Peroxiredoxin II is an essential antioxidant enzyme that prevents the oxidative inactivation of VEGF receptor-2 in vascular endothelial cells.

Cellular antioxidant enzymes play crucial roles in aerobic organisms by eliminating detrimental oxidants and maintaining the intracellular redox homeostasis. Therefore, the function of antioxidant enzymes is inextricably linked to the redox-dependent activities of multiple proteins and signaling pathways. Here, we report that the VEGFR2 RTK has an oxidation-sensitive cysteine residue whose reduced state is preserved specifically by peroxiredoxin II (PrxII) in vascular endothelial cells. In the absence of PrxII, the cellular H(2)O(2) level is markedly increased and the VEGFR2 becomes inactive, no longer responding to VEGF stimulation. Such VEGFR2 inactivation is due to the formation of intramolecular disulfide linkage between Cys1199 and Cys1206 in the C-terminal tail. Interestingly, the PrxII-mediated VEGFR2 protection is achieved by association of two proteins in the caveolae. Furthermore, PrxII deficiency suppresses tumor angiogenesis in vivo. This study thus demonstrates a physiological function of PrxII as the residential antioxidant safeguard specific to the redox-sensitive VEGFR2.

Caveolae-specific activation loop between CaMKII and L-type Ca2+ channel aggravates cardiac hypertrophy in α1-adrenergic stimulation.

Activation of CaMKII induces a myriad of biological processes and plays dominant roles in cardiac hypertrophy. Caveolar microdomain contains many calcium/calmodulin-dependent kinase II (CaMKII) targets, including L-type Ca2+ channel (LTCC) complex, and serves as a signaling platform. The location of CaMKII activation is thought to be critical; however, the roles of CaMKII in caveolae are still elusive due to lack of methodology for the assessment of caveolae-specific activation. Our aim was to develop a novel tool for the specific analysis of CaMKII activation in caveolae and to determine the functional role of caveolar CaMKII in cardiac hypertrophy. To assess the caveolae-specific activation of CaMKII, we generated a fusion protein composed of phospholamban and caveolin-3 (cPLN-Cav3) and GFP fusion protein with caveolin-binding domain fused to CaMKII inhibitory peptide (CBD-GFP-AIP), which inhibits CaMKII activation specifically in caveolae. Caveolae-specific activation of CaMKII was detected using phosphospecific antibody for PLN (Thr17). Furthermore, adenoviral overexpression of LTCC ß2a-subunit (ß2a) in NRCMs showed its constitutive phosphorylation by CaMKII, which induces hypertrophy, and that both phosphorylation and hypertrophy are abolished by CBD-GFP-AIP expression, indicating that ß2a phosphorylation occurs specifically in caveolae. Finally, ß2a phosphorylation was observed after phenylephrine stimulation in ß2a-overexpressing mice, and attenuation of cardiac hypertrophy after chronic phenylephrine stimulation was observed in nonphosphorylated mutant of ß2a-overexpressing mice. We developed novel tools for the evaluation and inhibition of caveolae-specific activation of CaMKII. We demonstrated that phosphorylated ß2a dominantly localizes to caveolae and induces cardiac hypertrophy after α1-adrenergic stimulation in mice.NEW & NOTEWORTHY While signaling in caveolae is thought to be important in cardiac hypertrophy, direct evidence is missing due to lack of tools to assess caveolae-specific signaling. This is the first study to demonstrate caveolae-specific activation of CaMKII signaling in cardiac hypertrophy induced by α1-adrenergic stimulation using an originally developed tool.

N-acetylcysteine attenuates myocardial dysfunction and postischemic injury by restoring caveolin-3/eNOS signaling in diabetic rats.

BACKGROUND: Patients with diabetes are prone to develop cardiac hypertrophy and more susceptible to myocardial ischemia-reperfusion (I/R) injury, which are concomitant with hyperglycemia-induced oxidative stress and impaired endothelial nitric oxide (NO) synthase (eNOS)/NO signaling. Caveolae are critical in the transduction of eNOS/NO signaling in cardiovascular system. Caveolin (Cav)-3, the cardiomyocytes-specific caveolae structural protein, is decreased in the diabetic heart in which production of reactive oxygen species are increased. We hypothesized that treatment with antioxidant N-acetylcysteine (NAC) could enhance cardiac Cav-3 expression and attenuate caveolae dysfunction and the accompanying eNOS/NO signaling abnormalities in diabetes. METHODS: Control or streptozotocin-induced diabetic rats were either untreated or treated with NAC (1.5 g/kg/day, NAC) by oral gavage for 4 weeks. Rats in subgroup were randomly assigned to receive 30 min of left anterior descending artery ligation followed by 2 h of reperfusion. Isolated rat cardiomyocytes or H9C2 cells were exposed to low glucose (LG, 5.5 mmol/L) or high glucose (HG, 25 mmol/L) for 36 h before being subjected to 4 h of hypoxia followed by 4 h of reoxygenation (H/R). RESULTS: NAC treatment ameliorated myocardial dysfunction and cardiac hypertrophy, and attenuated myocardial I/R injury and post-ischemic cardiac dysfunction in diabetic rats. NAC attenuated the reductions of NO, Cav-3 and phosphorylated eNOS and mitigated the augmentation of O2-, nitrotyrosine and 15-F2t-isoprostane in diabetic myocardium. Immunofluorescence analysis demonstrated the colocalization of Cav-3 and eNOS in isolated cardiomyocytes. Immunoprecipitation analysis revealed that diabetic conditions decreased the association of Cav-3 and eNOS in isolated cardiomyocytes, which was enhanced by treatment with NAC. Disruption of caveolae by methyl-ß-cyclodextrin or Cav-3 siRNA transfection reduced eNOS phosphorylation. NAC treatment attenuated the reductions of Cav-3 expression and eNOS phosphorylation in HG-treated cardiomyocytes or H9C2 cells. NAC treatment attenuated HG and H/R induced cell injury, which was abolished during concomitant treatment with Cav-3 siRNA or eNOS siRNA. CONCLUSIONS: Hyperglycemia-induced inhibition of eNOS activity might be consequences of caveolae dysfunction and reduced Cav-3 expression. Antioxidant NAC attenuated myocardial dysfunction and myocardial I/R injury by improving Cav-3/eNOS signaling.

The N-terminal leucine-zipper motif in PTRF/cavin-1 is essential and sufficient for its caveolae-association.

PTRF/cavin-1 is a protein of two lives. Its reported functions in ribosomal RNA synthesis and in caveolae formation happen in two different cellular locations: nucleus vs. plasma membrane. Here, we identified that the N-terminal leucine-zipper motif in PTRF/cavin-1 was essential for the protein to be associated with caveolae in plasma membrane. It could counteract the effect of nuclear localization sequence in the molecule (AA 235-251). Deletion of this leucine-zipper motif from PTRF/cavin-1 caused the mutant to be exclusively localized in nuclei. The fusion of this leucine-zipper motif with histone 2A, which is a nuclear protein, could induce the fusion protein to be exported from nucleus. Cell migration was greatly inhibited in PTRF/cavin-1(-/-) mouse embryonic fibroblasts (MEFs). The inhibited cell motility could only be rescued by exogenous cavin-1 but not the leucine-zipper motif deleted cavin-1 mutant. Plasma membrane dynamics is an important factor in cell motility control. Our results suggested that the membrane dynamics in cell migration is affected by caveolae associated PTRF/cavin-1.

Cyclophilin A is required for angiotensin II-induced p47phox translocation to caveolae in vascular smooth muscle cells.

OBJECTIVE: Angiotensin II (AngII) signal transduction in vascular smooth muscle cells (VSMC) is mediated by reactive oxygen species (ROS). Cyclophilin A (CyPA) is a ubiquitously expressed cytosolic protein that possesses peptidyl-prolyl cis-trans isomerase activity, scaffold function, and significantly enhances AngII-induced ROS production in VSMC. We hypothesized that CyPA regulates AngII-induced ROS generation by promoting translocation of NADPH oxidase cytosolic subunit p47phox to caveolae of the plasma membrane. APPROACH AND RESULTS: Overexpression of CyPA in CyPA-deficient VSMC (CyPA(-/-)VSMC) significantly increased AngII-stimulated ROS production. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors (VAS2870 or diphenylene iodonium) significantly attenuated AngII-induced ROS production in CyPA and p47phox-overexpressing CyPA(-/-)VSMC. Cell fractionation and sucrose gradient analyses showed that AngII-induced p47phox plasma membrane translocation, specifically to the caveolae, was reduced in CyPA(-/-)VSMC compared with wild-type-VSMC. Immunofluorescence studies demonstrated that AngII increased p47phox and CyPA colocalization and translocation to the plasma membrane. In addition, immunoprecipitation of CyPA followed by immunoblotting of p47phox and actin showed that AngII increased CyPA and p47phox interaction. AngII-induced p47phox and actin cell cytoskeleton association was attenuated in CyPA(-/-)VSMC. Mechanistically, inhibition of p47phox phosphorylation and phox homology domain deletion attenuated CyPA and p47phox interaction. Finally, cyclosporine A and CyPA-peptidyl-prolyl cis-trans isomerase mutant, R55A, inhibited AngII-stimulated CyPA and p47phox association in VSMC, suggesting that peptidyl-prolyl cis-trans isomerase activity was required for their interaction. CONCLUSIONS: These findings provide the mechanism by which CyPA is an important regulator for AngII-induced ROS generation in VSMC through interaction with p47phox and cell cytoskeleton, which enhances the translocation of p47phox to caveolae.

Endothelial caveolar subcellular domain regulation of endothelial nitric oxide synthase.

Complex regulatory processes alter the activity of endothelial nitric oxide synthase (eNOS) leading to nitric oxide (NO) production by endothelial cells under various physiological states. These complex processes require specific subcellular eNOS partitioning between plasma membrane caveolar domains and non-caveolar compartments. Translocation of eNOS from the plasma membrane to intracellular compartments is important for eNOS activation and subsequent NO biosynthesis. We present data reviewing and interpreting information regarding: (i) the coupling of endothelial plasma membrane receptor systems in the caveolar structure relative to eNOS trafficking; (ii) how eNOS trafficking relates to specific protein-protein interactions for inactivation and activation of eNOS; and (iii) how these complex mechanisms confer specific subcellular location relative to eNOS multisite phosphorylation and signalling. Dysfunction in the regulation of eNOS activation may contribute to several disease states, in particular gestational endothelial abnormalities (pre-eclampsia, gestational diabetes etc.), that have life-long deleterious health consequences that predispose the offspring to develop hypertensive disease, Type 2 diabetes and adiposity.

Novel role of p66Shc in ROS-dependent VEGF signaling and angiogenesis in endothelial cells.

p66Shc, a longevity adaptor protein, is demonstrated as a key regulator of reactive oxygen species (ROS) metabolism involved in aging and cardiovascular diseases. Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration and proliferation primarily through the VEGF receptor-2 (VEGFR2). We have shown that ROS derived from Rac1-dependent NADPH oxidase are involved in VEGFR2 autophosphorylation and angiogenic-related responses in ECs. However, a role of p66Shc in VEGF signaling and physiological responses in ECs is unknown. Here we show that VEGF promotes p66Shc phosphorylation at Ser36 through the JNK/ERK or PKC pathway as well as Rac1 binding to a nonphosphorylated form of p66Shc in ECs. Depletion of endogenous p66Shc with short interfering RNA inhibits VEGF-induced Rac1 activity and ROS production. Fractionation of caveolin-enriched lipid raft demonstrates that p66Shc plays a critical role in VEGFR2 phosphorylation in caveolae/lipid rafts as well as downstream p38MAP kinase activation. This in turn stimulates VEGF-induced EC migration, proliferation, and capillary-like tube formation. These studies uncover a novel role of p66Shc as a positive regulator for ROS-dependent VEGFR2 signaling linked to angiogenesis in ECs and suggest p66Shc as a potential therapeutic target for various angiogenesis-dependent diseases.

Enhanced phosphorylation of caveolar PKC-α limits peptide internalization in lung endothelial cells.

We previously reported that the vasoactive peptide 1 (P1, "SSWRRKRKESS") modulates the tension of pulmonary artery vessels through caveolar endothelial nitric oxide synthase (eNOS) activation in intact lung endothelial cells (ECs). Since PKC-α is a caveolae resident protein and caveolae play a critical role in the peptide internalization process, we determined whether modulation of caveolae and/or caveolar PKC-α phosphorylation regulates internalization of P1 in lung ECs. Cell monolayers were incubated in culture medium containing Rhodamine red-labeled P1 (100 µM) for 0-120 min. Confocal examinations indicate that P1 internalization is time-dependent and reaches a plateau at 60 min. Caveolae disruption by methyl-ß-cyclodextrin (CD) and filipin (FIL) inhibited the internalization of P1 in ECs suggesting that P1 internalizes via caveolae. P1-stimulation also enhances phosphorylation of caveolar PKC-α and increases intracellular calcium (Ca(2+)) release in intact cells suggesting that P1 internalization is regulated by PKC-α in ECs. To confirm the roles of increased phosphorylation of PKC-α and Ca(2+) release in internalization of P1, PKC-α modulation by phorbol ester (PMA), PKC-α knockdown, and Ca(2+) scavenger BAPTA-AM model systems were used. PMA-stimulated phosphorylation of caveolar PKC-α is associated with significant reduction in P1 internalization. In contrast, PKC-α deficiency and reduced phosphorylation of PKC-α enhanced P1 internalization. P1-mediated increased phosphorylation of PKC-α appears to be associated with increased intracellular calcium (Ca(2+)) release since the Ca(2+) scavenger BAPTA-AM enhanced P1 internalization. These data indicate that caveolar integrity and P1-mediated increased phosphorylation of caveolar PKC-α play crucial roles in the regulation of P1 internalization in lung ECs.

Subcellular localization of oxidants and redox modulation of endothelial nitric oxide synthase.

Reactive oxygen species (ROS) have long been viewed as deleterious chemicals that lead to oxidative stress. More recently, ROS, especially the stable ROS hydrogen peroxide (H(2)O(2)), have been shown to have roles in normal physiological responses in vascular cells. Endothelial nitric oxide synthase (eNOS) is dynamically targeted to plasmalemmal caveolae, and represents the principal enzymatic source of nitric oxide (NO(•)) in the vascular wall. eNOS maintains normal vascular tone and inhibits the clinical expression of many cardiovascular diseases. Increases in oxidative stress are associated with eNOS dysfunction. In a paradigm shift in the conceptual framework linking redox biochemistry and vascular function, H(2)O(2) has been established as a physiological mediator in signaling pathways, yet the intracellular sources of H(2)O(2) and their regulation remain incompletely understood. The subcellular distributions of ROS and of ROS-modified proteins critically influence the redox-sensitive regulation of eNOS-dependent pathways. ROS localization in specific subcellular compartments can lead to selective oxidative modifications of eNOS and eNOS-associated proteins. Likewise, the dynamic targeting of eNOS and other signaling proteins influences their interactions with reactive nitrogen species and ROS that are also differentially distributed within the cell. Thus, the subcellular distribution both of eNOS and redox-active biomolecules serves as a critical basis for the control of the "redox switch" that influences NO(•)- and oxidant-regulated signaling pathways. Here we discuss the biochemical factors, cellular determinants, and molecular mechanisms that modulate redox-sensitive regulation of eNOS and NO(•) signaling under normal and pathological conditions.

Regulation of eNOS in caveolae.

Caveolae are a specialized subset of lipid domains that are prevalent on the plasma membrane of endothelial cells. They compartmentalize signal transduction molecules which regulate multiple endothelial functions including the production of nitric oxide (NO) by the caveolae resident enzyme endothelial NO synthase (eNOS). eNOS is one of the three isoforms of the NOS enzyme which generates NO upon the conversion of L-arginine to L-citrulline and it is regulated by multiple mechanisms. Caveolin negatively impact eNOS activity through direct interaction with the enzyme. Circulating factors known to modify cardiovascular disease risk also influence the activity of the enzyme. In particular, high density lipoprotein cholesterol (HDL) maintains the lipid environment in caveolae, thereby promoting the retention and function of eNOS in the domain and it also causes direct activation of eNOS via scavenger receptor class B, Type I (SR-BI)-induced kinase signaling. Estrogen binding to estrogen receptors (ER) in caveolae also activates eNOS and this occurs through G protein coupling and kinase activation. Discrete domains within SR-BI and ER mediating signal initiation in caveolae have been identified. Counteracting the promodulatory actions of HDL and estrogen, C-reactive protein (CRP) antagonizes eNOS through FcγRIIB, which is the sole inhibitory receptor for IgG. Through their actions on eNOS, estrogen and CRP also regulate endothelial cell growth and migration. Thus, signaling events in caveolae invoked by known circulating cardiovascular disease risk factors have major impact on eNOS and endothelial cell phenotypes of importance to cardiovascular health and disease.

Comparative properties of caveolar and noncaveolar preparations of kidney Na+/K+-ATPase.

To evaluate previously proposed functions of renal caveolar Na(+)/K(+)-ATPase, we modified the standard procedures for the preparation of the purified membrane-bound kidney enzyme, separated the caveolar and noncaveolar pools, and compared their properties. While the subunits of Na(+)/K(+)-ATPase (α,ß,γ) constituted most of the protein content of the noncaveolar pool, the caveolar pool also contained caveolins and major caveolar proteins annexin-2 tetramer and E-cadherin. Ouabain-sensitive Na(+)/K(+)-ATPase activities of the two pools had similar properties and equal molar activities, indicating that the caveolar enzyme retains its ion transport function and does not contain nonpumping enzyme. As minor constituents, both caveolar and noncaveolar pools also contained Src, EGFR, PI3K, and several other proteins known to be involved in stimulous-induced signaling by Na(+)/K(+)-ATPase, indicating that signaling function is not limited to the caveolar pool. Endogenous Src was active in both pools but was not further activated by ouabain, calling into question direct interaction of Src with native Na(+)/K(+)-ATPase. Chemical cross-linking, co-immunoprecipitation, and immunodetection studies showed that in the caveolar pool, caveolin-1 oligomers, annexin-2 tetramers, and oligomers of the α,ß,γ-protomers of Na(+)/K(+)-ATPase form a large multiprotein complex. In conjunction with known roles of E-cadherin and the ß-subunit of Na(+)/K(+)-ATPase in cell adhesion and noted intercellular ß,ß-contacts within the structure of Na(+)/K(+)-ATPase, our findings suggest that interacting caveolar Na(+)/K(+)-ATPases located at renal adherens junctions maintain contact of two adjacent cells, conduct essential ion pumping, and are capable of locus-specific signaling in junctional cells.

Palmitoylation of R-Ras by human DHHC19, a palmitoyl transferase with a CaaX box.

Mammalian proteins that contain an aspartate-histidine-histidine-cysteine-(DHHC) motif have been recently identified as a group of membrane-associated palmitoyl acyltransferases (PATs). Among the several protein substrates known to become palmitoylated by DHHC PATs are small GTPases prenylated at their carboxy-terminal end, such as H-Ras or N-Ras, eNOS, kinases myristoylated at their N-terminal end, such as Lck, and many transmembrane proteins and channels. We have focused our studies on the product of the human gene DHHC19, a putative palmitoyl transferase that, interestingly, displays a conserved CaaX box at its carboxy-terminal end. We show herein that the amino acid sequence present at the carboxy-terminus of DHHC19 is able to exclude a green fluorescent protein (GFP) reporter from the nucleus and direct it towards perinuclear regions. Transfection of full-length DHHC19 in COS7 cells reveals a perinuclear distribution, in analogy to other palmitoyl transferases, with a strong colocalization with the trans-Golgi markers Gal-T and TGN38. We have tested several small GTPases that are known to be palmitoylated as possible substrates of DHHC19. Although DHHC19 failed to increase the palmitoylation of H-Ras, N-Ras, K-Ras4A, RhoB or Rap2 it increased the palmitoylation of R-Ras approximately two-fold. The increased palmitoylation of R-Ras cotransfected with DHHC19 is accompanied by an augmented association with membranes as well as with rafts/caveolae. Finally, using both wild-type and an activated GTP bound form of R-Ras (G38V), we also show that the increased palmitoylation of R-Ras due to DHHC19 coexpression is accompanied by an enhanced viability of the transfected cells.

Caveolae facilitate but are not essential for platelet-activating factor-mediated calcium mobilization and extracellular signal-regulated kinase activation.

Certain proteins, including receptors and signaling molecules, are known to be enriched in caveolae and lipid rafts. Caveolin-1, the major structural protein of caveolae, specifically interacts with many signaling molecules and, thus, caveolae and lipid rafts are often seen as preassembled signaling platforms. A potential binding site for caveolin-1 is present in the platelet-activating factor receptor (PAFR) sequence, and many downstream signaling components of PAFR activation preferentially localize in caveolae. The aim of this study was to investigate whether the PAFR was localized in caveolae/lipid raft domains and, if so, what would be the significance of such localization for PAFR signaling. In this study, we demonstrate that PAFR localizes within membrane microdomains, in close proximity to caveolin-1 in living cells, with potential interaction through a caveolin-1-binding sequence in the PAFR C terminus. Caveolin-1, however, is not essential for PAFR localization in lipid rafts. Disruption of caveolae/lipid rafts with methyl-beta-cyclodextrin markedly reduced PAF-triggered inositol phosphate production and cytosolic calcium flux, suggesting that PAFR signaling through the Galphaq protein was critically dependent on integrity of lipid rafts and/or caveolae. Interestingly, whereas in caveolin-1-expressing cells lipid raft disruption markedly decreased PAFR-mediated activation of the ERK/MAPK pathway, in cells lacking caveolae, such as leukocytes, lipid raft disruption had either the same inhibitory effect (Ramos B cells) or no effect (monocytes) on PAFR capacity to signal through the ERK/MAPK pathway. In conclusion, PAFR appears to localize within caveolae or lipid rafts in different cell types, and this location may be important for specific signaling events.

Kinase-regulated quantal assemblies and kiss-and-run recycling of caveolae.

A functional genomics approach has revealed that caveolae/raft-mediated endocytosis is subject to regulation by a large number of kinases. Here we explore the role of some of these kinases in caveolae dynamics. We discover that caveolae operate using principles different from classical membrane trafficking. First, each caveolar coat contains a set number (one 'quantum') of caveolin-1 molecules. Second, caveolae are either stored as in stationary multi-caveolar structures at the plasma membrane, or undergo continuous cycles of fission and fusion with the plasma membrane in a small volume beneath the surface, without disassembling the caveolar coat. Third, a switch mechanism shifts caveolae from this localized cycle to long-range cytoplasmic transport. We have identified six kinases that regulate different steps of the caveolar cycle. Our observations reveal new principles in caveolae trafficking and suggest that the dynamic properties of caveolae and their transport competence are regulated by different kinases operating at several levels.

Lung vascular targeting using antibody to aminopeptidase P: CT-SPECT imaging, biodistribution and pharmacokinetic analysis.

BACKGROUND/AIMS: Aminopeptidase P (APP) is specifically enriched in caveolae on the luminal surface of pulmonary vascular endothelium. APP antibodies bind lung endothelium in vivo and are rapidly and actively pumped across the endothelium into lung tissue. Here we characterize the immunotargeting properties and pharmacokinetics of the APP-specific recombinant antibody 833c. METHODS: We used in situ binding, biodistribution analysis and in vivo imaging to assess the lung targeting of 833c. RESULTS: More than 80% of 833c bound during the first pass through isolated perfused lungs. Dynamic SPECT acquisition showed that 833c rapidly and specifically targeted the lungs in vivo, reaching maximum levels within 2 min after intravenous injection. CT-SPECT imaging revealed specific targeting of 833c to the thoracic cavity and co-localization with a lung perfusion marker, Tc99m-labeled macroaggregated albumin. Biodistribution analysis confirmed lung-specific uptake of 833c which declined by first-order kinetics (t(½) = 110 h) with significant levels of 833c still present 30 days after injection. CONCLUSION: These data show that APP expressed in endothelial caveolae appears to be readily accessible to circulating antibody rather specifically in lung. Targeting lung-specific caveolar APP provides an extraordinarily rapid and specific means to target pulmonary vasculature and potentially deliver therapeutic agents into the lung tissue.

Modulation of PP2A activity by Jacalin: is it through caveolae and ER chaperones?

Plant lectins have been reported to affect the proliferation of different human cancer cell line probably by binding to the specific carbohydrate moieties. In the present study, Badan labeled single cysteine mutant (present in the caveolin-1 binding motif) of jacalin (rJacalin) was found to penetrate the target membrane, indicating a protein-protein or protein-membrane interaction apart from its primary mode of binding i.e. protein-carbohydrate interaction. Further, Jacalin treatment has resulted in the movement of the GFP-Caveolin-1 predominantly at the cell-cell contact region with much restricted dynamics. Jacalin treatment has resulted in the perinuclear accumulation of PP2A and dissociation of the PHAP1/PP2A complex. PP2A was found to act as a negative regulator of ERK signaling and a significant decrease in the phosphorylation level of MEK and AKT (T308) in A431. In addition, we have also identified several ER resident proteins including molecular chaperones like ORP150, Hsp70, Grp78, BiP of A431 cells, which were bound to the Jacalin-sepharose column. Among various ER chaperones that were identified, ORP150 was found to present on the cell surface of A431 cells.

Nuclear alpha1-adrenergic receptors signal activated ERK localization to caveolae in adult cardiac myocytes.

We previously identified an alpha1-AR-ERK (alpha1A-adrenergic receptor-extracellular signal-regulated kinase) survival signaling pathway in adult cardiac myocytes. Here, we investigated localization of alpha1-AR subtypes (alpha1A and alpha1B) and how their localization influences alpha1-AR signaling in cardiac myocytes. Using binding assays on myocyte subcellular fractions or a fluorescent alpha1-AR antagonist, we localized endogenous alpha1-ARs to the nucleus in wild-type adult cardiac myocytes. To clarify alpha1 subtype localization, we reconstituted alpha1 signaling in cultured alpha1A- and alpha1B-AR double knockout cardiac myocytes using alpha1-AR-green fluorescent protein (GFP) fusion proteins. Similar to endogenous alpha1-ARs and alpha1A- and alpha1B-GFP colocalized with LAP2 at the nuclear membrane. alpha1-AR nuclear localization was confirmed in vivo using alpha1-AR-GFP transgenic mice. The alpha1-signaling partners Galphaq and phospholipase Cbeta1 also colocalized with alpha1-ARs only at the nuclear membrane. Furthermore, we observed rapid catecholamine uptake mediated by norepinephrine-uptake-2 and found that alpha1-mediated activation of ERK was not inhibited by a membrane impermeant alpha1-blocker, suggesting alpha1 signaling is initiated at the nucleus. Contrary to prior studies, we did not observe alpha1-AR localization to caveolae, but we found that alpha1-AR signaling initiated at the nucleus led to activated ERK localized to caveolae. In summary, our results show that nuclear alpha1-ARs transduce signals to caveolae at the plasma membrane in cardiac myocytes.

The Tom1L1-clathrin heavy chain complex regulates membrane partitioning of the tyrosine kinase Src required for mitogenic and transforming activities.

Compartmentalization of Src tyrosine kinases (SFK) plays an important role in signal transduction induced by a number of extracellular stimuli. For example, Src mitogenic signaling induced by platelet-derived growth factor (PDGF) is initiated in cholesterol-enriched microdomain caveolae. How this Src subcellular localization is regulated is largely unknown. Here we show that the Tom1L1-clathrin heavy chain (CHC) complex negatively regulates the level of SFK in caveolae needed for the induction of DNA synthesis. Tom1L1 is both an interactor and a substrate of SFK. Intriguingly, it stimulates Src activity without promoting mitogenic signaling. We found that, upon association with CHC, Tom1L1 reduced the level of SFK in caveolae, thereby preventing its association with the PDGF receptor, which is required for the induction of mitogenesis. Similarly, the Tom1L1-CHC complex reduced also the level of oncogenic Src in cholesterol-enriched microdomains, thus affecting both its capacity to induce DNA synthesis and cell transformation. Conversely, Tom1L1, when not associated with CHC, accumulated in caveolae and promoted Src-driven DNA synthesis. We concluded that the Tom1L1-CHC complex defines a novel mechanism involved in negative regulation of mitogenic and transforming signals, by modulating SFK partitioning at the plasma membrane.

Recruitment of protein phosphatase 2A to dorsal ruffles by platelet-derived growth factor in smooth muscle cells: dephosphorylation of Hsp27.

In this study, we investigated the mechanism underlying Hsp27 dephosphorylation in smooth muscle cells. We found that protein phosphatase 2A (PP2A) dephosphorylates Hsp27. In addition, Hsp27 dephosphorylation was regulated by membrane cholesterol content. We showed that PDGF induced a three-fold increase in the proportion of PP2A activity regulated by cholesterol in the Triton-insoluble fraction of cell lysates. Moreover, cholesterol depletion decreased the amount of PP2A recovered in Triton-insoluble fraction. Thus, PDGF might regulate a small pool of PP2A associated with lipid rafts. Isolation of detergent-resistant membrane fragments by Optiprep-gradient density indicated that this pool of PP2A was not associated with caveolae, but was recovered in a higher density fraction (DRM-H) with ganglioside GM1, alpha-actinin, Hsp27 and p34, a component of Arp2/3 complex. These proteins were also present in dorsal ruffles containing GM1 but not caveolin-1. Phosphorylated Hsp27 levels detected in dorsal ruffles were variable. Cholesterol depletion, which inhibits dorsal ruffle formation, decreased PP2A levels and increased the Hsp27-P to Hsp27 ratio in DRM-H. These findings suggest that Hsp27 is dephosphorylated by PP2A in dorsal ruffles, in non-caveolar lipid raft microdomains. However, similarly to p34, non-phosphorylated Hsp27 is associated to non-raft membrane domains at the leading edge of lamellipodia.

Lipid accumulation controls the balance between surface connection and scission of caveolae.

Caveolae are bulb-shaped invaginations of the plasma membrane (PM) that undergo scission and fusion at the cell surface and are enriched in specific lipids. However, the influence of lipid composition on caveolae surface stability is not well described or understood. Accordingly, we inserted specific lipids into the cell PM via membrane fusion and studied their acute effects on caveolae dynamics. We demonstrate that sphingomyelin stabilizes caveolae to the cell surface, whereas cholesterol and glycosphingolipids drive caveolae scission from the PM. Although all three lipids accumulated specifically in caveolae, cholesterol and sphingomyelin were actively sequestered, whereas glycosphingolipids diffused freely. The ATPase EHD2 restricts lipid diffusion and counteracts lipid-induced scission. We propose that specific lipid accumulation in caveolae generates an intrinsically unstable domain prone to scission if not restrained by EHD2 at the caveolae neck. This work provides a mechanistic link between caveolae and their ability to sense the PM lipid composition.