Increase In Phosphotyrosine Content Research Articles

Discoidin Domain Receptor 2–microRNA 196a–Mediated Negative Feedback against Excess Type I Collagen Expression Is Impaired in Scleroderma Dermal Fibroblasts

Systemic sclerosis (SSc) is characterized by excess collagen deposition in the skin, due to intrinsic transforming growth factor-β (TGF-β) activation. We tried to determine the expression and the role of discoidin domain receptor 2 (DDR2) in SSc. The expression of DDR2 mRNA and protein was significantly decreased in SSc dermal fibroblasts, which was recovered by knocking down TGF-β. The knockdown of DDR2 in normal fibroblasts induced microRNA-196a expression, which led to type I collagen downregulation, indicating that DDR2 itself has a negative effect on microRNA-196a expression and inducible effect on collagen expression. In SSc fibroblasts, however, the DDR2 knockdown did not affect TGF-β signaling and microRNA-196a expression. The microRNA-196a levels were significantly decreased in normal fibroblasts treated with TGF-β and in SSc fibroblasts. Taken together our data indicate that, in SSc fibroblasts, intrinsic TGF-β stimulation induces type I collagen expression, and also downregulates DDR2 expression. This probably acts as a negative feedback mechanism against excess collagen expression, as a decreased DDR2 expression is supposed to stimulate the microRNA-196a expression and further change the collagen expression. However, in SSc fibroblasts the microRNA-196a expression was downregulated by TGF-β signaling. DDR2-microRNA-196a pathway may be a previously unreported negative feedback system, and its impairment may be involved in the pathogenesis of SSc.

Mediators of the JAK/STAT Signaling Pathway in Human Spermatozoa1

In their journey to acquire the ability to fertilize the egg, numerous intracellular signaling systems are activated in spermatozoa, leading to an increase in protein tyrosine phosphorylation. Although the JAK/STAT signaling pathway is usually associated with the activation of transcription of specific genes, our laboratory previously demonstrated the presence of the IL6 receptor (IL6R) and the Janus kinase 1 (JAK1) in human spermatozoa, a cell that is mostly transcriptionally inactive. In order to determine the importance of the JAK/STAT signaling pathway, our objectives were to identify and characterize the mediators of this system in human sperm. Cell fractionation and surface biotinylation assays clearly demonstrated that IL6R is expressed at the sperm membrane surface. The kinase JAK1 is enriched in membrane fractions and is activated during human sperm capacitation as suggested by its increase in phosphotyrosine content. Many signal transducer and activator of transcription (STAT) proteins are expressed in human sperm, including STAT1, STAT3, STAT4, STAT5, and STAT6. Among them, only STAT1 and STAT5 were detected in the cytosolic fraction. All the detected STAT proteins were enriched in the cytoskeletal structures. STAT4 was present in the perinuclear theca, whereas JAK1, STAT1, and STAT5 were detected in the fibrous sheath. Indirect immunofluorescence studies showed that JAK1 and STAT1 colocalized in the neck region and that STAT4 is present at the equatorial segment and flagella. The presence of STAT proteins in sperm structural components suggests that their role is different from their well-known transcription factor activity in somatic cells, but further investigations are required to determine their role in sperm function.

Role of CrkII in Fcγ Receptor-mediated Phagocytosis

Phagocytosis of IgG-opsonized pathogens by Fcgamma receptors requires extensive remodeling of the actin cytoskeleton, a process regulated by the small GTPase Rac. Vav was thought to be the guanine nucleotide exchange factor responsible for the activation of Rac, but recent evidence indicates that Fcgamma receptor-mediated phagocytosis is unaffected in macrophages lacking all three isoforms of Vav. We therefore tested whether another GEF, DOCK180, participates in Fcgamma receptor-initiated phagocytosis. DOCK180 associates with the adaptor protein Crk, which mediates recruitment of the GEF to sites of tyrosine phosphorylation. CrkII and DOCK180 were found to accumulate at the phagocytic cup. Knockdown of Crk or DOCK180 in murine macrophages using small interfering RNA inhibited phagocytosis of IgG-opsonized particles. Moreover, transfection of dominant negative CrkII prevented both recruitment of DOCK180 and the activation of Rac at the phagocytic cup. This is the first report of a role for either Crk or DOCK180 in Fcgamma receptor-mediated phagocytosis. The Crk-DOCK180 complex is involved in the clearance of apoptotic cells, which unlike the ingestion of IgG-opsonized particles, is an anti-inflammatory process. The finding that CrkII-DOCK180 is also responsible, at least in part, for the effects of Fcgamma receptors implies that additional, parallel pathways must account for the associated pro-inflammatory effect.

Cysteine-Rich Domains of Muc3 Intestinal Mucin Promote Cell Migration, Inhibit Apoptosis, and Accelerate Wound Healing

Muc3 intestinal mucin contains an extracellular cysteine-rich domain with 2 epidermal growth factor (EGF)-like motifs. The aim of this study was to determine the functional properties of Muc3 proteins. Glutathione S-transferase-fusion proteins containing both Muc3 EGF-like domains (m3EGF1,2) or truncated versions (m3EGF1 and m3EGF2) were purified from Escherichia coli. Mouse colon (young adult mouse colon) and human A431 and LoVo cells were examined for migration and tyrosine phosphorylation in response to recombinant proteins. LoVo cells were transfected with a human MUC3A transmembrane-EGF1,2 construct and a stable clone was isolated (LhM3c14). Endogenous MUC3A in LoVo was inhibited by specific small interfering RNA transfection. Apoptosis was quantitated by nuclear morphology or terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling assay. Colitis was induced in mice by oral 5% dextran sodium sulfate or rectal 5% acetic acid, followed by enema treatments. m3EGF1,2 stimulated cell migration in all cell lines, but did not induce proliferation. Migration was inhibited by a tyrosine phosphorylation inhibitor, genistein, but not by the EGF receptor inhibitor, tyrphostin (AG1478). Inhibition of endogenous MUC3A in LoVo reduced baseline migration. Tyrosine phosphorylation of ErbB receptors was not observed after treatment of cells with m3EGF1,2. LoVo cells pretreated with m3EGF1,2 and transfected LhM3c14 cells showed reduced apoptosis in response to tumor necrosis factor alpha or Fas-receptor stimulation. Administration of m3EGF1,2 per rectum significantly reduced mucosal ulceration and apoptosis in experimental acute colitis. Truncated proteins m3EGF1 and m3EGF2 had no effect. The Muc3 mucin cysteine-rich domain plays an active role in epithelial restitution, and represents a potential novel therapeutic agent for intestinal wound healing.

Aged mice exhibit distinct B cell precursor phenotypes differing in activation, proliferation and apoptosis

Senescence in murine models is associated with a reduction, albeit heterogeneous, in bone marrow pre-B cells. We have categorized aged BALB/c mice into two phenotypes based on their patterns of pre-B/pro-B cell loss. Each phenotype is characterized by distinct responses to the growth cytokine IL-7 and capacity for survival in vitro. A ‘moderate’ loss of late-stage pre-B cells (25–80%) coincided with decline in proliferation to rmIL-7. This was also associated with a decrease in the frequency of pro-B cells which increased phosphotyrosine content upon IL-7 stimulation, an indicator of early activation events. A ‘severe’ loss of pre-B cells (>80%) resulted in a reduced pro-B cell pool which retained normal activation and proliferative responses to IL-7. B cell precursors from aged mice with severe alterations in B lymphopoiesis displayed increased susceptibility to apoptosis in comparison to both aged mice with moderate B cell precursor loss and young mice. Conceivably, during senescence, aged mice may initially accumulate B cell precursors which are poorly responsive to IL-7. Progressively, these refractory B cell precursors may be eliminated via apoptosis; however, the remaining limited pool of B cell precursors retains the capacity to respond to IL-7 stimulation.

Keratinocyte Growth Inhibition by High-Dose Epidermal Growth Factor Is Mediated by Transforming Growth Factor β Autoinduction: A Negative Feedback Mechanism for Keratinocyte Growth

The epidermal growth factor receptor and its ligands initiate a major signaling pathway that regulates keratinocyte growth in an autocrine manner. It is well known that high doses of epidermal growth factor receptor ligands inhibit keratinocyte growth. Recently, signal transducers and activators of transcription 1-dependent p21Waf1/Cip1 induction were reported to be involved in high-dose epidermal growth factor-dependent cell growth arrest in the A431 squamous cell carcinoma cell line; however, transfection of dominant-negative signal transducers and activators of transcription 1 adenovirus vector did not block epidermal growth factor-induced growth inhibition in normal human keratinocytes. As transforming growth factor beta is a potent inhibitor of keratinocyte proliferation, we hypothesized that transforming growth factor beta contributes to epidermal growth factor-mediated keratinocyte growth inhibition. Epidermal growth factor concentrations of 10 ng per ml enhanced transforming growth factor beta1 mRNA expression from 3 to 6 h poststimulation. Enzyme-linked immunosorbent assay analysis detected 150 pg per ml of transforming growth factor beta1 in the culture medium of keratinocytes incubated with 10 and 100 ng per ml epidermal growth factor, whereas 0.1 and 1.0 ng per ml epidermal growth factor slightly enhance transforming growth factor beta1 production. Epidermal growth factor (100 ng per ml) upregulated luciferase activity of p3TP-lux, which contains three tandem transforming growth factor beta-Smad signaling responsive elements, 6-fold compared with unstimulated cells. The epidermal growth factor-dependent induction of p3TP-lux luciferase activity was disrupted by transfection of the dominant negative form of transforming growth factor beta type I receptor adenovirus vector (AxdnALK5), which suggests that epidermal growth factor-induced transforming growth factor beta acts in an autocrine manner in keratinocytes. Moreover, transfection of AxdnALK5 completely blocked the growth inhibition induced by 100 ng per ml of epidermal growth factor in normal keratinocytes. These data demonstrate that an autocrine transforming growth factor beta1-ALK5 pathway is a negative feedback mechanism for epidermal growth factor-induced normal human keratinocyte growth.

Adhesion of Endothelial Cells to NOV Is Mediated by the Integrins αvβ3 and α5β1

NOV is a member of the CCN family of matricellular proteins. We have shown previously that Nov is strongly expressed by vascular smooth muscle cells (VSMCs) of the rat carotid artery. However, 7 days after injury, Nov expression is down-regulated, except near the luminal surface of the developing intima, where it is strongly expressed. These data suggested that NOV might be involved in the regulation of endothelial cell adhesion. NOV promoted the adhesion of human umbilical vein endothelial cells (HUVECs), which was abolished by anti-NOV antibody. HUVEC adhesion to NOV required divalent cations and was inhibited by GRGDS peptide, implicating integrins in the adhesion mechanism. Monoclonal antibodies (mAbs) against αvβ3 inhibited adhesion of HUVECs to NOV, and NOV was shown to bind to αvβ3. Anti-α5β1 mAbs also inhibited HUVEC adhesion to NOV, but adhesion via α5β1 was mediated by fibronectin. HUVEC adhesion to NOV caused intracellular signalling, as evidenced by increased phosphotyrosine content of focal adhesion kinase. Together with evidence that Nov expression in a variety of tissues is restricted to blood vessels containing VSMCs, these data are consistent with a role for NOV in endothelial cell adhesion in vascular homeostasis and in the response to injury.

Role of protein tyrosine phosphorylation in the thapsigargin-induced intracellular Ca(2+) store depletion during human sperm acrosome reaction.

During human sperm capacitation, an increase in phosphotyrosine content of specific proteins results partially from an increase in the intracellular free Ca(2+) concentrations. In the present study, the inter-regulation between protein phosphotyrosine content and the intracellular Ca(2+) concentration during the thapsigargin treatment of capacitated human sperm was investigated. The involvement of a tyrosine kinase pathway in the thapsigargin-induced acrosome reaction was also investigated. In response to thapsigargin, two sperm subpopulations, called LR (low responsive) and HR (high responsive), according to their increase in intracellular Ca(2+), were observed. In addition to their high increase in intracellular Ca(2+), sperm from the HR population expressed a higher protein phosphotyrosine content, and a higher proportion (P < 0.05) of them underwent the acrosome reaction in response to thapsigargin, as compared with LR sperm. Although the tyrosine kinase inhibitor PP2 abolished the thapsigargin-induced increase in protein phosphotyrosine content, it did not affect the intracellular Ca( 2+) concentration or the percentage of acrosome-reacted sperm. The inability of an src-related tyrosine kinase inhibitor to block the thapsigargin-mediated Ca(2+) increase and acrosomal exocytosis suggests that, during the acrosome reaction, the signalling pathway mediated by src-related tyrosine kinases is involved upstream of the capacitative Ca(2+) entry.

Regulation of the phosphotyrosine content of human sperm proteins by intracellular Ca2+: role of Ca2+-adenosine triphosphatases.

An increase in the concentration of intracellular free Ca2+ and in the phosphotyrosine content of specific proteins characterizes human sperm capacitation. Whether tyrosine phosphorylation regulates the intracellular free Ca2+ concentration through modulation of Ca2+-ATPase activity or the phosphotyrosine content is under Ca2+ regulation was investigated using Ca2+-ATPase modulators and tyrosine kinase inhibitors. The presence of the Ca2+-ATPase-inhibitor thapsigargin during human sperm capacitation caused an increase in the cytoplasmic free Ca2+ concentration and was associated with an increase in the phosphotyrosine content of specific sperm proteins. Conversely, a decrease in protein tyrosine phosphorylation was observed when gingerol, a Ca2+-ATPase activator, was present during the incubation period. On the other hand, thapsigargin had no effect on the phosphotyrosine content or the cytoplasmic Ca2+ concentration when spermatozoa were incubated in the presence of the phosphodiesterase-inhibitor 3-isobutyl-1-methylxanthine (IBMX). However, the effect of IBMX on phosphotyrosine-containing proteins appears to be a Ca2+-dependent phenomenon, because it was partly inhibited in spermatozoa pretreated with 1,2-bis-(o-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA-AM) even though, by itself, BAPTA-AM caused an increase in sperm protein phosphotyrosine content. Tyrosine kinase inhibitors prevented the increase in the phosphotyrosine content without affecting the cytoplasmic free Ca2+ concentration. Based on these findings, the present study suggests that Ca2+-ATPases are involved in the filling of internal Ca2+ stores, such as the acrosome, and are inhibited later during capacitation. Their inhibition allows an increase in cytoplasmic free Ca2+, which is involved in the subsequent increase in the phosphotyrosine content of specific sperm proteins.

Insulin Stimulates Tyrosine Phosphorylation and Inactivation of Protein-tyrosine Phosphatase 1B in Vivo

Protein-tyrosine phosphatase (PTP) 1B has been implicated in negative regulation of insulin action, although little is known of the ability of insulin to regulate PTP1B itself. The ability of insulin to regulate phosphorylation and activation of PTP1B was probed in vivo. Challenge with insulin in vivo provoked a transient, sharp increase in the phosphotyrosine content of PTP1B in fat and skeletal muscle that peaked within 15 min. Insulin stimulated a decline of 60--70% in PTP1B activity. In mouse adipocytes, the inhibition of PTP1B activity and increased tyrosine phosphorylation of the enzyme were blocked by the insulin receptor tyrosine kinase inhibitor AG1024. Phosphoserine content of PTP1B declined in response to insulin stimulation. Elevation of intracellular cyclic AMP provokes a sharp increase in PTP1B activity and leads to increased phosphorylation of serine residues and decreased tyrosine phosphorylation. Suppression of cyclic AMP levels or inhibition of protein kinase A leads to a sharp decline in PTP1B activity, a decrease in phosphoserine content, and an increase in PTP1B phosphotyrosine content. PTP1B appears to be a critical point for insulin and catecholamine counter-regulation.

Protein-tyrosine Phosphatase D1, a Potential Regulator and Effector for Tec Family Kinases

Etk, also named Bmx, is a member of the Tec tyrosine kinase family, which is characterized by a multimodular structure including a pleckstrin homology (PH) domain, an SH3 domain, an SH2 domain, and a catalytic domain. The signaling mechanisms regulating Etk kinase activity remain largely unknown. To identify factor(s) regulating Etk activity, we used the PH domain and a linker region of Etk as a bait for a yeast two-hybrid screen. Three independent clones encoding protein-tyrosine phosphatase D1 (PTPD1) fragments were isolated. The binding of PTPD1 to Etk is specific since PTPD1 cannot associate with either the Akt PH domain or lamin. In vitro and in vivo binding studies demonstrated that PTPD1 can interact with Etk and that residues 726-848 of PTPD1 are essential for this interaction. Deletion analysis of Etk indicated that the PH domain is essential for PTPD1 interaction. Furthermore, the Etk-PTPD1 interaction stimulated the kinase activity of Etk, resulting in an increased phosphotyrosine content in both factors. The Etk-PTPD1 interaction also increased Stat3 activation. The effect of PTPD1 on Etk activation is specific since PTPD1 cannot potentiate Jak2 activity upon Stat3 activation. In addition, Tec (but not Btk) kinase can also be activated by PTPD1. Taken together, these findings indicate that PTPD1 can selectively associate with and stimulate Tec family kinases and modulate Stat3 activation.

ROCK mediates thrombin's endothelial barrier dysfunction.

Thrombin-induced endothelial monolayer hyperpermeability is thought to result from increased F-actin stress fiber-related contractile tension, a process regulated by the small GTP-binding protein Rho. We tested whether this process was dependent on the Rho-associated protein kinase, ROCK, using a specific ROCK inhibitor, Y-27632. The effects of Y-27632 on thrombin-induced myosin light chain phosphorylation (MLCP) and tyrosine phosphorylation of p125 focal adhesion kinase (p125(FAK)) and paxillin were measured by Western blotting. F-actin organization and content were analyzed by digital imaging, and endothelial monolayer permeability was measured in bovine pulmonary artery endothelial cell (EC) monolayers using a size-selective permeability assay. Y-27632 enhanced EC monolayer barrier function due to a decline in small-pore number that was associated with increased EC surface area, reduced F-actin content, and reorganization of F-actin to beta-catenin-containing cell-cell adherens junctions. Although Y-27632 prevented thrombin-induced MLCP, stress fiber formation, and the increased phosphotyrosine content of paxillin and p125(FAK), it attenuated but did not prevent the thrombin-induced formation of large paracellular holes. These data indicate that thrombin-induced stress fiber formation is ROCK dependent. In contrast, thrombin-induced paracellular hole formation occurs in a ROCK-independent manner, whereas thrombin-induced monolayer hyperpermeability appears to be partially ROCK dependent.

Platelet-derived Growth Factor-stimulated Migration of Murine Fibroblasts Is Associated with Epidermal Growth Factor Receptor Expression and Tyrosine Phosphorylation

Previous studies have shown that epidermal growth factor (EGF) synergizes with various extracellular matrix components in promoting the migration of B82L fibroblasts expressing wild-type EGF receptors and that functional EGF receptors are critical for the conversion of B82L fibroblasts to a migratory cell type (). In the present study, we examined the effects of platelet-derived growth factor (PDGF) on the motility of B82L fibroblasts using a microchemotaxis chamber. We found that PDGF can enhance fibronectin-induced migration of B82L fibroblasts expressing wild-type EGF receptors (B82L-clone B3). However, B82L cells that lack the EGF receptor (B82L-parental) or that express an EGF receptor that is kinase-inactive (B82L-K721M) or C-terminally truncated (B82L-c'973) exhibit little PDGF-stimulated migration. In addition, none of these three cell lines exhibit the capacity to migrate to fibronectin alone. These observations indicate that, similar to cell migration toward fibronectin, PDGF-induced cell migration of B82L fibroblasts is augmented by the expression of an intact EGF receptor kinase. The loss of PDGF-stimulated motility in B82L cells that do not express an intact EGF receptor does not appear to result from a gross dysfunction of PDGF receptors, because ligand-stimulated tyrosine phosphorylation of the PDGF-beta receptor and the activation of mitogen-activated protein kinases are readily detectable in these cells. Moreover, an interaction between EGF and PDGF receptor systems is supported by the observation that the EGF receptor exhibits an increase in phosphotyrosine content in a time-dependent fashion upon the addition of PDGF. Altogether, these studies demonstrate that the expression of EGF receptor is critical for PDGF-stimulated migration of murine B82L fibroblasts and suggest a role for the EGF receptor downstream of PDGF receptor activation in the signaling events that lead to PDGF-stimulated cell motility.

Inhibitory effects of trapidil on PDGF signaling in balloon-injured rat carotid artery

Trapidil, which was originally developed as an anti-platelet agent, is among the few agents thus far proven to be clinically effective in preventing restenosis after percutaneous coronary interventions. Trapidil was previously shown to inhibit platelet-derived growth factor (PDGF)-induced cellular responses in vitro in cultured cells. However, its mechanism of action is poorly understood. In this study, we investigated by using a rat carotid balloon-injury model whether and how trapidil inhibited the in vivo action of PDGF, which is regarded as a most important growth factor implicated in proliferation and migration of vascular smooth muscle cells. The combination of both oral and topical administration of trapidil reduced the intimal lesion size by more than 70 % and nearly completely suppressed injury-induced increases in phosphotyrosine content of PDGF α- and β- receptors of carotid artery. Moreover, trapidil was found to decrease mRNA levels of PDGF α- and β- receptors strongly and of PDGF A- and B- chains moderately in injured arteries. These results indicate that trapidil potently suppresses the action of PDGF with inhibition of neointima formation in injured artery, which is mediated at least in part through decreasing the expression of both PDGF ligands and their receptors.

RhoA inactivation enhances endothelial barrier function.

The modulation of endothelial barrier function is thought to be a function of contractile tension mediated by the cell cytoskeleton, which consists of actomyosin stress fibers (SF) linked to focal adhesions (FA). We tested this hypothesis by dissociating SF/FA with Clostridium botulinum exoenzyme C3 transferase (C3), an inhibitor of the small GTP-binding protein RhoA. Bovine pulmonary artery endothelial cell (EC) monolayers given C3, C3 + thrombin, thrombin, or no treatment were examined using a size-selective permeability assay and quantitative digital imaging measurements of SF/FA. C3 treatment disassembled SF/FA, stimulated diffuse myosin II immunostaining, and reduced the phosphotyrosine (PY) content of paxillin and 130- to 140-kDa proteins that included p125(FAK). C3-treated monolayers displayed a 60-85% decline in F-actin content and a 170-300% increase in EC surface area with enhanced endothelial barrier function. This activity correlated with reorganization of F-actin and PY protein(s) to beta-catenin-containing cell-cell junctions. Because C3 prevented the thrombin-induced formation of myosin ribbons, SF/FA, and the increased PY content of proteins, these characteristics were Rho dependent. Our data show that C3 inhibition of Rho proteins leads to cAMP-like characteristics of reduced SF/FA and enhanced endothelial barrier function.

Tyrosine phosphorylation in peripheral T cells of kidney transplant recipients: analyses of baseline levels and response to T cell receptor stimulation.

Impaired immunosurveillance in recipients of organ transplants has been attributed to alleviation of T cell functions. We analyzed the phosphorylation of tyrosine residues in T cells of peripheral blood, and after T cell receptor (TCR) stimulation. The TCR was stimulated by OKT3 monoclonal antibody (mAb) in non-separated heparinized blood specimens of patients (n=64) and healthy controls (n=25). After fixation and red cell lysis, lymphocytes were permeabilized by saponin. Subsequently, intracellular phosphotyrosine residues and surface CD3 antigen were stained simultaneously with specific mAbs. We analyzed transplant recipients and healthy donors for baseline levels of total cellular tyrosine phosphorylation and for increase in phosphotyrosine content following stimulation by OKT3. Phosphotyrosine levels were significantly lower in non-stimulated T cells of kidney transplant recipients compared to controls (p=0.004). There was a marked variability in the levels of tyrosine phosphorylation among transplanted patients (p=0.02). T cell receptor stimulation by OKT3 mAb in vitro led to a strong increase of tyrosine phosphorylation in all specimens of patients and healthy controls. In conclusion, we demonstrated decreased phosphotyrosine levels in T cells of kidney transplant recipients compared to healthy donors. However, increase in tyrosine phosphorylation was not impaired in all patients as a result of TCR stimulation.

Activation of Nuclear Factor of Activated T Cells-(NFAT) and Activating Protein 1 (AP-1) by Oncogenic 70Z Cbl Requires an Intact Phosphotyrosine Binding Domain but Not Crk(L) or p85 Phosphatidylinositol 3-Kinase Association

The Cbl proto-oncogene product is a complex adapter protein that functions as a negative regulator of protein tyrosine kinases. It is rapidly tyrosine-phosphorylated and associates with Crk(L) and p85 phosphatidylinositol 3-kinase (PI3K) upon engagement of numerous receptors linked to tyrosine kinases. Elucidation of the mechanism(s) underlying Cbl deregulation is therefore of considerable interest. The 70Z Cbl oncoprotein shows increased baseline tyrosine phosphorylation in fibroblasts and enhances nuclear factor of activated T cells (NFAT) activity in Jurkat T cells. Its transforming ability has been proposed to relate to its increased phosphotyrosine content. We demonstrate that 70Z Cbl shows increased basal and activation-induced tyrosine phosphorylation and association with Crk(L) and p85 PI3K in Jurkat T cells. 70Z Cbl, however, retains the ability to enhance NFAT and activating protein 1 (AP1) activity in the absence of Crk(L)/p85 PI3K association. In contrast, the G306E mutation, which inactivates the phosphotyrosine binding domain of Cbl, blocks NFAT/AP1 activation by 70Z Cbl. We conclude that 70Z Cbl-induced NFAT/AP1 activation requires the phosphotyrosine binding domain but not Crk(L)/p85 PI3K association. We hypothesize that 70Z Cbl acts as a dominant negative by blocking the negative regulatory function of the Cbl phosphotyrosine binding domain on protein-tyrosine kinases.

Tyrosine phosphorylation of paxillin/pp125FAK and microvascular endothelial barrier function.

The transendothelial movement of solutes is a dynamic process controlled by a complex interaction between the cytoskeleton and adhesion proteins. The aim of this study was to examine whether protein tyrosine phosphorylation is involved in the regulation of endothelial barrier function. The apparent permeability coefficient of albumin (Pa) was measured in isolated and perfused coronary venules. Tyrosine phosphatase inhibitors, including phenylarsine oxide and sodium orthovanadate, dose and time dependently increased basal Pa. Western blot analysis of cultured coronary venular endothelial cells revealed that inhibition of tyrosine phosphatase induced an increase in phosphotyrosine content in a number of proteins, including bands at 65-70 and 120-130 kDa, which were identified as paxillin and focal adhesion kinase (pp125FAK), respectively. The time course and dose responsiveness of protein tyrosine phosphorylation were tightly correlated with those of increases in Pa. Furthermore, stimulation of endothelial cells with histamine or phorbol myristate acetate (PMA) enhanced tyrosine phosphorylation of paxillin and pp125FAK, which was blocked by the tyrosine kinase inhibitor damnacanthal. Correspondingly, the increases in venular permeability elicited by histamine and PMA were abolished in damnacanthal-treated venules. Taken together, the data suggest a possible involvement of protein tyrosine phosphorylation in the control of endothelial barrier function. Paxillin and its associated focal adhesion proteins may play a specific role in agonist-induced hyperpermeability responses in the endothelium of exchange vessels.

Intracellular acidosis activates c-Src.

The purpose of the present studies was to determine whether acidosis activates protein tyrosine kinase pathways. Incubation of MCT cells, a renal proximal tubule cell line, in acid media caused increased phosphotyrosine content of 60- to 70- and 120-kDa cytosolic proteins. Media acidification induced a twofold increase in c-Src activity that occurred within 30 s. Significant activation occurred with media pH changes as small as 0.07 pH unit accompanied by cell acidification of 0.06 pH unit. Sodium propionate addition, NH4Cl prepulse, and nigericin addition, maneuvers that decrease intracellular pH in the absence of changes in extracellular pH, activated c-Src. Significant activation by sodium propionate was seen with cell pH changes as small as 0.07 pH unit. Sodium orthovanadate, a protein tyrosine phosphatase inhibitor, prevented c-Src activation by media acidification but did not prevent protein tyrosine phosphorylation. In summary, decreased intracellular pH activates c-Src. Acid activation of c-Src represents a novel mechanism of c-Src activation that may be relevant to many cellular responses to acidosis.

5-Hydroxytryptamine induces TIS8/egr-1 and c-fos expression in PC12 cells. Involvement of tyrosine protein phosphorylation.

The TIS8/egr-1 gene is a member of the class of immediate early genes. Originally discovered as a mitogen-induced gene, it can also be induced by synaptic activity. We report here the induction of the TIS8/egr-1 gene by the neurotransmitter 5-hydroxytryptamine (5-HT) in cultured rat phaeochromocytoma PC12 cells. Induction was maximal 40 min after addition of 5-HT to the cells, and declined very rapidly to reach the basal level after 90 min. The electrophoretic mobility-shift assay showed that induction of the TIS8/egr-1 gene by 5-HT was accompained by increased Egr-1 protein binding to its DNA consensus sequence. We found an overall correlation of 5-HT-induced egr-1 expression with that of c-fos expression. The kinetics of the ability of both gene products to bind to their respective DNA consensus sequence was also similar. The 5-HT-induced activation in Egr-1 binding was inhibited by ketanserin and mesulergine, indicating that 5-HT exerted its action via a 5-HT2 receptor subtype. The tyrosine protein kinase inhibitor genistein abolished induction of the TIS8/egr-1 gene, suggesting that tyrosine kinase activity is required for the induction of early genes by 5-HT. Genistein also inhibited 5-HT-induced Egr-1 binding activity. The increase in phosphotyrosine content of focal adhesion kinase we noticed upon addition of 5-HT to cells suggests that this cytoplasmic tyrosine protein kinase mediates the effect of 5-HT in eliciting early gene induction.