Biomedical subjects

Hyangkyu Lee

Publications and source records attributed to Hyangkyu Lee.

16 recordsLinked to original sources

PubMed2006

IFN-gamma upregulates expression of the mouse complement C1rA gene in keratinocytes via IFN-regulatory factor-1.

We examined the expression of the mouse complement component C1rA (mC1rA) in IFN-gamma-stimulated mouse keratinocytes (Pam 212) and found that it was upregulated. To analyze the mechanism involved, we cloned the 2,150 bp 5'-flanking region of mC1rA by the vectorette-PCR technique, and identified the transcription start site of mC1rA by rapid amplification of complementary DNA ends. Analysis of the 5' sequence revealed putative binding sites for activator protein 1, CCAAT/enhancer binding protein (C/EBP), signal transducer and activator of transcription 1 (STAT-1), IFN-regulatory factor-1 (IRF-1), and others. We detected transcriptional activation dependent on this upstream region in reporter gene assays and Northern blots. To identify the cis-acting regulatory elements involved, we analyzed serial deletion constructs of the promoter using luciferase reporters. The -80 to -19 bp region, which contains a putative IRF-1 binding site, was required for both basal promoter activity and responses to IFN-gamma. The use of site-directed point mutations, electrophoresis mobility shift assays, and supershift assays indicated that the putative IRF-1 binding site was essential for both IFN-gamma-dependent and -independent transcriptional activity of the mC1rA promoter. We conclude that IFN-gamma stimulates mC1rA gene expression via IRF-1 in mouse keratinocytes.

Animals↗

PubMed2006

Identification of phosphocaveolin-1 as a novel protein tyrosine phosphatase 1B substrate.

Protein tyrosine phosphatase 1B (PTP1B) is implicated in a number of signaling pathways including those mediated by insulin, epidermal growth factor (EGF), and the Src family kinases. The scaffolding protein caveolin-1 is also a participant in these pathways and is specifically phosphorylated on tyrosine 14, when these pathways are activated. Here, we provide evidence that PTP1B can efficiently catalyze the removal of the phosphoryl group from phosphocaveolin-1. Overexpression of PTP1B decreases tyrosine 14 phosphorylation in caveolin-1, while expression of the substrate-trapping mutant PTP1B/D181A causes the accumulation of phosphocaveolin-1 and prevents its dephosphorylation by endogenous PTPs. We further demonstrate that PTP1B physically associates with caveolin-1. Finally, we show that inhibition of PTP1B activity with a potent and specific small molecule PTP1B inhibitor blocks the PTP1B-catalyzed caveolin-1 dephosphorylation both in vitro and in vivo. Taken together, the results strongly suggest that caveolin-1 is a specific substrate for PTP1B. Identification of caveolin-1 as a PTP1B substrate represents an important new step in further understanding the signaling pathways regulated by PTP1B.

Animals↗

PubMed2006

Stromal and epithelial caveolin-1 both confer a protective effect against mammary hyperplasia and tumorigenesis: Caveolin-1 antagonizes cyclin D1 function in mammary epithelial cells.

Here, we investigate the role of caveolin-1 (Cav-1) in breast cancer onset and progression, with a focus on epithelial-stromal interactions, ie, the tumor microenvironment. Cav-1 is highly expressed in adipocytes and is abundant in mammary fat pads (stroma), but it remains unknown whether loss of Cav-1 within mammary stromal cells affects the differentiated state of mammary epithelia via paracrine signaling. To address this issue, we characterized the development of the mammary ductal system in Cav-1-/- mice and performed a series of mammary transplant studies, using both wild-type and Cav-1-/- mammary fat pads. Cav-1-/- mammary epithelia were hyperproliferative in vivo, with dramatic increases in terminal end bud area and mammary ductal thickness as well as increases in bromodeoxyuridine incorporation, extracellular signal-regulated kinase-1/2 hyperactivation, and up-regulation of STAT5a and cyclin D1. Consistent with these findings, loss of Cav-1 dramatically exacerbated mammary lobulo-alveolar hyperplasia in cyclin D1 Tg mice, whereas overexpression of Cav-1 caused reversion of this phenotype. Most importantly, Cav-1-/- mammary stromal cells (fat pads) promoted the growth of both normal mammary ductal epithelia and mammary tumor cells. Thus, Cav-1 expression in both epithelial and stromal cells provides a protective effect against mammary hyperplasia as well as mammary tumorigenesis.

Adipose Tissue↗

PubMed2005

Adipocyte-derived collagen VI affects early mammary tumor progression in vivo, demonstrating a critical interaction in the tumor/stroma microenvironment.

The interactions of transformed cells with the surrounding stromal cells are of importance for tumor progression and metastasis. The relevance of adipocyte-derived factors to breast cancer cell survival and growth is well established. However, it remains unknown which specific adipocyte-derived factors are most critical in this process. Collagen VI is abundantly expressed in adipocytes. Collagen(-/-) mice in the background of the mouse mammary tumor virus/polyoma virus middle T oncogene (MMTV-PyMT) mammary cancer model demonstrate dramatically reduced rates of early hyperplasia and primary tumor growth. Collagen VI promotes its growth-stimulatory and pro-survival effects in part by signaling through the NG2/chondroitin sulfate proteoglycan receptor expressed on the surface of malignant ductal epithelial cells to sequentially activate Akt and beta-catenin and stabilize cyclin D1. Levels of the carboxyterminal domain of collagen VIalpha3, a proteolytic product of the full-length molecule, are dramatically upregulated in murine and human breast cancer lesions. The same fragment exerts potent growth-stimulatory effects on MCF-7 cells in vitro. Therefore, adipocytes play a vital role in defining the ECM environment for normal and tumor-derived ductal epithelial cells and contribute significantly to tumor growth at early stages through secretion and processing of collagen VI.

Adipocytes↗

PubMed2005

Type VII collagen is required for Ras-driven human epidermal tumorigenesis.

Type VII collagen defects cause recessive dystrophic epidermolysis bullosa (RDEB), a blistering skin disorder often accompanied by epidermal cancers. To study the role of collagen VII in these cancers, we examined Ras-driven tumorigenesis in RDEB keratinocytes. Cells devoid of collagen VII did not form tumors in mice, whereas those retaining a specific collagen VII fragment (the amino-terminal noncollagenous domain NC1) were tumorigenic. Forced NC1 expression restored tumorigenicity to collagen VII-null epidermis in a non-cell-autonomous fashion. Fibronectin-like sequences within NC1 (FNC1) promoted tumor cell invasion in a laminin 5-dependent manner and were required for tumorigenesis. Tumor-stroma interactions mediated by collagen VII thus promote neoplasia, and retention of NC1 sequences in a subset of RDEB patients may contribute to their increased susceptibility to squamous cell carcinoma.

Adolescent↗

PubMed2005

Drosophila peptidoglycan recognition protein LC (PGRP-LC) acts as a signal-transducing innate immune receptor.

Drosophila peptidoglycan recognition protein LC (PGRP-LC), a transmembrane protein required for the response to bacterial infection, acts at the top of a cytoplasmic signaling cascade that requires the death-domain protein Imd and an IkappaB kinase to activate Relish, an NF-kappaB family member. It is not clear how binding of peptidoglycan to the extracellular domain of PGRP-LC activates intracellular signaling because its cytoplasmic domain has no homology to characterized proteins. Here, we demonstrate that PGRP-LC binds Imd and that its cytoplasmic domain is critical for its activity, suggesting that PGRP-LC acts as a signal-transducing receptor. The PGRP-LC cytoplasmic domain is also essential for the formation of dimers, and results suggest that dimerization may be required for receptor activation. The PGRP-LC cytoplasmic domain can mediate formation of heterodimers between different PGRP-LC isoforms, thereby potentially expanding the diversity of ligands that can be recognized by the receptor.

Animals↗

PubMed2004

Muscle-specific interaction of caveolin isoforms: differential complex formation between caveolins in fibroblastic vs. muscle cells.

It is generally well accepted that caveolin-3 expression is muscle specific, whereas caveolin-1 and -2 are coexpressed in a variety of cell types, including adipocytes, endothelial cells, epithelial cells, and fibroblasts. Caveolin-1 and -2 are known to form functional hetero-oligomeric complexes in cells where they are coexpressed, whereas caveolin-3 forms homo-oligomeric high molecular mass complexes. Although caveolin-2 might be expected to interact in a similar manner with caveolin-3, most studies indicate that this is not the case. However, this view has recently been challenged as it has been demonstrated that caveolin-2 and -3 are coexpressed in primary cultures of cardiac myocytes, where these two proteins can be coimmunoprecipitated. Thus it remains controversial whether caveolin-2 interacts with caveolin-3. Here, we directly address the issue of caveolin isoform protein-protein interactions by means of three distinct molecular genetic approaches. First, using caveolin-1-deficient mouse embryonic fibroblasts, in which we have stably expressed caveolin-1, -2, or -3, we find that caveolin-1 interacts with caveolin-2 in this setting, whereas caveolin-3 does not, in agreement with most published observations. Next, we used a transfected L6 myoblast cell system expressing all three caveolin proteins. Surprisingly, we found that caveolin-1, -2, and -3 all coimmunoprecipitate in this cell type, suggesting that this interaction is muscle cell specific. Similar results were obtained when the skeletal muscle of caveolin-1 transgenic animals was analyzed for caveolin-1 and caveolin-3 coimmunoprecipitation. Thus we conclude that all three caveolins can interact to form a discrete hetero-oligomeric complex, but that such complex formation is clearly muscle specific.

Animals↗

PubMed2004

Tyrosine phosphorylation of caveolin-2 at residue 27: differences in the spatial and temporal behavior of phospho-Cav-2 (pY19 and pY27).

Caveolin-2 is an accessory molecule and the binding partner of caveolin-1. Previously, we showed that c-Src expression leads to the tyrosine phosphorylation of Cav-2 at position 19. To further investigate the tyrosine phosphorylation of Cav-2, we have now generated a novel phospho-specific antibody directed against phospho-Cav-2 (pY27). Here, we show that Cav-2 is phosphorylated at both tyrosines 19 and 27. We reconstituted this phosphorylation event by recombinantly coexpressing c-Src and Cav-2. We generated a series of Cav-2 constructs harboring the mutation of each tyrosine to alanine, singly or in combination, i.e., Cav-2 Y19A, Y27A, and Y19A/Y27A. Recombinant expression of these mutants in Cos-7 cells demonstrated that neither tyrosine is the unique phosphorylation site, and that double mutation of tyrosines 19 and 27 to alanine abrogates Cav-2 tyrosine phosphorylation. Immunofluorescence analysis of NIH 3T3 cells revealed that the two tyrosine-phosphorylated forms of Cav-2 exhibited some distinct properties. Phospho-Cav-2 (pY19) is concentrated at cell edges and at cell-cell contacts, whereas phospho-Cav-2 (pY27) is distributed in a dotlike pattern throughout the cell surface and cytoplasm. Further functional analysis revealed that tyrosine phosphorylation of Cav-2 has no effect on its targeting to lipid rafts, but clearly disrupts the hetero-oligomerization of Cav-2 with Cav-1. In an attempt to identify upstream mediators, we investigated Cav-2 tyrosine phosphorylation in an endogenous setting. We found that in A431 cells, EGF stimulation is sufficient to induce Cav-2 phosphorylation at tyrosines 19 and 27. However, the behavior of the two phosphorylated forms of Cav-2 diverges upon EGF stimulation. First, phospho-Cav-2 (pY19) and phospho-Cav-2 (pY27) display different localization patterns. In addition, the temporal response to EGF stimulation appears to be different. Cav-2 is phosphorylated at tyrosine 19 in a rapid and transient fashion, whereas phosphorylation at tyrosine 27 is sustained over time. Three SH2 domain-containing proteins, c-Src, Nck, and Ras-GAP, were found to associate with Cav-2 in a phosphorylation-dependent manner. However, phosphorylation at tyrosine 27 appears to be more critical than phosphorylation at tyrosine 19 for this binding to occur. Taken together, these results suggest that, in addition to the common characteristics that these two sites appear to share, phospho-Cav-2 (pY19) and phospho-Cav-2 (pY27) may each possess a set of unique functional roles.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Combined loss of INK4a and caveolin-1 synergistically enhances cell proliferation and oncogene-induced tumorigenesis: role of INK4a/CAV-1 in mammary epithelial cell hyperplasia.

Tumorigenesis is a multistep process that involves a series of genetic changes or "multiple hits," leading to alterations in signaling, proliferation, immortalization, and transformation. Many of the molecular factors that govern tumor initiation and progression remain unknown. Here, we evaluate the transformation suppressor potential of caveolin-1 (Cav-1) and its ability to cooperate with a well established tumor suppressor, the INK4a locus. To study the effects of loss of caveolin-1 on cellular transformation, we established immortalized primary mouse embryonic fibroblasts (MEFs) expressing and lacking caveolin-1 by interbreeding Cav-1 (+/+) and Cav-1 (-/-) mice with INK4a (-/-) mice. Analysis of these cells reveals that loss of caveolin-1 confers a significant growth advantage, as measured via cellular proliferation and cell cycle analysis. Loss of caveolin-1 in the INK4a (-/-) genetic background results in constitutive hyperactivation of the p42/44 MAP kinase cascade, decreased expression of p21(Cip1), as well as cyclin D1 and PCNA overexpression, consistent with their hyperproliferative phenotype. Importantly, in cells lacking Cav-1 expression, transformation by activated oncogenes (H-Ras(G12V) or v-Src) results in increased tumor growth in vivo (up to >40-fold). Finally, INK4a (-/-)/Cav-1 (-/-) mice demonstrate disturbed mammary epithelial ductal morphology, with hyperplasia, increased side-branching, and fibrosis. Our results provide important new evidence for the transformation suppressor properties of Cav-1 and the first molecular genetic evidence that Cav-1 cooperates with a tumor suppressor, namely the INK4a genetic locus.

Animals↗

PubMed2003

Caveolin-1 null (-/-) mice show dramatic reductions in life span.

Caveolae are 50-100 nm flask-shaped invaginations of the plasma membrane found in most cell types. Caveolin-1 is the principal protein component of caveolae membranes in nonmuscle cells. The recent development of Cav-1-deficient mice has allowed investigators to study the in vivo functional role of caveolae in the context of a whole animal model, as these mice lack morphologically detectable caveolae membrane domains. Surprisingly, Cav-1 null mice are both viable and fertile. However, it remains unknown whether loss of caveolin-1 significantly affects the overall life span of these animals. To quantitatively determine whether loss of Cav-1 gene expression confers any survival disadvantages with increasing age, we generated a large cohort of mice (n = 180), consisting of Cav-1 wild-type (+/+) (n = 53), Cav-1 heterozygous (+/-) (n = 70), and Cav-1 knockout (-/-) (n = 57) animals, and monitored their long-term survival over a 2 year period. Here, we show that Cav-1 null (-/-) mice exhibit an approximately 50% reduction in life span, with major declines in viability occurring between 27 and 65 weeks of age. However, Cav-1 heterozygous (+/-) mice did not show any changes in long-term survival, indicating that loss of both Cav-1 alleles is required to mediate a reduction in life span. Mechanistically, these dramatic reductions in life span appear to be secondary to a combination of pulmonary fibrosis, pulmonary hypertension, and cardiac hypertrophy in Cav-1 null mice. Taken together, our results provide the first demonstration that loss of Cav-1 gene expression and caveolae organelles dramatically affects the long-term survival of an organism. In addition, aged Cav-1 null mice may provide a new animal model to study the pathogenesis and treatment of progressive hypertrophic cardiomyopathy and sudden cardiac death syndrome.

Aging↗

PubMed2003

Genetic ablation of caveolin-1 confers protection against atherosclerosis.

OBJECTIVE: The development of atherosclerosis is a process characterized by the accumulation of lipids in the form of modified lipoproteins in the subendothelial space. This initiating step is followed by the subsequent recruitment and proliferation of other cell types, including monocytes/macrophages and smooth muscle cells. Here, we evaluate the potential role of caveolae membrane domains in the pathogenesis of atherosclerosis by using apolipoprotein E-deficient (ApoE-/-) mice as a model system. METHODS AND RESULTS: Caveolin-1 (Cav-1) is a principal structural protein component of caveolae membrane domains. To directly assess the in vivo role of caveolae and Cav-1 in atherosclerosis, we interbred Cav-1-/- mice with ApoE-/- mice. Interestingly, loss of Cav-1 resulted in a dramatic >2-fold increase in non-HDL plasma cholesterol levels in the ApoE-/- background. However, despite this hypercholesterolemia, we found that loss of Cav-1 gene expression was clearly protective against the development of aortic atheromas, with up to an approximately 70% reduction in atherosclerotic lesion area. Mechanistically, we demonstrated that loss of Cav-1 resulted in the dramatic downregulation of certain proatherogenic molecules, namely, CD36 and vascular cell adhesion molecule-1. CONCLUSIONS: Taken together, our results indicate that loss of Cav-1 can counteract the detrimental effects of atherogenic lipoproteins. Thus, Cav-1 is a novel target for drug development in the pharmacologic prevention of atheroma formation. Our current data also provide the first molecular genetic evidence to support the hypothesis that caveolar transcytosis of modified lipoproteins (from the blood to the sub-endothelial space) is a critical initiating step in atherosclerosis.

Animals↗

PubMed2003

Caveolin-1 knockout mice show an impaired angiogenic response to exogenous stimuli.

Recent studies have shown that caveolin-1 (Cav-1) plays an important role as a regulator of angiogenesis in vitro. Here, we use Cav-1 knockout (KO) mice as a model system to examine the in vivo relevance of these findings. A primary mediator of angiogenesis is basic fibroblast growth factor (bFGF). Thus, we studied bFGF-induced angiogenesis in Cav-1 KO mice using a reconstituted basement membrane system, ie, Matrigel plugs, supplemented with bFGF. In Cav-1 KO mice, implanted Matrigel plugs showed a dramatic reduction in both vessel infiltration and density, as compared with identical plugs implanted in wild-type control mice. We also examined the necessity of Cav-1 to support the angiogenic response of an exogenous tumor by subcutaneously injecting Cav-1 KO mice with the melanoma cell line, B16-F10. We show that tumor weight, volume, and vessel density are all reduced in Cav-1 KO mice, consistent with diminished angiogenesis. Ultrastructural analysis of newly formed capillaries within the exogenous tumors reveals a lack of endothelial caveolae and incomplete capillary formation in Cav-1 KO mice. These results provide novel evidence that Cav-1 and caveolae play an important positive role in the process of pathological angiogenesis in vivo.

Animals↗

PubMed2002

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.

3T3 Cells↗

PubMed2002

Caveolin-1 mutations (P132L and null) and the pathogenesis of breast cancer: caveolin-1 (P132L) behaves in a dominant-negative manner and caveolin-1 (-/-) null mice show mammary epithelial cell hyperplasia.

Caveolin-1 (Cav-1) is the principal structural protein of caveolae membranes that are found in most cells types, including mammary epithelial cells. Recently, we mapped the human CAV1 gene to a suspected tumor suppressor locus (7q31.1/D7S522) that is deleted in a variety of human cancers, as well as mammary tumors. In addition, the CAV1 gene is mutated (P132L) in up to approximately 16% of human breast cancers. The mechanism by which deletion or mutation of the Cav-1 gene contributes to mammary tumorigenesis remains unknown. To understand the role of the Cav-1 (P132L) mutation in the pathogenesis of human breast cancers, we generated the same mutation in wild-type (WT) Cav-1 and studied its behavior in cultured cells. Interestingly, the P132L mutation leads to formation of misfolded Cav-1 oligomers that are retained within the Golgi complex and are not targeted to caveolae or the plasma membrane. To examine whether the Cav-1 (P132L) mutant behaves in a dominant-negative manner, we next co-transfected cells with Cav-1 (P132L) and WT Cav-1, and evaluated their caveolar targeting. Our results indicate that Cav-1 (P132L) behaves in a dominant-negative manner, causing the mislocalization and intracellular retention of WT Cav-1. Virtually identical results were obtained when Cav-1 (P132L) was stably expressed at physiological levels in a nontransformed human mammary epithelial cell line (hTERT-HME1). These data provide a molecular explanation for why only a single mutated CAV1 allele is found in patients with breast cancer. Thus, we next investigated if functional inactivation of Cav-1 gene expression leads to mammary tumorigenesis in vivo. For this purpose, we performed mammary gland analysis on Cav-1-deficient mice (-/-) that harbor a targeted disruption of the Cav-1 gene (a null mutation). Interestingly, we show that inactivation of Cav-1 gene expression leads to mammary epithelial cell hyperplasia, even in 6-week-old virgin female mice. These data clearly implicate loss of functional Cav-1 in the pathogenesis of mammary epithelial cell hyperplasia, and suggest that Cav-1-null mice represent a novel animal model to study premalignant mammary disease.

Animals↗

PubMed2002

Caveolin-1-deficient mice show accelerated mammary gland development during pregnancy, premature lactation, and hyperactivation of the Jak-2/STAT5a signaling cascade.

It is well established that mammary gland development and lactation are tightly controlled by prolactin signaling. Binding of prolactin to its cognate receptor (Prl-R) leads to activation of the Jak-2 tyrosine kinase and the recruitment/tyrosine phosphorylation of STAT5a. However, the mechanisms for attenuating the Prl-R/Jak-2/STAT5a signaling cascade are just now being elucidated. Here, we present evidence that caveolin-1 functions as a novel suppressor of cytokine signaling in the mammary gland, akin to the SOCS family of proteins. Specifically, we show that caveolin-1 expression blocks prolactin-induced activation of a STAT5a-responsive luciferase reporter in mammary epithelial cells. Furthermore, caveolin-1 expression inhibited prolactin-induced STAT5a tyrosine phosphorylation and DNA binding activity, suggesting that caveolin-1 may negatively regulate the Jak-2 tyrosine kinase. Because the caveolin-scaffolding domain bears a striking resemblance to the SOCS pseudosubstrate domain, we examined whether Jak-2 associates with caveolin-1. In accordance with this homology, we demonstrate that Jak-2 cofractionates and coimmunoprecipitates with caveolin-1. We next tested the in vivo relevance of these findings using female Cav-1 (-/-) null mice. If caveolin-1 normally functions as a suppressor of cytokine signaling in the mammary gland, then Cav-1 null mice should show premature development of the lobuloalveolar compartment because of hyperactivation of the prolactin signaling cascade via disinhibition of Jak-2. In accordance with this prediction, Cav-1 null mice show accelerated development of the lobuloalveolar compartment, premature milk production, and hyperphosphorylation of STAT5a (pY694) at its Jak-2 phosphorylation site. In addition, the Ras-p42/44 MAPK cascade is hyper-activated. Because a similar premature lactation phenotype is observed in SOCS1 (-/-) null mice, we conclude that caveolin-1 is a novel suppressor of cytokine signaling.

Amino Acid Sequence↗

PubMed2002

Intracellular retention of glycosylphosphatidyl inositol-linked proteins in caveolin-deficient cells.

The relationship between glycosylphosphatidyl inositol (GPI)-linked proteins and caveolins remains controversial. Here, we derived fibroblasts from Cav-1 null mouse embryos to study the behavior of GPI-linked proteins in the absence of caveolins. These cells lack morphological caveolae, do not express caveolin-1, and show a approximately 95% down-regulation in caveolin-2 expression; these cells also do not express caveolin-3, a muscle-specific caveolin family member. As such, these caveolin-deficient cells represent an ideal tool to study the role of caveolins in GPI-linked protein sorting. We show that in Cav-1 null cells GPI-linked proteins are preferentially retained in an intracellular compartment that we identify as the Golgi complex. This intracellular pool of GPI-linked proteins is not degraded and remains associated with intracellular lipid rafts as judged by its Triton insolubility. In contrast, GPI-linked proteins are transported to the plasma membrane in wild-type cells, as expected. Furthermore, recombinant expression of caveolin-1 or caveolin-3, but not caveolin-2, in Cav-1 null cells complements this phenotype and restores the cell surface expression of GPI-linked proteins. This is perhaps surprising, as GPI-linked proteins are confined to the exoplasmic leaflet of the membrane, while caveolins are cytoplasmically oriented membrane proteins. As caveolin-1 normally undergoes palmitoylation on three cysteine residues (133, 143, and 156), we speculated that palmitoylation might mechanistically couple caveolin-1 to GPI-linked proteins. In support of this hypothesis, we show that palmitoylation of caveolin-1 on residues 143 and 156, but not residue 133, is required to restore cell surface expression of GPI-linked proteins in this complementation assay. We also show that another lipid raft-associated protein, c-Src, is retained intracellularly in Cav-1 null cells. Thus, Golgi-associated caveolins and caveola-like vesicles could represent part of the transport machinery that is necessary for efficiently moving lipid rafts and their associated proteins from the trans-Golgi to the plasma membrane. In further support of these findings, GPI-linked proteins were also retained intracellularly in tissue samples derived from Cav-1 null mice (i.e., lung endothelial and renal epithelial cells) and Cav-3 null mice (skeletal muscle fibers).

3T3 Cells↗