Vascular endothelial growth factor activates STAT proteins in aortic endothelial cells.

Vascular endothelial growth factor (VEGF) intracellular signaling in endothelial cells is initiated by the activation of distinct tyrosine kinase receptors, VEGFR1 (Flt-1) and VEGFR2 (Flk-1/KDR). Because the tyrosine kinase-dependent transcription factors known as STAT (signal transducers and activators of transcription) proteins are important modulators of cell growth responses induced by other growth factor receptors, we have determined the effects VEGF of on STAT activation in BAEC (bovine aortic endothelial cells). Here, we show that VEGF induces tyrosine phosphorylation and nuclear translocation of STAT1 and STAT6. VEGF also stimulates STAT3 tyrosine phosphorylation, but nuclear translocation does not occur. We found that placenta growth factor, which selectively activates VEGFR1, has no effect on the STATs. However, upon VEGF stimulation, STAT1 associates with the VEGFR2 in a tyrosine kinase-dependent manner, indicating that VEGF-induced STAT1 activation is mediated primarily by VEGFR2. Thus, our study shows for the first time that VEGF activates the STAT pathway through VEGFR2. Because the growth-promoting activity of VEGF depends upon VEGFR2 activation, these findings suggest a role for the STATs in the regulation of gene expression associated with the angiogenic effects of VEGF.

Interleukin-4 mediates STAT6 activation in 3T3-L1 preadipocytes but not adipocytes.

STAT6 is abundantly expressed in 3T3-L1 preadipocytes and adipocytes but activating ligands are not well defined. In this report, we provide evidence that interleukin 4 (IL-4) induced JAK2-mediated STAT6 tyrosine phosphorylation and DNA binding in 3T3-L1 preadipocytes but not in 3T3-L1 adipocytes. Loss of IL-4-mediated STAT6 tyrosine phosphorylation occurred 2 days after preadipocytes were induced to differentiate into adipocytes but when cells remained phenotypically preadipocytes. 3T3-L1 adipocytes were still responsive to IL-4 through tyrosine phosphorylation of other cellular proteins. We conclude that IL-4 signals through STAT6 in 3T3-L1 preadipocytes but not in 3T3-L1 adipocytes. This differentiation-dependent loss of STAT6 activation may be critical for distinct biological effects of IL-4 in 3T3-L1 preadipocytes and adipocytes.

Hyperglycemia enhances angiotensin II-induced janus-activated kinase/STAT signaling in vascular smooth muscle cells.

We have shown previously that angiotensin II (Ang II) activates the janus-activated kinase (JAK)/signal transducers and activators of transcription (STAT) pathway in vascular smooth muscle cells (VSMCs) and that activation of the JAK/STAT pathway is required for Ang II induction of VSMC proliferation. In the present study, we examined the effects of hyperglycemia (HG) on Ang II-induced JAK/STAT signaling events in cultured VSMCs. HG increases Ang II-induced JAK2 tyrosine phosphorylation and promotes a partial tyrosine phosphorylation of the enzyme under basal conditions. In addition, HG increases both basal and Ang II-induced complex formation of JAK2 with the Ang II AT(1) receptor. The extent of STAT1 and STAT3 tyrosine and serine phosphorylation are also increased under HG conditions. Furthermore, the tyrosine phosphorylation and activities of the SHP-1 and SHP-2 tyrosine phosphatases, enzymes that regulate Ang II-induced JAK2 tyrosine phosphorylation, are altered by HG. SHP-1, which is responsible for JAK2 tyrosine dephosphorylation in VSMC, is completely deactivated in HG, resulting in a prolonged duration of JAK2 phosphorylation under HG conditions. HG also enhances Ang II induction of VSMC proliferation. Taken together, these data suggest that HG augments Ang II induction of VSMC proliferation by increasing signal transduction through the JAK/STAT pathway.

Endothelial nitric oxide synthase interactions with G-protein-coupled receptors.

The endothelial nitric oxide synthase (eNOS) is activated in response to stimulation of endothelial cells by a number of vasoactive substances including, bradykinin (BK), angiotensin II (Ang II), endothelin-1 (ET-1) and ATP. In the present study we have used in vitro activity assays of purified eNOS and in vitro binding assays with glutathione S-transferase fusion proteins to show that the capacity to bind and inhibit eNOS is a common feature of membrane-proximal regions of intracellular domain 4 of the BK B2, the Ang II AT1 and the ET-1 ETB receptors, but not of the ATP P2Y2 receptor. Phosphorylation of serine or tyrosine residues in the eNOS-interacting region of the B2 receptor results in a loss of eNOS inhibition due to a decrease in the binding affinity of the receptor domain for the eNOS enzyme. Furthermore, the B2 receptor is transiently phosphorylated on tyrosine residues in cultured endothelial cells in response to BK stimulation. Phosphorylation occurs during the time in which eNOS transiently dissociates from the receptor accompanied by a transient increase in nitric oxide production. Taken together, these data support the hypotheses that eNOS is regulated in endothelial cells by reversible and inhibitory interactions with G-protein-coupled receptors and that these interactions can be modulated by receptor phosphorylation.

Vascular endothelial growth factor signals endothelial cell production of nitric oxide and prostacyclin through flk-1/KDR activation of c-Src.

Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific mitogen that promotes angiogenesis, vascular hyperpermeability, and vasodilation by autocrine mechanisms involving nitric oxide (NO) and prostacyclin (PGI(2)) production. These experiments used immunoprecipitation and immunoassay procedures to characterize the signaling pathways by which VEGF induces NO and PGI(2) formation in cultured endothelial cells. The data showed that VEGF stimulates complex formation of the flk-1/kinase-insert domain-containing receptor (KDR) VEGF receptor with c-Src and that Src activation is required for VEGF induction of phospholipase C gamma1 activation and inositol 1,4,5-trisphosphate formation. Reporter cell assays showed that VEGF promotes a approximately 50-fold increase in NO formation, which peaks at 5-20 min. This effect is mediated by a signaling cascade initiated by flk-1/KDR activation of c-Src, leading to phospholipase C gamma1 activation, inositol 1,4,5-trisphosphate formation, release of [Ca(2+)](i) and nitric oxide synthase activation. Immunoassays of VEGF-induced 6-keto prostaglandin F(1alpha) formation as an indicator of PGI(2) production revealed a 3-4-fold increase that peaked at 45-60 min. The PGI(2) signaling pathway follows the NO pathway through release of [Ca(2+)](i), but diverges prior to NOS activation and also requires activation of mitogen-activated protein kinase. These results suggest that NO and PGI(2) function in parallel in mediating the effects of VEGF.

Regulation of angiotensin II-induced phosphorylation of STAT3 in vascular smooth muscle cells.

Ligand binding to the angiotensin II (Ang II) AT1 receptor on vascular smooth muscle cells (VSMCs) activates the Janus-activated kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. We have shown previously that the JAK2 tyrosine kinase and the Src family p59 Fyn tyrosine kinase are required for Ang II-induced STAT1 tyrosine phosphorylation in VSMCs. The mitogen-activated protein kinase phosphatase, MKP-1, is required for STAT1 tyrosine dephosphorylation. In the present study, using specific enzyme inhibitors and antisense oligonucleotides, we show that Ang II-induced tyrosine phosphorylation and nuclear translocation of STAT3 in VSMCs is mediated by p60 c-Src, whereas tyrosine dephosphorylation is mediated by calcineurin. Calcineurin is activated in response to Ang II stimulation of VSMCs and is translocated to the nucleus. In addition, we show that Ang II-induced serine phosphorylation of STAT3 in VSMCs is mediated by mitogen-activated protein kinase and that dephosphorylation is mediated by protein phosphatase 2A (PP2A). PP2A translocates to the nucleus in response to Ang II stimulation of VSMCs and forms a complex with STAT3 in an Ang II-dependent manner.

Immunolocalization of phospholipase C isoforms in rat kidney.

BACKGROUND: Phospholipase C (PLC) is an important factor in signal transduction because this enzyme is activated by several hormones and growth factors. Eight PLC isoforms have been described raising the possibility that different cells express a single isoform or activate specific isoforms in different cells. Therefore, the goal of this study was to determine which PLC isoforms are expressed in specific regions of rat kidney. METHODS: Western blot analysis was performed in microdissected nephron segments of rat kidney, while immunohistochemical analysis was performed on whole rat kidney slices using PLC isoform-specific antibodies. RESULTS: All three families of PLC isoforms (beta, gamma, and delta) were present throughout the cortical and medullary regions of the kidney. Only the PLC-beta1 isoform was observed in the brush border of the proximal tubule, but all isoforms were present in glomeruli and in the cytoplasm of tubular epithelial cells. In addition, only the PLC-gamma1 isoform was expressed in the internal elastic lamina of the renal artery, while vasa recta expressed PLC-beta1 most intensely. Medullary thick ascending limbs showed an intense level of expression of all three isoforms. CONCLUSION: Multiple PLC isoforms are present in glomeruli, renal tubules, and renal vasculature in vivo, but with some segment-specific differences. These findings suggest that the response of a specific cell is not determined by expression of only one PLC isoform, with the exception of the brush border of the proximal tubule and the renal arteries. Instead, the presence of multiple PLC isoforms in specific regions of the kidney suggests that hormonal regulation in vivo involves mechanisms beyond cell-specific isoforms of PLC.

Regulation of angiotensin II-induced JAK2 tyrosine phosphorylation: roles of SHP-1 and SHP-2.

Angiotensin II (ANG II) exerts its effects on vascular smooth muscle cells through G protein-coupled AT1 receptors. ANG II stimulation activates the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway by inducing tyrosine phosphorylation, activation, and association of JAK2 with the receptor. Association appears to be required for JAK2 phosphorylation. In the present study, electroporation experiments with neutralizing anti-Src homology phosphatase-1 (SHP-1) and anti-SHP-2 antibodies and time course determinations of SHP-1 and SHP-2 activation and complexation with JAK2 suggest that the tyrosine phosphatases, SHP-1 and SHP-2, have opposite roles in ANG II-induced JAK2 phosphorylation. SHP-1 appears responsible for JAK2 dephosphorylation and termination of the ANG II-induced JAK/STAT cascade. SHP-2 appears to have an essential role in JAK2 phosphorylation and initiation of the ANG II-induced JAK/STAT cascade leading to cell proliferation. The motif in the AT1 receptor that is required for association with JAK2 is also required for association with SHP-2. Furthermore, SHP-2 is required for JAK2-receptor association. SHP-2 may thus play a role as an adaptor protein for JAK2 association with the receptor, thereby facilitating JAK2 phosphorylation and activation.

Angiotensin II-induced tyrosine phosphorylation of signal transducers and activators of transcription 1 is regulated by Janus-activated kinase 2 and Fyn kinases and mitogen-activated protein kinase phosphatase 1.

Angiotensin II (Ang II) AT1 receptors on vascular smooth muscle cells (VSMCs) are coupled to the Janus-activated kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. We have shown previously that Ang II stimulation of VSMCs results in the tyrosine phosphorylation of JAK2 and STAT1 and the translocation of STAT1 to the nucleus. In the present study, we demonstrate using specific enzyme inhibitors and antisense oligonucleotides that both JAK2 and p59 Fyn tyrosine kinases are required for the Ang II-induced tyrosine phosphorylation and nuclear translocation of STAT1 in VSMCs. Neither tyrosine kinase, however, appears to function upstream from the other in a phosphorylation cascade. Rather, p59 Fyn functions as an Ang II-activated docking protein for both JAK2 and STAT1, a docking interaction that may facilitate JAK2-mediated STAT1 tyrosine phosphorylation. In this study, we have also identified the nuclear dual-specificity phosphatase mitogen-activated protein kinase phosphatase 1 as the enzyme responsible for STAT1 tyrosine dephosphorylation in VSMCs.

Inhibition by the JAK/STAT pathway of IFNgamma- and LPS-stimulated nitric oxide synthase induction in vascular smooth muscle cells.

Inducible nitric oxide synthase (iNOS) is induced in many cell types by cytokines and lipopolysaccharide (LPS). Cytokine signal transduction is believed to be mediated primarily through the JAK/STAT pathway. We therefore examined the effects of a JAK2-specific inhibitor, an antisense oligonucleotide to JAK2, and electroporation of neutralizing anti-STAT1 and anti-STAT3 antibodies on IFNgamma- and LPS-stimulated induction of iNOS in vascular smooth muscle cells. Unexpectedly, we found that the JAK/STAT pathway suppresses IFNgamma- and LPS-stimulated iNOS induction in these cells. In contrast, the JAK/STAT pathway appears to have a positive role in iNOS induction in RAW 264.7 macrophages.

Inhibitory interactions of the bradykinin B2 receptor with endothelial nitric-oxide synthase.

It has been shown previously that the endothelial nitric-oxide synthase (eNOS) interacts reversibly with the plasmalemmal caveolae structural protein, caveolin-1. The eNOS-caveolin-1 interaction inhibits eNOS catalytic activity. In the present study, we show that eNOS also participates in reversible inhibitory interactions with the G protein-coupled bradykinin B2 receptor. eNOS and the B2 receptor are coimmunoprecipitated from endothelial cell lysates by antibodies directed against either of the two proteins. A glutathione S-transferase fusion protein containing intracellular domain 4 of the receptor is bound by purified recombinant eNOS in in vitro binding assays. The fusion protein selectively inhibits the activity of purified eNOS. A synthetic peptide corresponding to membrane-proximal residues 310-334 in intracellular domain 4 also potently inhibits eNOS activity (IC50 < 1 microM). Treatment of cultured endothelial cells with bradykinin or Ca2+ ionophore promotes a rapid dissociation of the eNOS.B2 receptor complex. These data demonstrate that the bradykinin B2 receptor physically associates with eNOS in a ligand- and Ca2+-dependent manner. Reversible and inhibitory membrane-docking interactions of eNOS, therefore, are not restricted to those with caveolin-1 but also occur with the bradykinin B2 receptor.

Phosphorylation of p130Cas by angiotensin II is dependent on c-Src, intracellular Ca2+, and protein kinase C.

p130Cas is a signaling molecule that was initially found to be tyrosine-phosphorylated in v-Crk and v-Src transformed cells. We characterized the regulation of p130Cas tyrosine phosphorylation in vascular smooth muscle cells by angiotensin II (Ang II). This ligand induced a transient increase in p130Cas tyrosine phosphorylation, which was sensitive to the actin polymerization inhibitor cytochalasin D and to the intracellular Ca2+ chelator BAPTA-AM but not the Ca2+ channel blocker verapamil. The Ang II-induced tyrosine phosphorylation of p130Cas was also dependent on an active Src family tyrosine kinase, since it could be blocked by the Src kinase inhibitors geldanamycin and PP1. Ang II treatment resulted in the ability of p130Cas to bind at least 11 different phosphate-containing proteins. Analysis of these proteins revealed that protein kinase Calpha and the cell adhesion signaling molecule pp120 formed temporal associations with p130Cas in response to Ang II. c-Src was found to associate with p130Cas in a manner that was independent of Ang II treatment. Inhibition of protein kinase C by either calphostin C or phorbol 12-myristate 13-acetate downregulation inhibited the Ang II-induced tyrosine phosphorylation of p130Cas. These results are the first to demonstrate that the tyrosine phosphorylation of p130Cas by Ang II is transduced by the Src, intracellular Ca2+, protein kinase C signaling pathway.

Characterization of fatty acid synthase in cell lines derived from experimental mammary tumors.

The lipogenic enzyme fatty acid synthase (FAS) is elevated in various human primary cancers and certain human cancer cell lines. FAS overexpression in human neoplasia has clinical relevance because of its association with tumor aggression and potential chemotherapeutic intervention. Here, we surveyed FAS in cell lines established from normal murine mammary epithelium (NMuMG) and from mammary tumors induced by either rodent polyoma (Py) virus or murine mammary tumor virus (MMTV). Western blotting revealed greater content of FAS in Py-transformed A1-1 and T1 than NMuMG or MMTV-transformed Mm5MT, RIIIMT and MMT060562. These data suggest that signaling events mediated by Py transformation may increase cellular amounts of FAS. Although FAS content was elevated to similar levels in A1-1 and T1, specific activities were significantly different as enzyme activity in T1 was 3-fold higher than A1-1. Likewise, FAS activity in NMuMG was about 0.5-fold higher than the MMTV-transformed lines, even though enzyme content was similar. Immunoprecipitation studies employing anti-phosphoamino acid antibodies followed by immunoblot analysis with anti-FAS antisera (and vice versa) were used to characterize the constitutive phosphorylation state of the enzyme. Phosphoserine and phosphothreonine residues were detected in the more active FAS from T1 and NMuMG, but not in the less active FAS from Mm5MT or A1-1. Discovery of phosphorylated FAS suggests that the enzyme may have more immediate control over lipogenesis than previously thought. High-dose (10-4 M) dexamethasone induced FAS content and activity in NMuMG and MMTV-transformed lines but not Py-transformed cells. Lower concentrations (10-8, 10-6 M) of dexamethasone also activated FAS but without concomitant elevation of its protein content, which was consistent with a phosphorylated form of FAS. Finally, cell lines were treated with the FAS inhibitor cerulenin: almost all breast cancer lines were growth inhibited at significantly lower amounts of drug than normal cell lineages, suggesting that FAS plays a greater role in viability of tumor cells than normal cells. Pretreatment with palmitate (a primary end-product of FAS) prior to cerulenin rescued A1-1 cells only slightly from growth inhibition, whereas pretreatment with oleate (a monounsaturated fatty acid synthesized from palmitate) synergized cerulenin's cytotoxic effects.

Bradykinin stimulates the tyrosine phosphorylation and bradykinin B2 receptor association of phospholipase C gamma 1 in vascular endothelial cells.

Bradykinin (BK) B2 receptor signaling involves activation of phospholipase C (PLC). PLC activation by other receptors consists of either allosteric activation of PLC beta isoforms by G-proteins or tyrosine phosphorylation of PLC gamma isoforms. Because the B2 receptor is a G-protein-coupled receptor, it has been assumed that the receptor signals through PLC beta. In the present study, however, we have found that BK stimulation of IP3 production and the Ca2+ signal in endothelial cells is dependent on tyrosine phosphorylation. Furthermore, stimulation of B2 receptors in these cells is accompanied by a transient tyrosine phosphorylation of PLC gamma 1. Phosphorylation is correlated with increased IP3 production and association of PLC gamma 1 with the C-terminal intracellular domain of the B2 receptor. The B2 receptor can thus physically associate with intracellular proteins other than G-proteins. Activation of PLC gamma isoforms, rather than PLC beta isoforms, may, therefore, be primarily responsible for BK-stimulated IP3 generation in endothelial cells.

Erythropoietin receptor-operated Ca2+ channels: activation by phospholipase C-gamma 1.

Erythropoietin (EPO) increases Ca2+ influx in vascular smooth muscle cells and acts both as a direct vasoconstrictor and vascular growth factor (that is, angiogenesis). However, the mechanism by which EPO promotes extracellular Ca2+ entry in contractile cells has not been elucidated. In hematopoietic cells, EPO induces tyrosine kinase (TK)-dependent activation of phospholipase C (PLC)-gamma 1 and Ca2+ influx via a voltage-independent Ca2+ conductance. In contractile mesangial cells, we have recently characterized a voltage-independent, 1 pS Ca2+ channel that is dependent on both TK and PLC-gamma 1 activity. Therefore, we examined cultured rat glomerular mesangial cells after timed exposure to recombinant human EPO (20 U/ml). Erythropoietin increased the tyrosine phosphorylation of PLC-gamma 1, promoted membrane complex formation between PLC-gamma 1 and the EPO receptor itself, and raised the levels of intracellular inositol 1,4,5-trisphosphate and intracellular Ca2+. Consistent with our previous studies, 1 pS Ca2+ channel activity was extremely low under basal, unstimulated conditions in cell-attached patches, but was dramatically increased when EPO was present in the patch pipette. Tyrosine kinase inhibition with 100 micron genistein or 1 micron PP1 (Src; selective tyrosine kinase inhibitor) prevented all of these EPO-induced responses. We conclude that: (1) EPO-induced stimulation of 1 pS Ca2+ channels is mediated via a cytosolic Src TK in glomerular mesangial cells. (2) Stimulation of this Ca2(+)-activated, Ca2(+)-permeable channel is dependent on the tyrosine phosphorylation/activation of PLC-gamma 1. (3) This cascade provides a possible mechanism for the vasoconstriction and hypertension observed with clinical EPO use for the treatment of chronic anemias.

Angiotensin II-induced association of phospholipase Cgamma1 with the G-protein-coupled AT1 receptor.

An early event in signaling by the G-protein-coupled angiotensin II (Ang II) AT1 receptor in vascular smooth muscle cells is the tyrosine phosphorylation and activation of phospholipase Cgamma1 (PLCgamma1). In the present study, we show that stimulation of this event by Ang II in vascular smooth muscle cells is accompanied by binding of PLCgamma1 to the AT1 receptor in an Ang II- and tyrosine phophorylation-dependent manner. The PLCgamma1-AT1 receptor interaction appears to depend on phosphorylation of tyrosine 319 in a YIPP motif in the C-terminal intracellular domain of the AT1 receptor and binding of the phosphorylated receptor by the most C-terminal of two Src homology 2 domains in PLCgamma1. PLCgamma1 thus binds to the same site in the receptor previously identified for binding by the SHP-2 phosphotyrosine phosphatase.JAK2 tyrosine kinase complex. A single site in the C-terminal tail of the AT1 receptor can, therefore, be bound in a ligand-dependent manner by two different downstream effector proteins. These data demonstrate that G-protein-coupled receptors can physically associate with intracellular proteins other than G proteins, creating membrane-delimited signal transduction complexes similar to those observed for classic growth factor receptors.

Dependence on the motif YIPP for the physical association of Jak2 kinase with the intracellular carboxyl tail of the angiotensin II AT1 receptor.

Angiotensin II is the effector molecule of the renin-angiotensin system. Virtually all of its biochemical actions are mediated through a single class of cell-surface receptors called AT1. These receptors contain the structural features of the seven-transmembrane, G-protein-coupled receptor superfamily. Angiotensin II, acting through the AT1 receptor, also stimulates the Jak/STAT pathway by inducing ligand-dependent Jak2 tyrosine phosphorylation and activation. Here, we show that a glutathione S-transferase fusion protein containing the carboxyl-terminal 54 amino acids of the rat AT1A receptor physically binds to Jak2 in an angiotensin II-dependent manner. Deletional analysis, using both in vitro protocols and cell transfection analysis, showed that this association is dependent on the AT1A receptor motif YIPP (amino acids 319-322). The wild-type AT1A receptor can induce Jak2 tyrosine phosphorylation. In contrast, an AT1A receptor lacking the YIPP motif is unable to induce ligand-dependent phosphorylation of Jak2. Competition experiments with synthetic peptides suggest that each of the YIPP amino acids, including tyrosine 319, is important in Jak2 binding to the AT1A receptor. The binding of the AT1A receptor to the intracellular tyrosine kinase Jak2 supports the concept that the seven-transmembrane superfamily of receptors can physically associate with enzymatically active intracellular proteins, creating a signaling complex mechanistically similar to that observed with growth factor and cytokine receptors.

Role of Janus kinase/signal transducer and activator of transcription and mitogen-activated protein kinase cascades in angiotensin II- and platelet-derived growth factor-induced vascular smooth muscle cell proliferation.

In vascular smooth muscle cells, the induction of early growth response genes involves the Janus kinase (JAK)/signal transducer and activators of transcription (STAT) and the Ras/Raf-1/mitogen-activated protein kinase cascades. In the present study, we found that electroporation of antibodies against MEK1 or ERK1 abolished vascular smooth muscle cell proliferation in response to either platelet-derived growth factor or angiotensin II. However, anti-STAT1 or -STAT3 antibody electroporation abolished proliferative responses only to angiotensin II and not to platelet-derived growth factor. AG-490, a specific inhibitor of the JAK2 tyrosine kinase, prevented proliferation of vascular smooth muscle cells, complex formation between JAK2 and Raf-1, the tyrosine phosphorylation of Raf-1, and the activation of ERK1 in response to either angiotensin II or platelet-derived growth factor. However, AG-490 had no effect on angiotensin II- or platelet-derived growth factor-induced Ras/Raf-1 complex formation. Our results indicate that: 1) STAT proteins play an essential role in angiotensin II-induced vascular smooth muscle cell proliferation, 2) JAK2 plays an essential role in the tyrosine phosphorylation of Raf-1, and 3) convergent mitogenic signaling cascades involving the cytosolic kinases JAK2, MEK1, and ERK1 mediate vascular smooth muscle cell proliferation in response to both growth factor and G protein-coupled receptors.

Caveolin-1 detergent solubility and association with endothelial nitric oxide synthase is modulated by tyrosine phosphorylation.

Caveolin-1 and endothelial nitric oxide synthase (eNOS) are associated within endothelial caveolae. We have shown previously that eNOS is translocated to the detergent-insoluble, cytoskeletal fraction of bovine aortic endothelial cells (BAEC) in response to bradykinin (BK)-stimulation or tyrosine phosphatase inhibition. In the present study, we have examined whether caveolin-1 is similarly translocated in response to these or other stimuli. Exposure of BAEC to the eNOS-activating agonists, BK, histamine, or ATP produces transient increases in the amounts of detergent-insoluble caveolin-1. Increases in insolubility are blocked by tyrosine kinase inhibitors and are potently mimicked by tyrosine phosphatase inhibitors. Increased insolubility is accompanied by an increased association of caveolin-1 with eNOS and inhibition of eNOS catalytic activity. Agonist-activation of eNOS in endothelial cells thus appears to involve tyrosine phosphorylation-dependent changes in the interaction of eNOS with caveolin-1. Increased interaction of eNOS with caveolin-1 may deactivate the enzyme subsequent to its activation by Ca2+/calmodulin.