RESUMO
IL-22 is a cytokine that plays a pivotal role in regulating tissue homeostasis at barrier surfaces and is produced by activated CD4+ Th cells. Currently, the molecular mechanisms regulating Il22 gene expression are still unclear. In this study, we have identified a crucial cis-regulatory element located 32 kb upstream of the mouse Il22 promoter, termed conserved noncoding sequence (CNS)-32. We demonstrated that CNS-32 acts as an enhancer in reporter assays and contains binding motifs for Runt-related transcription factor (Runx)1 and retinoic acid-related orphan receptor γt (RORγt). Mutation of these motifs significantly abrogated the reporter activity, suggesting a role for both factors in the control of enhancer-mediated Il22 expression. Runx1 and RORγt occupancy and elevated histone H4 acetylation at CNS-32 were evident, as naive T cells differentiated into IL-22-producing Th22 cells. Overexpression of Runx1 promoted IL-22 production by inducing RORγt and IL-23 receptor, all critical to Th22 cell induction. Although Runx1 alone enhanced IL-22 production in Th22 cells, it was further enhanced in the presence of RORγt. Conversely, short hairpin RNA-mediated knockdown of core-binding factor ß, a cofactor essential for Runx1 activity, was effective in limiting IL-22 production. Collectively, our results suggest that IL-22 production is controlled by a regulatory circuit in which Runx1 induces RORγt and then partners with RORγt to direct Il22 expression through their targeting of the Il22 enhancer.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Interleucinas/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Motivos de Aminoácidos/genética , Animais , Diferenciação Celular , Células Cultivadas , Sequência Conservada/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Elementos Facilitadores Genéticos/genética , Interleucinas/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutação/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Ligação Proteica , RNA Interferente Pequeno/genética , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Regulação para Cima , Interleucina 22RESUMO
The complement system, a part of the innate immune system, can be activated via three different pathways. In the alternative pathway, a factor D (FD) plays essential roles in both the initiation and the amplification loop and circulates as an active form. Mannose-binding lectin-associated serine proteases (MASPs) are key enzymes of the lectin pathway, and MASP-1 and/or MASP-3 are reported to be involved in the activation of FD. In the current study, we generated mice monospecifically deficient for MASP-1 or MASP-3 and found that the sera of the MASP-1-deficient mice lacked lectin pathway activity, but those of the MASP-3-deficient mice lacked alternative pathway activity with a zymogen FD. Furthermore, the results indicate that MASP-3 but not MASP-1 activates the zymogen FD under physiological conditions and MASP-3 circulates predominantly as an active form. Therefore, our study illustrates that, in mice, MASP-3 orchestrates the overall complement reaction through the activation of FD.
Assuntos
Fator D do Complemento/imunologia , Via Alternativa do Complemento/imunologia , Proteínas do Sistema Complemento/imunologia , Serina Proteases Associadas a Proteína de Ligação a Manose/imunologia , Animais , Ativação do Complemento/imunologia , Lectina de Ligação a Manose da Via do Complemento/imunologia , Feminino , Sistema Imunitário/imunologia , Lectinas/imunologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
How cell type-specific differences in chromatin conformation are achieved and their contribution to gene expression are incompletely understood. Here we identify a cryptic upstream orchestrator of interferon-gamma (IFNG) transcription, which is embedded within the human IL26 gene, compromised of a single CCCTC-binding factor (CTCF) binding site and retained in all mammals, even surviving near-complete evolutionary deletion of the equivalent gene encoding IL-26 in rodents. CTCF and cohesins occupy this element in vivo in a cell type-nonspecific manner. This element is juxtaposed to two other sites located within the first intron and downstream of Ifng, where CTCF, cohesins, and the transcription factor T-bet bind in a T helper 1 (Th1) cell-specific manner. These interactions, close proximity of other elements within the locus to each other and to the gene encoding interferon-gamma, and robust murine Ifng expression are dependent on CTCF and T-bet. The results demonstrate that cooperation between architectural (CTCF) and transcriptional enhancing (T-bet) factors and the elements to which they bind is required for proper Th1 cell-specific expression of Ifng.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Interferon gama/metabolismo , Proteínas Repressoras/metabolismo , Proteínas com Domínio T/metabolismo , Células Th1/imunologia , Animais , Fator de Ligação a CCCTC , Linfócitos T CD4-Positivos/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/imunologia , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Cromatina/imunologia , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/imunologia , Proteínas Cromossômicas não Histona/metabolismo , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucinas/genética , Interleucinas/imunologia , Interleucinas/metabolismo , Íntrons/genética , Íntrons/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ratos , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/imunologia , Células Th1/metabolismo , CoesinasRESUMO
The complement system plays an important role in host defense and is activated via three different activation pathways. We have previously reported that mannose-binding lectin-associated serine protease (MASP)-3, unlike its splicing variant MASP-1, circulates in an active form and is essential for the activation of the alternative pathway (AP) via the activation of complement factor D (FD). On the other hand, like MASP-1 and MASP-2 of the lectin pathway (LP), MASP-3 forms a complex with the pattern recognition molecules (PRMs) of the LP (LP-PRMs). Both MASP-1 and MASP-2 can be activated efficiently when the LP-PRMs complexed with them bind to their ligands. On the other hand, it remains unclear how MASP-3 is activated, or whether complex formation of MASP-3 with LP-PRMs is involved in activation of MASP-3 or its efficiency in the circulation. To address these issues, we generated wild-type (WT) and four mutant recombinant mouse MASP-3 proteins fused with PA (human podoplanin dodecapeptide)-tag (rmMASP-3-PAs), the latter of which have single amino acid substitution for alanine in the CUB1 or CUB2 domain responsible for binding to LP-PRMs. The mutant rmMASP-3-PAs showed significantly reduced in-vivo complex formation with LP-PRMs when compared with WT rmMASP-3-PA. In the in-vivo kinetic analysis of MASP-3 activation, both WT and mutant rmMASP-3-PAs were cleaved into the active forms as early as 30 minutes in the circulation of mice, and no significant difference in the efficiency of MASP-3 cleavage was observed throughout an observation period of 48 hours after intravenous administration. All sera collected 3 hours after administration of each rmMASP-3-PA showed full restoration of the active FD and AP activity in MASP-3-deficient mouse sera at the same levels as WT mouse sera. Unexpectedly, all mutant rmMASP-3-PAs showed faster clearance from the circulation than the WT rmMASP-3-PA. To our knowledge, the current study is the first to show in-vivo kinetics of MASP-3 demonstrating rapid activation and clearance in the circulation. In conclusion, our results demonstrated that the complex formation of MASP-3 with LP-PRMs is not required for in-vivo activation of MASP-3 or its efficiency, but may contribute to the long-term retention of MASP-3 in the circulation.
Assuntos
Lectina de Ligação a Manose da Via do Complemento , Serina Proteases Associadas a Proteína de Ligação a Manose , Animais , Lectina de Ligação a Manose da Via do Complemento/fisiologia , Proteínas do Sistema Complemento , Humanos , Cinética , Lectinas/genética , Lectinas/metabolismo , Serina Proteases Associadas a Proteína de Ligação a Manose/genética , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Camundongos , Mutação , Proteínas Recombinantes/metabolismoRESUMO
Vimentin exhibits a complex pattern of tissue-specific and developmentally regulated expression, but the mechanisms underlying the complex transcriptional regulation remain poorly understood. Here we examined whether vimentin expression can be regulated by CpG methylation of the vimentin promoter. Two subclones of the rat C6 glioma cells were established with (C6vim+) and without (C6vim-) vimentin. Bisulfite genomic sequencing revealed that the vicinity of the transcription start site within the vimentin promoter is highly methylated in C6vim- cells but not in C6vim+ cells. Treatment of C6vim- cells with a demethylating agent, 5-aza-2'-deoxycytidine, restored vimentin expression, indicating that hypermethylation of the promoter region correlates with transcriptional silencing of the vimentin gene. Electrophoretic mobility shift assay (EMSA) and transient transfection experiments demonstrated that YY1 is a key transcriptional activator regulating vimentin expression and that CpG methylation is sufficient to prevent the binding of YY1 to the vimentin promoter. These data suggest that the inability of YY1 to access the hypermethylated promoter may be one of the mechanisms that mediate vimentin downregulation.
Assuntos
Ilhas de CpG , Metilação de DNA , Ativação Transcricional , Vimentina/genética , Fator de Transcrição YY1/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Regulação para Baixo , Ensaio de Desvio de Mobilidade Eletroforética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ratos , Fator de Transcrição YY1/genéticaRESUMO
Newly synthesized secretory proteins are released into the lumen of the endoplasmic reticulum (ER). The secretory proteins are surrounded by coat protein complex II (COPII) vesicles, and transported from the ER and reach their destinations through the Golgi apparatus. Sec12p is a guanine nucleotide exchange factor for Sar1p, which initiates COPII vesicle budding from the ER. The activation of Sar1p by Sec12p and the subsequent COPII coat assembly have been well characterized, but the events that take place upstream of Sec12p remain unclear. In this study, we isolated the novel extragenic suppressor of sec12-4, PIN4/MDT1, a cell cycle checkpoint target. A yeast two-hybrid screening was used to identify Pin4/Mdt1p as a binding partner of the casein kinase I isoform Hrr25p, which we have previously identified as a modulator of Sec12p function. Deletion of PIN4 suppressed both defects of temperature-sensitive growth and the partial protein transport observed in sec12-4 mutants. The results of this study suggest that Pin4p provides novel aspects of Sec12p modulations.
Assuntos
Mutação , Transporte Proteico/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transporte Biológico , Caseína Quinase I/genética , Retículo Endoplasmático/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
MBD2 is a member of the methyl-CpG-binding protein family that plays an important role in methylated DNA silencing. We have recently identified a novel zinc finger protein, MIZF, as an MBD2-binding partner. To understand the physiological function of MIZF in MBD2-mediated gene silencing, we investigated the DNA-binding properties of MIZF and its potential target genes. Using a cyclic amplification and selection of targets technique, the consensus sequence CGGACGTT, which contains a conserved CGGAC core, was determined as sufficient for MIZF binding. Deletion of individual zinc fingers revealed that five of the seven zinc fingers are required for DNA binding. Reporter assays demonstrated that MIZF represses transcription from the promoter including this DNA sequence. A database search indicated that a variety of human genes, including Rb, contain this sequence in their promoter region. MIZF actually bound to its recognition sequence within the Rb promoter and repressed Rb transcription. These results suggest that MIZF, through its DNA-binding activity, acts as a sequence-specific transcriptional repressor likely involved in MBD2-mediated epigenetic gene silencing.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Inativação Gênica , Proteínas Repressoras/metabolismo , Elementos de Resposta/genética , Transcrição Gênica/genética , Dedos de Zinco , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/química , Linhagem Celular , Sequência Consenso/genética , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/química , Genes do Retinoblastoma/genética , Humanos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Repressoras/química , Especificidade por SubstratoRESUMO
BACKGROUND: We recently demonstrated that phospholipase C (PLC)-delta1 activity in cultured skin fibroblasts obtained from patients with coronary spastic angina (CSA) is enhanced. We tested the hypothesis that structural abnormality in PLC-delta1 isoform is a cause of the enhanced activity. METHODS AND RESULTS: Sequence analysis of the cDNA coding for PLC-delta1 obtained from fibroblasts revealed that one conversion of guanine to adenine (A) was present at nucleotide position 864 in one CSA patient, resulting in the amino acid replacement of arginine 257 by histidine (R257H). The incidence of 864A/A in genomic DNA, analyzed by single-strand conformation polymorphism, was greater in patients with CSA than in male control subjects (6 of 57 patients with CSA versus 1 of 62 control subjects, P<0.05). The activity of the variant PLC-delta1 protein under free calcium concentration between 10(-8) and 10(-7) mol/L was 2-fold higher than that of the wild-type protein. Baseline intracellular calcium concentration ([Ca2+]i) in human embryonic kidney 293 cells transfected with the variant PLC-delta1 was higher than that in cells with the wild type. The peak increase in [Ca2+]i in response to acetylcholine at 10(-6) and 10(-5) mol/L was greater in the cells with the variant PLC-delta1 than in those with the wild type. CONCLUSIONS: These findings indicate that the R257H variant in the PLC-delta1 gene detected in patients with CSA is associated with enhancement of enzyme activity, and they describe a novel mechanism for the enhanced coronary vasomotility in CSA.
Assuntos
Vasoespasmo Coronário/enzimologia , Isoenzimas/genética , Isoenzimas/metabolismo , Polimorfismo de Nucleotídeo Único , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo , Acetilcolina/farmacologia , Cálcio/metabolismo , Linhagem Celular , Células Cultivadas , Vasoespasmo Coronário/genética , DNA/análise , Ativação Enzimática , Feminino , Fibroblastos/enzimologia , Humanos , Masculino , Pessoa de Meia-Idade , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipase C delta , RNA Mensageiro/análise , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Naive CD4(+) T cells give rise to T-helper-cell subsets with functions that are tailored to their respective roles in host defence. The specification of T-helper-cell subsets is controlled by networks of lineage-specifying transcription factors, which bind to regulatory elements in genes that encode cytokines and other transcription factors. The nuclear context in which these transcription factors act is affected by epigenetic processes, which allow programmes of gene expression to be inherited by progeny cells that at the same time retain the potential for change in response to altered environmental signals. In this Review, we describe these epigenetic processes and discuss how they collaborate to govern the fate and function of T helper cells.
Assuntos
Epigênese Genética , Linfopoese/genética , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Cromatina/química , Cromatina/fisiologia , Metilação de DNA , Histonas/metabolismo , Humanos , Transcrição GênicaRESUMO
Unlike the well defined T helper type 2 cytokine locus, little is known about the regulatory elements that govern the expression of Ifng, which encodes the 'signature' T helper type 1 cytokine interferon-gamma. Here our evolutionary analysis showed that the mouse Ifng locus diverged from the ancestral locus as a result of structural rearrangements producing deletion of the neighboring gene encoding interleukin 26 and disrupting synteny 57 kilobases upstream of Ifng. Proximal to that disruption, we identified by high-resolution mapping many regions with CD4+ T cell subset-specific epigenetic modifications. A subset of those regions represented enhancers, whereas others blocked the activity of upstream enhancers or insulated Ifng from neighboring chromatin. Our findings suggest that proper expression of Ifng is maintained through the collective action of multiple distal regulatory elements present in a region of about 100 kilobases flanking Ifng.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Epigênese Genética/imunologia , Interferon gama/genética , Elementos Reguladores de Transcrição/imunologia , Animais , Linhagem Celular , Sequência Conservada , Metilação de DNA , Perfilação da Expressão Gênica , Humanos , Interferon gama/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transcrição Gênica/imunologia , Células Tumorais CultivadasRESUMO
Coreceptor expression is tightly regulated during thymocyte development. Deletion of specific Cd8 enhancers leads to variegated expression of CD8alphabeta heterodimers in double-positive thymocytes. Here we show CD8 variegation is correlated with an epigenetic 'off' state, linking Cd8 enhancer function with chromatin remodeling of the adjacent genes Cd8a and Cd8b1 (Cd8). The zinc finger protein MAZR bound the Cd8 enhancer and interacted with the nuclear receptor corepressor N-CoR complex in double-negative thymocytes. MAZR was downregulated in double-positive and CD8 single-positive thymocytes. 'Enforced' expression of MAZR led to impaired Cd8 activation and variegated CD8 expression. Our results demonstrate epigenetic control of the Cd8 loci and identify MAZR as an important regulator of Cd8 expression.
Assuntos
Antígenos CD8/biossíntese , Proteínas de Neoplasias/metabolismo , Proteínas Repressoras/metabolismo , Linfócitos T/imunologia , Animais , Antígenos CD8/genética , Antígenos CD8/imunologia , Cromatina/imunologia , Metilação de DNA , Regulação para Baixo , Ensaio de Desvio de Mobilidade Eletroforética , Elementos Facilitadores Genéticos/imunologia , Epigênese Genética , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Linfócitos T/citologia , Transcrição GênicaRESUMO
Hyperplasia of synovial lining cells is one of the main features of rheumatoid arthritis (RA). We previously reported that ERBB2 is highly expressed in RA synovial cells and that it plays an important role in their hyperproliferative growth. Recent findings have suggested that poly(ADP-ribose) polymerase-1 (PARP-1) is involved in the transactivation of NF-kappaB-dependent genes such as ERBB2. In the present study, we investigated the role of PARP-1 in ERBB2 transcription in RA synovial cells. The expression level of PARP-1 was significantly high in synovial cells derived from three patients with RA, compared with three patients with osteoarthritis (OA). Luciferase assays revealed that PARP-1 augments the transcription of the ERBB2 gene and that a region between -404 and -368 is responsible for this activation. A protein with an apparent molecular mass of 115 kDa was isolated mainly from nuclear extracts of RA synovial cells with an affinity matrix harboring a DNA fragment identical to the above region. Mass spectrometric analysis demonstrated this protein to be PARP-1. Southwestern blot analysis showed that PARP-1 binds to this region, but not to adjacent regions. PARP-1 associates directly with NF-kappaB, and a chromatin immunoprecipitation assay indicated that these proteins interact with this enhancer region in the ERBB2 gene. Treatment of RA synovial cells with PARP-1 small interfering RNA attenuated their ERBB2 expression, while an inhibitor of the polymerase activity of PARP-1 had no effect. PARP-1 DNA binding is not required for transcriptional activation. These findings suggest that PARP-1 is involved in the expression of ERBB2 in concert with NF-kappaB, which might be associated with the proliferation of RA synovial cells.
Assuntos
Artrite Reumatoide/metabolismo , Poli(ADP-Ribose) Polimerases/fisiologia , Receptor ErbB-2/metabolismo , Membrana Sinovial/metabolismo , Artrite Reumatoide/patologia , Sequência de Bases , Células Cultivadas , DNA/metabolismo , Elementos Facilitadores Genéticos , Humanos , Dados de Sequência Molecular , NF-kappa B/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Poli(ADP-Ribose) Polimerase-1 , Receptor ErbB-2/genética , Membrana Sinovial/patologia , Transcrição GênicaRESUMO
The induction of Rb gene expression is a key event in the process of myogenic differentiation. We recently demonstrated that transcription of the Rb gene is repressed by MIZF, previously characterized as an MBD2-binding partner. Here we show the new roles of MIZF as a regulator of myogenic differentiation. MIZF mRNA was detected in undifferentiated C2C12 myoblasts but its expression decreased during myogenesis, correlating with an increase in Rb mRNA. To examine the function of MIZF in regulating myogenic differentiation, we transduced C2C12 myoblasts with adenoviral vectors to constitutively produce MIZF at high levels. When switched to differentiation medium, these cells showed decreased expression of Rb as well as differentiation markers such as myogenin and Troponin-T, and consequently could not differentiate into multinucleated myotubes. These results suggest that transcriptional repression of Rb by MIZF could be one of the critical determinants in myogenic differentiation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Mioblastos/citologia , Mioblastos/fisiologia , Proteínas Repressoras/metabolismo , Proteína do Retinoblastoma/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Humanos , Camundongos , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteína do Retinoblastoma/genética , Dedos de Zinco/fisiologiaRESUMO
It is generally believed that active invasion by cancer cells is essential to the metastatic process. In this report, we describe a murine mammary tumor (MCH66) model of metastasis that does not require invasion into the vascular wall of both the primary tumor and the target organ, in this case, the lung. The process involves intravasation of tumor nests surrounded by sinusoidal blood vessels, followed by intravascular tumor growth in the lung, without penetration of the vascular wall during the process. Comparative studies using a nonmetastatic MCH66 clone (MCH66C8) and another highly invasive metastatic cell line (MCH416) suggested that high angiogenic activity and sinusoidal remodeling of tumor blood vessels were prerequisites for MCH66 metastasis. Differential cDNA analysis identified several genes that were overexpressed by MCH66, including genes for the angiogenesis factor pleiotrophin, and extracellular matrix-associated molecules that may modulate the microenvironment toward neovascularization. Our analyses suggest that tumor angiogenesis plays a role in the induction of invasion-independent metastasis. This model should prove useful in screening and development of new therapeutic agents for cancer metastasis.
Assuntos
Neoplasias Mamárias Experimentais/patologia , Metástase Neoplásica/patologia , Animais , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Citocinas/biossíntese , Citocinas/genética , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/genética , Feminino , Gelatinases/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Invasividade Neoplásica , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
A GTPase activating protein (GAP), p122, has previously been cloned as a phospholipase C (PLC)delta1-interacting protein. p122 shows a specific GAP activity for Rho and enhances the enzyme activity of PLCdelta1. In this study, we examined the localization and functions of p122/RhoGAP, using enhanced green fluorescent protein (EGFP)-tagged proteins. EGFP-p122 was observed as punctate structures at the plasma membrane of BHK (fibroblastic) cells and MDCK (epithelial) cells. This patchy distribution depended on membrane cholesterol levels and the C-terminal region of p122 containing the GAP domain was responsible for it. Sucrose density gradient centrifugation and immunostaining of caveolin-1 revealed that p122 was localized in caveolin-enriched membrane domains mainly via its GAP domain. We demonstrated that transient expression of EGFP-p122 caused internalization of caveolin-1. Moreover, when the EGFP-tagged GAP domain was introduced in another fibroblastic cell line, NRK cells, punctate fluorescent structures were co-localized with caveolin-1. In this case, caveolin-1-positive structures were found in patches of F-actin, unlike those of untransfected cells that formed linear arrays along with actin stress fibres. These results suggest that p122 is localized in caveolae and plays an important role in caveolin distribution through reorganization of the actin cytoskeleton.