced membrane deformation. Finally, we define the core structural domain of Cav1, constituting only 66 amino acids and of great potential to nanoengineering applications, which is required for caveolin-induced vesicle formation in a bacterial system. These results have significant implications for understanding the role of Cav1 in caveola formation and in regulating cellular signaling events.     

Negative regulation of protease-activated receptor 1-induced Src kinase activity by the association of phosphocaveolin-1 with Csk

Protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, has been correlated with cell proliferation. PAR1 is activated by the irreversibly proteolytic cleavage, internalized via clathrin-coated pits, and then sorted to lysosomes for degradation. Caveolae play important roles in both signaling transduction and internalization of several GPCRs. However, the role of caveolae in cellular signaling and trafficking of PAR1 is still unclear. In this study, we show that PAR1 was partially localized in caveolae. Disruption of caveolae by cholesterol depletion did not inhibit PAR1 internalization, indicating that internalization of PAR1 was not via caveolae. Of interest, activation of PAR1 resulted in the phosphorylation of caveolin-1, a principal component of caveolae, on tyrosine 14 by a Gi-linked Src kinase pathway and p38 mitogen-activated protein kinase. Analysis of immunoprecipitates from cells stimulated by PAR1 showed that phosphocaveolin-1 but not caveolin-1 with mutation at tyrosine 14 could bind to Csk. In addition, phosphocaveolin-1 could not bind to CskS109C mutant with the defective SH2 domain. These results indicated that phosphocaveolin-1 was associated with the SH2 domain of Csk in response to PAR1 activation. The association further resulted in a rapid decrease in Src kinase activity. Thus, PAR1-induced Src activation is negatively regulated by recruiting Csk through phosphocaveolin-1. Our results also reveal that phosphocaveolin-1 represents a novel effector of PAR1 to downregulate Src kinase activity. The downregulation of PAR1-induced Src activation mediated by phosphocaveolin-1 provides an additional mechanism for the termination of PAR1 signaling at its downstream molecules.     

Inhibition of protein kinase C reduces ischemia-induced tyrosine phosphorylation of the N-methyl-d-aspartate receptor

The role of protein kinase C (PKC) in tyrosine phosphorylation of the N‐methyl‐d ‐aspartate receptor (NMDAR) following transient cerebral ischemia was investigated. Transient (15 min) cerebral ischemia was produced in adult rats by four‐vessel occlusion and animals allowed to recover for 15 or 45 min. Following ischemia, tyrosine phosphorylation of NR2A and NR2B and activated Src‐family kinases (SFKs) and Pyk2 were increased in post‐synaptic densities (PSDs). Phosphorylation of NR2B on Y1472 by PSDs isolated from post‐ischemic forebrains was inhibited by the SFK specific inhibitor PP2, and by the PKC inhibitors GF109203X (GF), Gö6976 and calphostin C. Intravenous injection of GF immediately following the ischemic challenge resulted in decreased phosphorylation of NR1 on PKC phosphorylation sites and reduced ischemia‐induced increases in tyrosine phosphorylation of NR2A and NR2B without affecting the increase in total tyrosine phosphorylation of hippocampal proteins. Ischemia‐induced increases in activated Pyk2 and SFKs in PSDs, but not the translocation of PKC, Pyk2 or Src to the PSD, were also inhibited by GF. The inactive homologue of GF, bisindolylmaleimide V, had no effect on these parameters. The results are consistent with a role for PKC in the ischemia‐induced increase in tyrosine phosphorylation of the NMDAR, via a pathway involving Pyk2 and Src‐family kinases.     

Src-mediated tyrosine phosphorylation of caveolin-1 induces its association with membrane type 1 matrix metalloproteinase

We have recently shown that stimulation of endothelial cells with vascular endothelial growth factor (VEGF) induces dissociation of caveolin-1 from the VEGFR-2 receptor, followed by Src family kinase-dependent tyrosine phosphorylation of the protein (Labrecque, L., Royal, I., Surprenant, D. S., Patterson, C., Gingras, D., and Beliveau, R. (2003) Mol. Biol. Cell 14, 334-347). In this study, we provide evidence that the VEGF-dependent tyrosine phosphorylation of caveolin-1 induces interaction of the protein with the membrane-type 1 matrix metalloproteinase (MT1-MMP). This interaction requires the phosphorylation of caveolin-1 on tyrosine 14 by members of the Src family of protein kinases, such as Src and Fyn, because it is completely abolished by expression of a catalytically inactive Src mutant or by site-directed mutagenesis of tyrosine 14 of caveolin-1. Most interestingly, the association of MT1-MMP with phosphorylated caveolin-1 induced the recruitment of Src and a concomitant inhibition of the kinase activity of the enzyme, suggesting that this complex may be involved in the negative regulation of Src activity. The association of MT1-MMP with phosphorylated caveolin-1 occurs in caveolae membranes and involves the cytoplasmic domain of MT1-MMP because it was markedly reduced by mutation of Cys574 and Val582 residues of the cytoplasmic tail of the enzyme. Most interestingly, the reduction of the interaction between MT1-MMP and caveolin-1 by using these mutants also decreases MT1-MMP-dependent cell locomotion. Overall these results indicate that MT1-MMP associates with tyrosine-phosphorylated caveolin-1 and that this complex may play an important role in MT1-MMP regulation and function.     

P2Y12 receptor signalling towards PKB proceeds through IGF-I receptor cross-talk and requires activation of Src, Pyk2 and Rap1

Previously it was shown that stimulation of the P2Y12 receptor activates PKB signalling in C6 glioma cells [K. Van Kolen and H. Slegers, J. Neurochem. 89, 442.]. In the present study, the mechanisms involved in this response were further elucidated. In cells transfected with the Gbetagamma-scavenger beta-ARK1/GRK2 or Rap1GAPII, stimulation with 2MeSADP failed to enhance PKB phosphorylation demonstrating that the signalling proceeds through Gbetagamma-subunits and Rap1. Moreover, Rap1-GTP pull-down assays revealed that P2Y12 receptor stimulation induced a rapid activation of Rap1. Treatment of cells with the Ca2+ chelator BAPTA-AM and inhibition of Src and PLD2 with PP2 or 1-butanol, respectively, abrogated P2Y12 receptor-mediated activation of Rap1 and PKB. In addition inhibition of PKCzeta decreased basal and 2MeSADP-stimulated phosphorylation of PKB indicating a role for this PKC isoform in PKB signalling. Although the increased PKB phosphorylation was abolished in the presence of the IGF-I receptor tyrosine kinase inhibitor AG 1024, 2MeSADP did not significantly increase receptor phosphorylation. Nevertheless, phosphorylation of a 120 kDa IGF-I receptor-associated protein was observed. The latter protein was identified by MALDI-TOF/TOF-MS as the proline-rich tyrosine kinase 2 (Pyk2) that co-operates with Src in a PLD2-dependent manner. Consistent with the signalling towards Rap1 and PKB, activation of Pyk2 was abrogated by Ca2+ chelation, inhibition of PLD2 and IGF-I receptor tyrosine kinase activity. In conclusion, the data reveal a novel type of cross-talk between P2Y12 and IGF-I receptors that proceeds through Gbetagamma-, Ca2+-and PLD2-dependent activation of the Pyk2/Src pathway resulting in GTP-loading of Rap1 required for an increased PKB phosphorylation.     

Angiotensin II induces epithelial-to-mesenchymal transition in renal epithelial cells through reactive oxygen species/Src/caveolin-mediated activation of an epidermal growth factor receptor-extracellular signal-regulated kinase signaling pathway

Chronic activation of the renin-angiotensin system plays a deleterious role in progressive kidney damage, and the renal proximal tubule is known to play an important role in tubulointerstitial fibrosis; however, the underlying molecular mechanism is unclear. Here we report that in the proximal tubule-like LLCPKcl4 cells expressing angiotensin II (Ang II) type 1 receptor, Ang II induced changes in cell morphology and expression of epithelial-to-mesenchymal transition (EMT) markers, which were inhibited by the miotogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK)-activating kinase (MEK) inhibitor PD98059 or the Src kinase inhibitor PP2. Ang II-stimulated phosphorylation of caveolin-1 (Cav) at Y14 and epidermal growth factor receptor (EGFR) at Y845 and induced association of these phosphoproteins in caveolin-enriched lipid rafts, thereby leading to prolonged EGFR-ERK signaling that was inhibited by Nox4 small interfering RNA (siRNA) and Src siRNA. Two different antioxidants not only inhibited phosphorylation of Src at Y416 but also blocked the EGFR-ERK signaling. Moreover, erlotinib (the EGFR tyrosine kinase inhibitor), EGFR siRNA, and Cav siRNA all inhibited both prolonged EGFR-ERK signaling and phenotypic changes induced by Ang II. Thus, this report provides the first evidence that reactive oxygen species (ROS)/Src-dependent activation of persistent Cav-EGFR-ERK signaling mediates renal tubular cell dedifferentiation and identifies a novel molecular mechanism that may be involved in progressive renal injury caused by chronic exposure to Ang II.     

Muscarinic receptor stimulation induces TASK1 channel endocytosis through a PKC-Pyk2-Src pathway in PC12 cells

Muscarinic receptor stimulation or protein kinase C (PKC) activation in rat adrenal medullary and PC12 cells rapidly induces tyrosine phosphorylation of TWIK-related-acid-sensitive K⁺ 1 (TASK1) channels with the subsequent clathrin-dependent endocytosis. Our previous study suggested that the muscarinic signal is transmitted to the non-receptor tyrosine kinase Src through PKC and Pyk2. Although PKC activation is known to stimulate Pyk2 in certain types of cells, its molecular mechanism remains unclear. In this study, proximity ligation assay (PLA) and other molecular biological approaches were used to elucidate the details of this muscarinic signaling in PC12 cells. When green fluorescent protein (GFP)-TASK1 was expressed, the majority of GFP-TASK1 was located at the cell periphery. However, the simultaneous expression of GFP-TASK1 and PKCα, but not PKCδ, led to GFP-TASK1 internalization. Muscarinic receptor stimulation resulted in transient co-localization of Pyk2 and Src at the cell periphery, and expression of kinase dead (KD) Pyk2 and Src, but not Pyk2 and KD Src, resulted in GFP-TASK1 internalization. PLA analysis revealed that in response to muscarine, PKCα activates Pyk2 through phosphorylating its serine residues. These results indicate that muscarinic receptor stimulation induces TASK1 channel endocytosis sequentially through PKCα, Pyk2, and Src, and PKCα activates Pyk2 through phosphorylation.     

Critical Role of FLRT1 Phosphorylation in the Interdependent Regulation of FLRT1 Function and FGF Receptor Signalling

Background

Fibronectin leucine rich transmembrane (FLRT) proteins have dual properties as regulators of cell adhesion and potentiators of fibroblast growth factor (FGF) mediated signalling. The mechanism by which the latter is achieved is still unknown and is the subject of this investigation.

Principal Findings

Here we show that FLRT1 is a target for tyrosine phosphorylation mediated by FGFR1 and implicate a non-receptor Src family kinase (SFK). We identify the target tyrosine residues in the cytoplasmic domain of FLRT1 and show that these are not direct substrates for Src kinase suggesting that the SFK may exert effects via potentiation of FGFR1 kinase activity. We show that whilst FLRT1 expression results in a ligand-dependent elevation of MAP kinase activity, a mutant version of FLRT1, defective as an FGFR1 kinase substrate (Y3F-FLRT1), has the property of eliciting ligand-independent chronic activation of the MAP kinase pathway which is suppressed by pharmacological inhibition of either FGFR1 or Src kinase. Functional investigation of FGFR1 and FLRT1 signalling in SH-SY5Y neuroblastoma cells reveals that FLRT1 alone acts to induce a multi-polar phenotype whereas the combination of FLRT1 and FGFR activation, or expression of Y3F-FLRT1, acts to induce neurite outgrowth via MAPK activation. Similar results were obtained in a dendrite outgrowth assay in primary hippocampal neurons. We also show that FGFR1, FLRT1 and activated Src are co-localized and this complex is trafficked toward the soma of the cell. The presence of Y3F-FLRT1 rather than FLRT1 resulted in prolonged localization of this complex within the neuritic arbour.

Conclusions

This study shows that the phosphorylation state of FLRT1, which is itself FGFR1 dependent, may play a critical role in the potentiation of FGFR1 signalling and may also depend on a SFK-dependent phosphorylation mechanism acting via the FGFR. This is consistent with an ‘in vivo’ role for FLRT1 regulation of FGF signalling via SFKs. Furthermore, the phosphorylation-dependent futile cycle mechanism controlling FGFR1 signalling is concurrently crucial for regulation of FLRT1-mediated neurite outgrowth.     

Stimulation of M3 muscarinic receptors induces phosphorylation of the Cdc42 effector activated Cdc42Hs-associated kinase-1 via a Fyn tyrosine kinase signaling pathway

The tyrosine kinase, activated Cdc42Hs-associated kinase-1 (ACK-1), is a specific effector of the Rho family GTPase Cdc42. GTP-bound Cdc42 has been shown to facilitate neurite outgrowth elicited by activation of muscarinic cholinergic receptors (mAChRs). Because tyrosine kinase activity is a requirement for neuritogenesis in several cell systems, we investigated whether endogenous mAChRs (principally of the M3 subtype) expressed in human SH-SY5Y neuroblastoma cells would signal to ACK-1. Incubation of cells with the cholinergic agonist oxotremorine-M (Oxo-M) induced an approximately 6-fold increase in the tyrosine phosphorylation of ACK-1 which was inhibited by atropine. ACK-1 phosphorylation was blocked by Clostridium difficile toxin B, an inhibitor of Rho family GTPases. In contrast, disruption of the actin cytoskeleton with cytochalasin D stimulated ACK-1 phosphorylation, and moreover, addition of Oxo-M to cells preincubated with this agent elicited a further increase in phosphorylation, indicating that an intact cytoskeleton is not required for mAChR signaling to ACK-1. Although stimulation of M3 mAChRs induces both an increase in intracellular Ca2+ and activation of protein kinase C (PKC), neither of these second messenger pathways was required for receptor-stimulated ACK-1 phosphorylation. Instead, inhibition of PKC resulted in a 2-fold increase in Oxo-M-stimulated ACK-1 phosphorylation, whereas acute activation of PKC with phorbol ester decreased ACK-1 phosphorylation. The agonist-induced tyrosine phosphorylation of ACK-1 was blocked by inhibitors of Src family kinases, and ACK-1 was coprecipitated with Fyn (but not Src) in an agonist-dependent manner. Finally, scrape loading cells with glutathione S-transferase fusion proteins of either the Fyn-SH2 or Fyn-SH3 domain significantly attenuated mAChR-stimulated ACK-1 tyrosine phosphorylation. The data are the first to show phosphorylation of ACK-1 after stimulation of a receptor coupled to neurite outgrowth and indicate that a Rho family GTPase (i.e. Cdc42) and Fyn are essential upstream elements of this signaling pathway.     

Spreading by snail (Lymnaea stagnalis) defence cells is regulated through integrated PKC, FAK and Src signalling

Cell adhesion and spreading are vital to immune function. In molluscs, haemocytes (circulating phagocytes) are sentinels and effectors of the internal defence system; however, molecular mechanisms that regulate integrin-mediated spreading by haemocytes have not been characterised in detail. Visualisation of Lymnaea stagnalis haemocytes by scanning electron microscopy revealed membrane ruffling, formation of lamellipodia and extensive filopodia during early stages of cell adhesion and spreading. These events correlated with increased phosphorylation (activation) of protein kinase C (PKC) and focal adhesion kinase (FAK), sustained for 60 min. Treatment of haemocytes with the PKC inhibitors GF109203X or Gö 6976, or the Src/tyrosine kinase inhibitors SrcI or herbimycin A, attenuated haemocyte spread by 64, 46, 32 and 35%, respectively (P?≤?0.001); PKC or Src inhibition also prevented focal adhesion formation. Western blotting demonstrated that during spreading and adhesion these inhibitors also impaired PKC and FAK activation, with Gö 6976 or SrcI inhibiting FAK phosphorylation by at least 70% (P?≤?0.001), and herbimycin A or SrcI inhibiting PKC phosphorylation by at least 46% (P?≤?0.01). Confocal microscopy revealed phosphorylated PKC colocalised with focal adhesion sites, particularly during early phases of adhesion and spreading. Finally, fibronectin promoted PKC and FAK phosphorylation in suspended haemocytes demonstrating that activation can occur independent of cell adhesion. These novel data are consistent with PKC and FAK/Src playing an integrated role in integrin activation and integrin-mediated spreading by L. stagnalis haemocytes. We propose a model in which integrin engagement mediates association of PKC with FAK/Src complexes to promote focal adhesion assembly during immune recognition by these cells.     

Pleiotropic Effects of Cavin-1 Deficiency on Lipid Metabolism

Mice and humans lacking caveolae due to gene knock-out or inactivating mutations of cavin-1/PTRF have numerous pathologies including markedly aberrant fuel metabolism, lipodystrophy, and muscular dystrophy. We characterized the physiologic/metabolic profile of cavin-1 knock-out mice and determined that they were lean because of reduced white adipose depots. The knock-out mice were resistant to diet-induced obesity and had abnormal lipid metabolism in the major metabolic organs of white and brown fat and liver. Epididymal white fat cells from cavin-1-null mice were small and insensitive to insulin and β-adrenergic agonists resulting in reduced adipocyte lipid storage and impaired lipid tolerance. At the molecular level, the lipolytic defects in white fat were caused by impaired perilipin phosphorylation, and the reduced triglyceride accumulation was caused by decreased fatty acid uptake and incorporation as well as the virtual absence of insulin-stimulated glucose transport. The livers of cavin-1-null mice were mildly steatotic and did not accumulate more lipid after high-fat feeding. The brown adipose tissues of cavin-1-null mice exhibited decreased mitochondria protein expression, which was restored upon high fat feeding. Taken together, these data suggest that dysfunction in fat, muscle, and liver metabolism in cavin-1-null mice causes a pleiotropic phenotype, one apparently identical to that of humans lacking caveolae in all tissues.     

House dust mite, Dermatophagoides pteronissinus increases expression of MCP-1, IL-6, and IL-8 in human monocytic THP-1 cells

The house dust mite (Dermatophagoides pteronissinus) plays an important role in the pathogenesis of allergic diseases, including atopic dermatitis, and asthma. Monocyte chemotactic protein 1 (MCP-1/CCL2)/IL-6/IL-8 (CXCL8) plays a pivotal role in mediating the infiltration of various cells into the skin of atopic dermatitis and psoriasis. The aim of this study was to investigate the effect of D. pteronissinus extract (DpE) on expression of MCP-1/IL-6/IL-8 mRNA and protein and the signal transduction in the human monocytic cell line, THP-1. The mRNA and protein expression of MCP-1/CCL2, IL-6, and IL-8 were elevated by DpE in a time and dose-dependent manner in THP-1 cells. The increased expression of MCP-1, IL-6, and IL-8 was not affected by aprotinin (serine protease inhibitor) or E64 (cysteine protease inhibitor). We found that MCP-1 and IL-6 expression due to DpE was related to Src, protein kinase C δ (PKC δ), extracellular-signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) and IL-8 expression was involved in Src family tyrosine kinase, PKC δ, ERK. DpE increased the phosphorylation of ERK and p38 MAPK after 5 min and peaked at 30 min. The activation was significantly blocked by PP2, an inhibitor of Src family tyrosine kinase and rottlerin, an inhibitor of PKC δ (p < 0.01). DpE increases MCP-1, IL-6, and IL-8 expression and transduces its signal via Src family tyrosine kinase, PKC, and ERK in a protease-independent manner. This finding may contribute to the elucidation of the pathogenic mechanism triggered by DpE .     

The role of the Ca2+-sensitive tyrosine kinase Pyk2 and Src in thrombin signalling in rat astrocytes

We have recently demonstrated that multiple signalling pathways are involved in thrombin-induced proliferation in rat astrocytes. Thrombin acts by protease-activated receptor-1 (PAR-1) via mitogen-activated protein kinase activity. Signalling includes both Gi/(betagamma subunits)-phosphatidylinositol 3-kinase and a Gq-phospholipase C/Ca2+/protein kinase C (PKC) pathway. In the present study, we investigated the possible protein tyrosine kinases which might be involved in thrombin signalling cascades. We found that, in astrocytes, thrombin can evoke phosphorylation of proline-rich tyrosine kinase (Pyk2) via PAR-1. This process is dependent on the increase in intracellular Ca2+ and PKC activity. Moreover, in response to thrombin stimulation Pyk2 formed a complex with Src tyrosine kinase and adapter protein growth factor receptor-bound protein 2 (Grb2), which could be coprecipitated. Furthermore, both thrombin-induced Pyk2 phosphorylation and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation can be attenuated by Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. From these data we conclude that PAR-1 uses Ca2+- and PKC-dependent Pyk2 to activate Src, thereby leading to ERK1/2 activation, which predominantly recruits Grb2 in rat astrocytes.     

Stimulus-specific differences in protein kinase C delta localization and activation mechanisms in cardiomyocytes

Protein kinase C (PKC) isoforms play key roles in the regulation of cardiac contraction, ischemic preconditioning, and hypertrophy/failure. Models of PKC activation generally focus on lipid cofactor-induced PKC translocation to membranes. This study identifies tyrosine phosphorylation as an additional mechanism that regulates PKC delta actions in cardiomyocytes. Using immunoblot analysis with antibodies to total PKC delta and PKC delta-pY(311), we demonstrate that PKC delta partitions between soluble and particulate fractions (with little Tyr(311) phosphorylation) in resting cardiomyocytes. Phorbol 12-myristate 13-acetate (PMA) promotes PKC delta translocation to membranes and phosphorylation at Tyr(311). H(2)O(2) also increases PKC delta-pY(311) in association with its release from membranes. Both PMA- and H(2)O(2)-dependent increases in PKC delta-pY(311) are mediated by Src family kinases, but they occur via different mechanisms. The H(2)O(2)-dependent increase in PKC delta-pY(311) results from Src activation and increased Src-PKC delta complex formation. The PMA-dependent increase in PKC delta-pY(311) results from a lipid cofactor-induced conformational change that renders PKC delta a better substrate for phosphorylation by precomplexed Src kinases (without Src activation). PKC delta-Y(311) phosphorylation does not grossly alter the kinetics of PMA-dependent PKC delta down-regulation. Rather, tyrosine phosphorylation regulates PKC delta kinase activity. PKC delta is recovered from the soluble fraction of H(2)O(2)-treated cardiomyocytes as a tyrosine-phosphorylated, lipid-independent enzyme with altered substrate specificity. In vitro PKC delta phosphorylation by Src also increases lipid-independent kinase activity. The magnitude of this effect varies, depending upon the substrate, suggesting that tyrosine phosphorylation fine-tunes PKC delta substrate specificity. The stimulus-specific modes for PKC delta signaling identified in this study allow for distinct PKC delta-mediated phosphorylation events and responses during growth factor stimulation and oxidant stress in cardiomyocytes.