| 1. |
- Malmberg, Emily, 1978, et al.
(författare)
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Bcr (breakpoint cluster region) protein binds to PDZ-domains of scaffold protein PDZK1 and vesicle coat protein Mint3.
- 2004
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Ingår i: Journal of cell science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 117:Pt 23, s. 5535-41
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Tidskriftsartikel (refereegranskat)abstract
- The breakpoint cluster region protein (Bcr) is a large soluble oligomeric multidomain protein best known because of its involvement in chronic myelogenous leukemia (CML). A chromosomal translocation between its gene and that of the c-abl kinase ('Philadelphia chromosome') plays a major causative role in that malignancy. Thus most attention has been paid to the role of the protein in hemopoietic cells. However, Bcr is also expressed in other cell types including epithelia. Bcr is generally considered to be a cytoplasmic protein but in addition to its kinase and GTPase exchange and activating domains it contains potentially membrane-interacting pleckstrin homology and C2 domains as well as a PDZ-binding C terminus mediating an interaction with a PDZ-domain protein at intercellular junctions of epithelial cells. We have examined the ability of Bcr to interact with other epithelial PDZ proteins and found specific binding to both the apical PDZK1 protein and the Golgi-localized Mint3. The former is important in the organization of several apical functions and the latter in vesicular trafficking in the secretory pathway. Hence these findings extend the interactions and likely signaling impact of Bcr in epithelia from the cytosol to at least these two membrane compartments.
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| 2. |
- Malmberg, Emily, 1978, et al.
(författare)
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Increased levels of mucins in the cystic fibrosis mouse small intestine, and modulator effects of the Muc1 mucin expression.
- 2006
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Ingår i: American journal of physiology. Gastrointestinal and liver physiology. - : American Physiological Society. - 0193-1857 .- 1522-1547. ; 291:2
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Tidskriftsartikel (refereegranskat)abstract
- The mouse model (Cftr(tm1UNC)/Cftr(tm1UNC)) for cystic fibrosis (CF) shows mucus accumulation and increased Muc1 mucin mRNA levels due to altered splicing (Hinojosa-Kurtzberg AM, Johansson MEV, Madsen CS, Hansson GC, and Gendler SJ. Am J Physiol Gastrointest Liver Physiol 284: G853-G862, 2003). However, it is not known whether Muc1 is a major mucin contributing to the increased mucus and why CF/Muc1-/- mice show lower mucus accumulation. To address this, we have purified mucins from the small intestine of CF mice using guanidinium chloride extraction, ultracentrifugation, and gel filtration and analyzed them by slot blot, gel electrophoresis, proteomics, and immunoblotting. Normal and CF mice with wild-type (WT) Muc1 or Muc1-/- or that are transgenic for human MUC1 (MUC1.Tg, on a Muc1-/- background) were analyzed. The total amount of mucins, both soluble and insoluble in guanidinium chloride, increased up to 10-fold in the CF mice compared with non-CF animals, whereas the CF mice lacking Muc1 showed intermediate levels between the CF and non-CF mice. However, the levels of Muc3 (orthologue of human MUC17) were increased in the CF/Muc1-/- mice compared with the CF/MUC1.Tg animals. The amount of MUC1 mucin was increased several magnitudes in the CF mice, but MUC1 did still not appear to be a major mucin. The amount of insoluble mucus of the large intestine was also increased in the CF mice, an effect that was partially restored in the CF/Muc1-/- mice. The thickness of the firmly adherent mucus layer of colon in the Muc1-/- mice was significantly lower than that of WT mice. The results suggest that MUC1 is not a major component in the accumulated mucus of CF mice and that MUC1 can influence the amount of other mucins in a still unknown way.
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| 3. |
- Malmberg, Emily, 1978
(författare)
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Mucins, Cystic Fibrosis and PDZ protein interactions
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cystic Fibrosis (CF) is caused and characterized by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. The dominating feature of the disease is mucus accumulation on mucosal surfaces, but no direct functional connection between mucus and CFTR is known. Mouse models of CF suffer from intestinal mucus accumulation, but do not display the respiratory phenotype that dominates the human disease. Mucins are the major protein components of the mucus layer and they can be subdivided into transmembrane or gel forming. (MUC refers to human, while Muc refers to mouse mucins) We purified guanidinium chloride soluble or insoluble small intestinal mucins from CF mice (Cftrtm1UNC/ Cftrtm1UNC ) and wild type or CF mice that lack expression of the Muc1 transmembrane mucin. We previously suggested that the transmembrane mucin Muc3 is the orthologue of human MUC17 and named the mouse mucin Muc3(17). We demonstrate that the total amount of mucins is increased in CF small intestine. The individual mucins MUC1, Muc2 and Muc3(17) were also increased, with a drastic 1000-fold increase of MUC1. CF-Muc1-/- mice showed the largest amounts of Muc3(17) and in Muc-/- mouse large intestine, mucus thickness was altered. This indicates that Muc1 is able to modulate the biochemical composition and thickness of the mucus layer in the intestine. PDZK1 is a PDZ domain protein known to interact with CFTR. We wanted to identify additional proteins able to bind PDZK1 and study their biological function(s). By performing Glutathione-S Transferase pull down experiments, followed by MALDI-TOF or nanoHPLC mass spectrometry analysis and peptide fingerprinting we could identify MUC17 and the Breakpoint cluster region (Bcr) protein as PDZK1 interacting proteins. By immunofluorescence staining of Muc3(17) on Pdzk1-/- mouse jejunum we detected strong intracellular Muc3(17) staining compared to wild type mice where most of Muc3(17) was located at the apical brush border membrane. Thus, Pdzk1 has a strong influence on the subcellular localization of Muc3(17) in enterocytes, either by acting as an anchor at the apical membrane, or by influencing the apical targeting of the mucin. The facts that PDZK1 is a well known interaction partner of CFTR and that Pdzk1 binds Muc3(17) and stabilizes it at the apical membrane suggest, for the first time a direct connection between CFTR and mucins. The Bcr protein is multifunctional and participates in various signaling pathways. Besides PDZK1 we could identify the vesicle coat protein, Mint-3, as an interaction partner of Bcr. We also discovered two novel motifs in the Bcr amino acid sequence. A clathrin binding box and a consensus sequence for interaction with the AP-2 adapter complex. Both motifs are commonly present in proteins known to participate in clathrin mediated endocytosis. We show that Bcr interacts with both clathrin and á-adaptin and is enriched on clathrin coated vesicles. In BHK-21 cells stably overexpressing Bcr, endocytosis was enhanced and showed a preferred sorting of endocytosed material into lysosomes. It is possible that the interaction with PDZK1 serves as an anchor for Bcr at the plasma membrane where clathrin mediated endocytosis takes place.
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| 4. |
- Malmberg, Emily, 1978, et al.
(författare)
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The C-terminus of the transmembrane mucin MUC17 binds to the scaffold protein PDZK1 that stably localizes it to the enterocyte apical membrane in the small intestine
- 2008
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Ingår i: Biochemical Journal. - 1470-8728. ; 410, s. 283-289
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Tidskriftsartikel (refereegranskat)abstract
- The membrane bound mucins have a heavily O-glycosylated extracellular domain, a single pass membrane domain and a short cytoplasmic tail. Three of the membrane bound mucins, MUC3, MUC12 and MUC17, are clustered on chromosome 7 and found in the gastrointestinal tract. These mucins have C-terminal sequences typical for PDZ domain binding proteins. To identify PDZ proteins able to interact with the mucins, we screened PDZ domain arrays using YFP-tagged proteins. MUC17 exhibited a strong binding to PDZK1 whereas the binding to NHERF1 was weak. Furthermore, we showed weak binding of MUC12 to PDZK1, NHERF1 and NHERF2. GST pull-down experiments confirmed that the C-terminal tail of MUC17 co-precipitates with the scaffold protein PDZK1 as identified by mass spectrometry. This was mediated through the C-terminal PDZ-interaction site in MUC17 which was capable of binding to three of the four PDZ domains in PDZK1. Immunostaining of wild-type or Pdzk1-/- mouse jejunum with an antiserum against Muc3(17), the mouse orthologue of human MUC17, revealed strong brush border membrane staining in the wild-type mice compared to an intracellular Muc3(17) staining in the Pdzk1-/- mice. This suggests that Pdzk1 plays a specific role in stabilizing Muc3(17) in the apical membrane of small intestinal enterocytes.
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