Plasma membrane factor XIIIA transglutaminase activity regulates osteoblast matrix secretion and deposition by affecting microtubule dynamics

Transglutaminase activity, arising potentially from transglutaminase 2 (TG2) and Factor XIIIA (FXIIIA), has been linked to osteoblast differentiation where it is required for type I collagen and fibronectin matrix deposition. In this study we have used an irreversible TG-inhibitor to 'block -and-track' enzyme(s) targeted during osteoblast differentiation. We show that the irreversible TG-inhibitor is highly potent in inhibiting osteoblast differentiation and mineralization and reduces secretion of both fibronectin and type I collagen and their release from the cell surface. Tracking of the dansyl probe by Western blotting and immunofluorescence microscopy demonstrated that the inhibitor targets plasma membrane-associated FXIIIA. TG2 appears not to contribute to crosslinking activity on the osteoblast surface. Inhibition of FXIIIA with NC9 resulted in defective secretory vesicle delivery to the plasma membrane which was attributable to a disorganized microtubule network and decreased microtubule association with the plasma membrane. NC9 inhibition of FXIIIA resulted in destabilization of microtubules as assessed by cellular Glu-tubulin levels. Furthermore, NC9 blocked modification of Glu-tubulin into 150 kDa high-molecular weight Glu-tubulin form which was specifically localized to the plasma membrane. FXIIIA enzyme and its crosslinking activity were colocalized with plasma membrane-associated tubulin, and thus, it appears that FXIIIA crosslinking activity is directed towards stabilizing the interaction of microtubules with the plasma membrane. Our work provides the first mechanistic cues as to how transglutaminase activity could affect protein secretion and matrix deposition in osteoblasts and suggests a novel function for plasma membrane FXIIIA in microtubule dynamics.     

Palmitoylation of caveolin-1 at a single site (Cys-156) controls its coupling to the c-Src tyrosine kinase: targeting of dually acylated molecules (GPI-linked, transmembrane, or cytoplasmic) to caveolae effectively uncouples c-Src and caveolin-1 (TYR-14).

Caveolin-1 was initially identified as a phosphoprotein in Rous sarcoma virus-transformed cells. Previous studies have shown that caveolin-1 is phosphorylated on tyrosine 14 by c-Src and that lipid modification of c-Src is required for this phosphorylation event to occur in vivo. Phosphocaveolin-1 (Tyr(P)-14) localizes within caveolae near focal adhesions and, through its interaction with Grb7, augments anchorage-independent growth and epidermal growth factor-stimulated cell migration. However, the cellular factors that govern the coupling of caveolin-1 to the c-Src tyrosine kinase remain largely unknown. Here, we show that palmitoylation of caveolin-1 at a single site (Cys-156) is required for coupling caveolin-1 to the c-Src tyrosine kinase. Furthermore, upon evaluating a battery of nonreceptor and receptor tyrosine kinases, we demonstrate that the tyrosine phosphorylation of caveolin-1 by c-Src is a highly selective event. We show that Src-induced tyrosine phosphorylation of caveolin-1 can be inhibited or uncoupled by targeting dually acylated proteins (namely carcinoembryonic antigen (CEA), CD36, and the NH(2)-terminal domain of Galpha(i1)) to the exoplasmic, transmembrane, and cytoplasmic regions of the caveolae membrane, respectively. Conversely, when these proteins are not properly targeted or lipid-modified, the ability of c-Src to phosphorylate caveolin-1 remains unaffected. In addition, when purified caveolae preparations are preincubated with a myristoylated peptide derived from the extreme N terminus of c-Src, the tyrosine phosphorylation of caveolin-1 is abrogated; the same peptide lacking myristoylation has no inhibitory activity. However, an analogous myristoylated peptide derived from c-Yes also has no inhibitory activity. Thus, the inhibitory effects of the myristoylated c-Src peptide are both myristoylation-dependent and sequence-specific. Finally, we investigated whether phosphocaveolin-1 (Tyr(P)-14) interacts with the Src homology 2 and/or phosphotyrosine binding domains of Grb7, the only characterized downstream mediator of its function. Taken together, our data identify a series of novel lipid-lipid-based interactions as important regulatory factors for coupling caveolin-1 to the c-Src tyrosine kinase in vivo.     

The interaction of estrogen receptor α and caveolin-3 regulates connexin43 phosphorylation in metabolic inhibition-treated rat cardiomyocytes

Caveolin-3, the major caveolin isoform in cardiomyocytes, plays an important role in the rapid signaling pathways initiated by stimulation of the membrane-associated molecules. To examine the role of caveolin-3 in regulating estrogen receptor α in cardiomyocytes, we investigate whether the membrane estrogen receptor α associates with caveolin-3 and whether this association is linked to the 17β-estradiol-mediated signals. In control cardiomyocytes, following discontinuous sucrose gradient centrifugation, caveolin-3 was found predominantly in the lipid raft buoyant fractions, whereas it was distributed to both the buoyant and non-lipid raft heavy fractions following metabolic inhibition treatment. Confocal microscopy showed that estrogen receptor α co-localized with caveolin-3 on the plasma membrane of neonatal and adult rat cardiomyocytes. This membrane labeling of estrogen receptor α was not seen following treatment with the cholesterol-depleting agent methyl-β-cyclodextrin (5 mM), whereas metabolic inhibition had little effect on the membrane distribution of estrogen receptor α. Metabolic inhibition induced tyrosine phosphorylation of caveolin-3 and decreased its association with estrogen receptor α, both effects being mediated via a Src activation mechanism, since they were inhibited by the selective tyrosine kinase inhibitor PP2. Metabolic inhibition also induced tyrosine phosphorylation of connexin43 and increased its association with c-Src, both effects being prevented by 17β-estradiol (200 nM). The effect of 17β-estradiol on metabolic inhibition-induced tyrosine phosphorylation of connexin43 was inhibited by the specific estrogen receptor antagonist ICI182780. These data identify cardiac caveolin-3 as juxtamembrane scaffolding for estrogen receptor α docking at caveolae, which provide a unique compartment for conveying 17β-estradiol-elicited, rapid signaling to regulate connexin43 phosphorylation during ischemia.     

Plasma membrane Pdia3 and VDR interact to elicit rapid responses to 1α,25(OH)2D3

1α,25-Dihydroxyvitamin D3 (1α,25(OH)₂D₃) regulates osteoblasts through genomic and rapid membrane-mediated responses. Here we examined the interaction of protein disulfide isomerase family A, member 3 (Pdia3) and the traditional vitamin D receptor (VDR) in plasma membrane-associated responses to 1α,25(OH)₂D₃. We found that Pdia3 co-localized with VDR and the caveolae scaffolding protein, caveolin-1 on the surface of MC3T3-E1 osteoblasts. Immunoprecipitation showed that both Pdia3 and VDR interacted with caveolin-1. Pdia3 further interacted with phospholipase A2 activating protein (PLAA), whereas VDR interacted with c-Src. 1α,25(OH)₂D₃ changed the interactions and transport of the two receptors and rapidly activated phospholipase A2 (PLA2) and c-Src. Silencing either receptor or caveolin-1 inhibited both PLA2 and c-Src, indicating that the two receptors function interdependently. These two receptor dependent rapid responses to 1α,25(OH)₂D₃ regulated gene expression, proliferation and apoptosis of MC3T3-E1 cells. These data demonstrate the importance of both receptors and caveolin-1 in mediating membrane responses to 1α,25(OH)₂D₃ and subsequently regulating osteoblast biology.     

Serine 23 and 36 phosphorylation of caveolin-2 is differentially regulated by targeting to lipid raft/caveolae and in mitotic endothelial cells

In the present study, using a combination of reconstituted systems and endothelial cells endogenously expressing caveolins, we show that phosphorylation of caveolin-2 at serines 23 and 36 can be differentially regulated by caveolin-1 mediated subcellular targeting to lipid raft/caveolae and in endothelial cells synchronized in mitosis. Detergent insolubility and sucrose flotation gradient experiments revealed that serine 23 phosphorylation of caveolin-2 preferably occurs in detergent-resistant membranes (DRMs), while serine 36 phosphorylation takes place in non-DRMs. Furthermore, immunofluorescence microscopy studies determined that in the presence of caveolin-1, serine 23-phosphorylated caveolin-2 mostly localizes to plasma membrane, while serine 36-phosphorylated caveolin-2 primarily resides in intracellular compartments. To directly address the role of caveolin-1 in regulating phosphorylation of endogenous caveolin-2, we have used the siRNA approach. The specific knockdown of caveolin-1 in endothelial cells decreases caveolin-2 phosphorylation at serine 23 but not at serine 36. Thus, upregulation of serine 23 phosphorylation of caveolin-2 depends on caveolin-1-driven targeting to plasma membrane lipid rafts and caveolae. Interestingly, although serine 36 phosphorylation does not seem to be regulated in endothelial cells by caveolin-1, it can be selectively upregulated in endothelial cells synchronized in mitosis. The latter data suggests a possible involvement of serine 36-phosphorylated caveolin-2 in modulating mitosis.     

Filamin A Regulates Caveolae Internalization and Trafficking in Endothelial Cells

Transcytosis via caveolae is critical for maintaining vascular homeostasis by regulating the tissue delivery of macromolecules, hormones, and lipids. In the present study, we test the hypothesis that interactions between F-actin cross-linking protein filamin A and caveolin-1 facilitate the internalization and trafficking of caveolae. Small interfering RNA-mediated knockdown of filamin A, but not filamin B, reduced the uptake and transcytosis of albumin by ∼35 and 60%, respectively, without altering the actin cytoskeletal structure or cell–cell adherens junctions. Mobility of both intracellular caveolin-1–green fluorescent protein (GFP)-labeled vesicles measured by fluorescence recovery after photobleaching and membrane-associated vesicles measured by total internal reflection-fluorescence microscopy was decreased in cells with reduced filamin A expression. In addition, in melanoma cells that lack filamin A (M2 cells), the majority of caveolin-1-GFP was localized on the plasma membrane, whereas in cells in which filamin A expression was reconstituted (A7 cells and M2 cells transfected with filamin A-RFP), caveolin-1-GFP was concentrated in intracellular vesicles. Filamin A association with caveolin-1 in endothelial cells was confirmed by cofractionation of these proteins in density gradients, as well as by coimmunoprecipitation. Moreover, this interaction was enhanced by Src activation, associated with increased caveolin-1 phosphorylation, and blocked by Src inhibition. Taken together, these data suggest that filamin A association with caveolin-1 promotes caveolae-mediated transport by regulating vesicle internalization, clustering, and trafficking.     

Src-induced phosphorylation of caveolin-2 on tyrosine 19. Phospho-caveolin-2 (Tyr(P)19) is localized near focal adhesions,remains associated with lipid rafts/caveolae,but no longer forms a high molecular mass hetero-oligomer with caveolin-1

Caveolin-2 is the least well studied member of the caveolin gene family. It is believed that caveolin-2 is an "accessory protein" that functions in conjunction with caveolin-1. At the level of the ER, caveolin-2 interacts with caveolin-1 to form a high molecular mass hetero-oligomeric complex that is targeted to lipid rafts and drives the formation of caveolae. However, caveolin-2 is not required for caveolae formation, implying that it may fulfill some unknown regulatory role. Here, we present the first evidence that caveolin-2 is a phosphoprotein. We show that caveolin-2 undergoes Src-induced phosphorylation on tyrosine 19. To study this phosphorylation event in vivo, we generated a novel phospho-specific antibody probe that only recognizes phosphocaveolin-2 (Tyr(P)(19)). We then used NIH-3T3 cells stably overexpressing c-Src to examine the localization and biochemical properties of phosphocaveolin-2 (Tyr(P)(19)). Our results indicate that phosphocaveolin-2 (Tyr(P)(19)) is localized near focal adhesions, remains associated with lipid rafts/caveolae, but no longer forms a high molecular mass hetero-oligomer with caveolin-1. Instead, phosphocaveolin-2 (Tyr(P)(19)) behaves as a monomer/dimer in velocity gradients. Thus, we conclude that the tyrosine phosphorylation of caveolin-2 (Tyr(P)(19)) may function as a signal that is recognized by the cellular machinery to induce the dissociation of caveolin-2 from caveolin-1 oligomers. We also demonstrate that (i) insulin-stimulation of adipocytes and (ii) integrin ligation of endothelial cells can both induce the tyrosine phosphorylation of caveolin-2 (Tyr(P)(19)). During integrin ligation, phosphocaveolin-2 (Tyr(P)(19)) co-localizes with activated FAK at focal adhesions. Thus, phosphocaveolin-2 (Tyr(P)(19)) may function as a docking site for Src homology domain-2 (SH2) domain containing proteins during signal transduction. In support of this notion, we identify several SH2 domain containing proteins, namely c-Src, NCK, and Ras-GAP, that interact with caveolin-2 in a phosphorylation-dependent manner. Furthermore, our co-immunoprecipitation experiments show that caveolin-2 and Ras-GAP are constitutively associated in c-Src expressing NIH-3T3 cells, but not in untransfected NIH-3T3 cells.     

Exo70 Subunit of the Exocyst Complex Is Involved in Adhesion-Dependent Trafficking of Caveolin-1

Caveolae are specialized domains of the plasma membrane, which play key roles in signaling, endocytosis and mechanosensing. Using total internal reflection fluorescent microscopy (TIRF-M), we observe that the exocyst subunit Exo70 forms punctuate structures at the plasma membrane and partially localizes with caveolin-1, the main component of caveolae. Upon cell detachment, we found that Exo70 accumulates with caveolin-1-positive vesicular structures. Upon cell re-adhesion, caveolin-1 traffics back to the plasma membrane in a multistep process involving microtubules and actin cytoskeleton. In addition, silencing of Exo70 redirects caveolin-1 to focal adhesions identified by markers such as α5 integrin or vinculin. Based on these findings, we conclude that Exo70 is involved in caveolin-1 recycling to the plasma membrane during re-adhesion of the cells to the substratum.     

Microtubules and actin microfilaments regulate lipid raft/caveolae localization of adenylyl cyclase signaling components

Microtubules and actin filaments regulate plasma membrane topography, but their role in compartmentation of caveolae-resident signaling components, in particular G protein-coupled receptors (GPCR) and their stimulation of cAMP production, has not been defined. We hypothesized that the microtubular and actin cytoskeletons influence the expression and function of lipid rafts/caveolae, thereby regulating the distribution of GPCR signaling components that promote cAMP formation. Depolymerization of microtubules with colchicine (Colch) or actin microfilaments with cytochalasin D (CD) dramatically reduced the amount of caveolin-3 in buoyant (sucrose density) fractions of adult rat cardiac myocytes. Colch or CD treatment led to the exclusion of caveolin-1, caveolin-2, beta1-adrenergic receptors (beta1-AR), beta2-AR, Galpha(s), and adenylyl cyclase (AC)5/6 from buoyant fractions, decreasing AC5/6 and tyrosine-phosphorylated caveolin-1 in caveolin-1 immunoprecipitates but in parallel increased isoproterenol (beta-AR agonist)-stimulated cAMP production. Incubation with Colch decreased co-localization (by immunofluorescence microscopy) of caveolin-3 and alpha-tubulin; both Colch and CD decreased co-localization of caveolin-3 and filamin (an F-actin cross-linking protein), decreased phosphorylation of caveolin-1, Src, and p38 MAPK, and reduced the number of caveolae/mum of sarcolemma (determined by electron microscopy). Treatment of S49 T-lymphoma cells (which possess lipid rafts but lack caveolae) with CD or Colch redistributed a lipid raft marker (linker for activation of T cells (LAT)) and Galpha(s) from lipid raft domains. We conclude that microtubules and actin filaments restrict cAMP formation by regulating the localization and interaction of GPCR-G(s)-AC in lipid rafts/caveolae.     

Tyrosine-phosphorylated caveolin-1: immunolocalization and molecular characterization

Caveolin-1 was discovered as a major substrate for v-Src, but the effect of its tyrosine phosphorylation has not been known. We generated a specific antibody (PY14) to caveolin-1 phosphorylated at tyrosine 14 and studied the significance of the modification. By Western blotting of lysates of v-Src-expressing cells, PY14 recognized not only a 22-kDa band (the position of nonphosphorylated caveolin-1) but bands at 23-24 and 25 kDa. Bands of slower mobility were diminished by dephosphorylation and were also observed for mutant caveolin-1 lacking tyrosine 14. By immunofluorescence microscopy, PY14 did not label normal cells but detected large dots in v-Src-expressing cells. Immunoelectron microscopy revealed that the dots corresponded to aggregated caveolae and/or vesicles of various sizes; besides, the label was observed in intramembrane particle-free areas in the plasma membrane, which appeared to have been formed by fusion of flattened caveolae. A positive reaction with PY14 was found in normal cells after vanadate or pervanadate treatment; it occurred mainly at 22 kDa by Western blotting and was not seen as large dots by immunofluorescence microscopy. Detergent solubility, oligomerization, and association with caveolin-2 were observed similarly for caveolin-1 in normal and v-Src-expressing cells. The results indicate that phosphorylation of caveolin-1 in v-Src-expressing cells occurs at multiple residues and induces flattening, aggregation, and fusion of caveolae and/or caveolae-derived vesicles.     

Triacylglycerol is synthesized in a specific subclass of caveolae in primary adipocytes

A principal metabolic function of adipocytes is to synthesize triacylglycerol (TG) from exogenous fatty acids. The level of fatty acids has to be tightly controlled in the adipocyte, as they can act as detergents that rapidly dissolve the plasma membrane, causing cell lysis if allowed to accumulate. Fatty acids therefore have to be efficiently converted to TG and stored in the central lipid droplet. We report that in intact primary adipocytes exogenous oleic acid was taken up and directly converted to TG in the plasma membrane, in a novel subclass of caveolae that specifically contains the protein perilipin. Isolated caveolae catalyzed de novo TG synthesis from oleic acid and glycerol 3-phosphate. Electron microscopy revealed the presence of caveolin and perilipin in caveolae and in lipid-laden bulbs in the plasma membrane, and fluorescence microscopy demonstrated colocalization of fatty acids/TG with caveolin and perilipin at the plasma membrane. A second caveolae fraction was isolated, which lacked perilipin and the triacylglycerol synthesizing enzymes. Both caveolae fractions contained caveolin-1 and the insulin receptor. The findings demonstrate that specific subclasses of caveolae carry out specific functions in cell metabolism. In particular, triacylglycerol is synthesized at the site of fatty acid entry in one of these caveolae classes.     

N-terminal processing and modifications of caveolin-1 in caveolae from human adipocytes

Caveolin, the principal structural protein of caveolae membrane domains, has a cytosol-exposed N-terminal part that was cleaved off by trypsin treatment of caveolae vesicles isolated from primary human adipocytes. Sequencing of the released tryptic peptides by nanospray quadrupole time-of-flight mass spectrometry revealed that both caveolin-1alpha and caveolin-1beta were processed by excision of the starting methionines. The N-terminus of the mature caveolin-1alpha was acetylated, while caveolin-1beta was found in acetylated as well as in non-acetylated forms. Fractional phosphorylation of serine-36 in the mature caveolin-1alpha and of the homologous serine-5 in caveolin-1beta was identified. This is the first experimental evidence for in vivo phosphorylation of caveolin-1 at the consensus site for phosphorylation by protein kinase C. The phosphorylation was found in both the acetylated and non-acetylated variants of caveolin-1beta. This variability in modifications is consistent with critical involvement of the N-terminal domain of caveolin in the regulation of caveolae.     

Initiation of BMP2 signaling in domains on the plasma membrane

Bone morphogenetic protein 2 (BMP2) is a potent growth factor crucial for cell fate determination. It directs the differentiation of mesenchymal stem cells into osteoblasts, chondrocytes, adipocytes, and myocytes. Initiation of BMP2 signaling pathways occurs at the cell surface through type I and type II serine/threonine kinases housed in specific membrane domains such as caveolae enriched in the caveolin-1 beta isoform (CAV1β, caveolae) and clathrin-coated pits (CCPs). In order for BMP2 to initiate Smad signaling it must bind to its receptors on the plasma membrane resulting in the phosphorylation of the BMP type Ia receptor (BMPRIa) followed by activation of Smad signaling. The current model suggests that the canonical BMP signaling pathway, Smad, occurs in CCPs. However, several recent studies suggested Smad signaling may occur outside of CCPs. Here, we determined; (i) The location of BMP2 binding to receptors localized in caveolae, CCPs, or outside of these domains using AFM and confocal microscopy. (ii) The location of phosphorylation of BMPRIa on the plasma membrane using membrane fractionation, and (iii) the effect of down regulation of caveolae on Smad signaling. Our data indicate that BMP2 binds with highest force to BMP receptors (BMPRs) localized in caveolae. BMPRIa is phosphorylated in caveolae and the disruption of caveolae-inhibited Smad signaling in the presence of BMP2. This suggests caveolae are necessary for the initiation of Smad signaling. We propose an extension of the current model of BMP2 signaling, in which the initiation of Smad signaling is mediated by BMPRs in caveolae.     

p42/p44 MAP kinase activation is localized to caveolae-free membrane domains in airway smooth muscle

Caveolae are abundant plasma membrane invaginations in airway smooth muscle that may function as preorganized signalosomes by sequestering and regulating proteins that control cell proliferation, including receptor tyrosine kinases (RTKs) and their signaling effectors. We previously demonstrated, however, that p42/p44 MAP kinase, a critical effector for cell proliferation, does not colocalize with RTKs in caveolae of quiescent airway myocytes. Therefore, we investigated the subcellular sites of growth factor-induced MAP kinase activation. In quiescent myocytes, though epidermal growth factor receptor (EGFR) was almost exclusively found in caveolae, p42/p44 MAP kinase, Grb2, and Raf-1 were absent from these membrane domains. EGF induced concomitant phosphorylation of caveolin-1 and p42/p44 MAP kinase; however, EGF did not promote the localization of p42/p44 MAP kinase, Grb2, or Raf-1 to caveolae. Interestingly, stimulation of muscarinic M(2) and M(3) receptors that were enriched in caveolae-deficient membranes also induced p42/p44 MAP kinase phosphorylation, but this occurred in the absence of caveolin-1 phosphorylation. This suggests that the localization of receptors to caveolae and interaction with caveolin-1 is not directly required for p42/p44 MAP kinase phosphorylation. Furthermore, we found that EGF exposure induced rapid translocation of EGFR from caveolae to caveolae-free membranes. EGFR trafficking coincided temporally with EGFR and p42/p44 MAP kinase phosphorylation. Collectively, this indicates that although caveolae sequester some receptors associated with p42/p44 MAP kinase activation, the site of its activation is associated with caveolae-free membrane domains. This reveals that directed trafficking of plasma membrane EGFR is an essential element of signal transduction leading to p42/p44 MAP kinase activation.     

Participation of caveolae in beta1 integrin-mediated mechanotransduction

We previously reported that caveolin-1 is a key component in a beta1 integrin-dependent mechanotransduction pathway suggesting that caveolae organelles and integrins are functionally linked in their mechanotransduction properties. Here, we exposed BAEC monolayers to shear stress then isolated caveolae vesicles form the plasma membrane. While little beta1 integrin was detected in caveolae derived from cells kept in static culture, shear stress induced beta1 integrin transposition to the caveolae. To evaluate the significance of shear-induced beta1 integrin localization to caveolae, cells were pretreated with cholesterol sequestering compounds or caveolin-1 siRNA to disrupt caveolae structural domains. Cholesterol depletion attenuated integrin-dependent caveolin-1 phosphorylation, Src activation and Csk association with beta1 integrin. Reduction of both caveolin-1 protein and membrane cholesterol inhibited downstream shear-induced, integrin-dependent phosphorylation of myosin light chain. Taken together with our previous findings, the data supports the concept that beta1 integrin-mediated mechanotransduction is mediated by caveolae domains.     

A critical role of cavin (polymerase I and transcript release factor) in caveolae formation and organization

Cavin (PTRF) has been shown to be a highly abundant protein component of caveolae, but its functional role there is unknown. Here, we confirm that cavin co-localizes with caveolin-1 in adipocytes by confocal microscopy and co-distributes with caveolin-1 in lipid raft fractions by sucrose gradient flotation. However, cavin does not directly associate with caveolin-1 as solubilization of caveolae disrupts their interaction. Cholesterol depletion with beta-cyclodextrin causes a significant down-regulation of cavin from plasma membrane lipid raft fractions. Overexpression of cavin in HEK293-Cav-1 cells and knockdown of cavin in 3T3-L1 adipocytes enhances and diminishes caveolin-1 levels, respectively, indicating an important role for cavin in maintaining the level of caveolin-1. A truncated form of cavin, eGFP-cavin-1-322, which lacks 74 amino acids from the C-terminal, reveals a microtubular network localization by confocal microscopy. Disruption of cytoskeletal elements with latrunculin B or nocodazole diminishes cavin expression without affecting the caveolin-1 amount. We propose that the presence of cavin on the inside surface of caveolae stabilizes these structures, probably through interaction with the cytoskeleton, and cavin therefore plays an important role in caveolae formation and organization.     

Vesicle formation and trafficking in endothelial cells and regulation of endothelial barrier function

Endothelial barrier function is regulated in part by the transcellular transport of albumin and other macromolecules via endothelial caveolae (i.e., this process is defined as transcytosis). Using pulmonary microvascular endothelial cells, we have identified the specific interactions between a cell surface albumin-docking protein gp60 and caveolin-1 as well as components of the signaling machinery, heterotrimeric G protein (G(i))- and Src-family tyrosine kinase. Ligation of gp60 on the apical membrane induces the release of caveolae from the apical membrane and activation of endocytosis. The formed vesicles contain the gp60-bound albumin and also albumin and other solutes present in the fluid phase. Vesicles are transported in a polarized manner to the basolateral membrane, releasing their contents by exocytosis into the subendothelial space. The signaling functions of G(i) and Src are important in the release of caveolae from the plasma membrane. The Src-induced phosphorylation of caveolin-1 is crucial in regulating interactions of caveolin-1 with other components of the signaling machinery such as G(i), and key signaling entry of caveolae into the cytoplasm and endocytosis of albumin and other solutes. This review addresses the basis of transcytosis in endothelial cells, its central role as a determinant of endothelial barrier function, and signaling mechanisms involved in regulating fission of caveolae and trafficking of the formed vesicles from the luminal to abluminal side of the endothelial barrier.     

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

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

Sterol carrier protein-2 directly interacts with caveolin-1 in vitro and in vivo

HDL-mediated reverse-cholesterol transport as well as phosphoinositide signaling are mediated through plasma membrane microdomains termed caveolae/lipid rafts. However, relatively little is known regarding mechanism(s) whereby these lipids traffic to or are targeted to caveolae/lipid rafts. Since sterol carrier protein-2 (SCP-2) binds both cholesterol and phosphatidylinositol, the possibility that SCP-2 might interact with caveolin-1 and caveolae was examined. Double immunolabeling and laser scanning fluorescence microscopy showed that a small but significant portion of SCP-2 colocalized with caveolin-1 primarily at the plasma membrane of L-cells and more so within intracellular punctuate structures in hepatoma cells. In SCP-2 overexpressing L-cells, SCP-2 was detected in close proximity to caveolin, 48 +/- 4 A, as determined by fluorescence resonance energy transfer (FRET) and immunogold electron microscopy. Cell fractionation of SCP-2 overexpressing L-cells and Western blotting detected SCP-2 in purified plasma membranes, especially in caveolae/ lipid rafts as compared to the nonraft fraction. SCP-2 and caveolin-1 were coimmunoprecipitated from cell lysates by anti-caveolin-1 and anti-SCP-2. Finally, a yeast two-hybrid assay demonstrated that SCP-2 directly interacts with caveolin-1 in vivo. These interactions of SCP-2 with caveolin-1 were specific since a functionally related protein, phosphatidyinositol transfer protein (PITP), colocalized much less well with caveolin-1, was not in close proximity to caveolin-1 (i.e., >120 A), and was not coimmunoprecipitated by anti-caveolin-1 from cell lysates. In summary, it was shown for the first time that SCP-2 (but not PITP) selectively interacted with caveolin-1, both within the cytoplasm and at the plasma membrane. These data contribute significantly to our understanding of the role of SCP-2 in cholesterol and phosphatidylinositol targeted from intracellular sites of synthesis in the endoplasmic reticulum to caveolae/lipid rafts at the cell surface plasma membrane.     

Cellular stress induces the tyrosine phosphorylation of caveolin-1 (Tyr(14)) via activation of p38 mitogen-activated protein kinase and c-Src kinase. Evidence for caveolae, the actin cytoskeleton, and focal adhesions as mechanical sensors of osmotic stress

Environmental stressors have been recently shown to activate intracellular mitogen-activated protein (MAP) kinases, such as p38 MAP kinase, leading to changes in cellular functioning. However, little is known about the downstream elements in these signaling cascades. In this study, we show that caveolin-1 is phosphorylated on tyrosine 14 in NIH 3T3 cells after stimulation with a variety of cellular stressors (i.e. high osmolarity, H2O2, and UV light). To detect this phosphorylation event, we employed a phosphospecific monoclonal antibody probe that recognizes only tyrosine 14-phosphorylated caveolin-1. Since p38 MAP kinase and c-Src have been previously implicated in the stress response, we next assessed their role in the tyrosine phosphorylation of caveolin-1. Interestingly, we show that the p38 inhibitor (SB203580) and a dominant-negative mutant of c-Src (SRC-RF) both block the stress-induced tyrosine phosphorylation of caveolin-1 (Tyr(P)(14)). In contrast, inhibition of the p42/44 MAP kinase cascade did not affect the tyrosine phosphorylation of caveolin-1. These results indicate that extracellular stressors can induce caveolin-1 tyrosine phosphorylation through the activation of well established upstream elements, such as p38 MAP kinase and c-Src kinase. However, heat shock did not promote the tyrosine phosphorylation of caveolin-1 and did not activate p38 MAP kinase. Finally, we show that after hyperosmotic shock, tyrosine-phosphorylated caveolin-1 is localized near focal adhesions, the major sites of tyrosine kinase signaling. In accordance with this localization, disruption of the actin cytoskeleton dramatically potentiates the tyrosine phosphorylation of caveolin-1. Taken together, our results clearly define a novel signaling pathway, involving p38 MAP kinase activation and caveolin-1 (Tyr(P)(14)). Thus, tyrosine phosphorylation of caveolin-1 may represent an important downstream element in the signal transduction cascades activated by cellular stress.