Biomedical subjects

Oddmund Bakke

Publications and source records attributed to Oddmund Bakke.

14 recordsLinked to original sources

PubMed2006

Toll-like receptor 3 associates with c-Src tyrosine kinase on endosomes to initiate antiviral signaling.

Double-stranded RNA (dsRNA) is produced during the replication cycle of most viruses and triggers antiviral immune responses through Toll-like receptor 3 (TLR3). However, the molecular mechanisms and subcellular compartments associated with dsRNA-TLR3-mediated signaling are largely unknown. Here we show that c-Src tyrosine kinase is activated by dsRNA in human monocyte-derived dendritic cells, and is recruited to TLR3 in a dsRNA-dependent manner. DsRNA-induced activation of interferon-regulatory factor 3 and signal transducer and activator of transcription 1 was abolished in Src kinase-deficient cells, and restored by adding back c-Src, suggesting a central role of c-Src in antiviral immunity. We also provide evidence that TLR3 is localized in the endoplasmic reticulum of unstimulated cells, moves to dsRNA-containing endosomes in response to dsRNA, and colocalizes with c-Src on endosomes containing dsRNA in the lumen. These results provide novel insight into the molecular mechanisms of TLR3-mediated signaling, which may contribute to the understanding of innate immune responses during viral infections.

Animals↗

PubMed2006

Re-routing of the invariant chain to the direct sorting pathway by introduction of an AP3-binding motif from LIMP II.

AP3 is a heteromeric adaptor protein complex involved in the biogenesis of late endosomal/lysosomal structures. It recognizes tyrosine- and leucine-based sorting signals present in the cytoplasmic tails or loops of a number of proteins and is thought to be responsible for the direct transport of these proteins from the Golgi network to late endosomal/lysosomal structures. We have previously reported (Rodionov, Höning, Silye, Kongsvik, von Figura, Bakke, 2002. Structural requirements for interactions between leucine-sorting signals and clathrin-associated adaptor protein complex AP3. J. Biol. Chem. 277, 47436-47443) that in vitro binding of AP3 to the leucine signals is dependent on the nature of three residues immediately upstream of the leucine signal and suggested that these three amino acids define whether the protein is sorted to endosomes via the plasma membrane (PM) or traffics directly to the late endosomes/lysosomes. In this paper, we show in vivo evidence that residues favoring AP3 binding introduced into a protein that is transported via the PM such as the invariant chain can re-route such protein into direct sorting to late endosomal/lysosomal structures.

Adaptor Protein Complex 3↗

PubMed2005

Characterization of a novel chemokine-containing storage granule in endothelial cells: evidence for preferential exocytosis mediated by protein kinase A and diacylglycerol.

We have recently shown that several proinflammatory chemokines can be stored in secretory granules of endothelial cells (ECs). Subsequent regulated exocytosis of such chemokines may then enable rapid recruitment of leukocytes to inflammatory sites. Although IL-8/CXCL8 and eotaxin-3/CCL26 are sorted to the rod-shaped Weibel-Palade body (WPB), we found that GROalpha/CXCL1 and MCP-1/CCL2 reside in small granules that, similarly to the WPB, respond to secretagogue stimuli. In the present study, we report that GROalpha and MCP-1 colocalized in 50- to 100-nm granules, which occur throughout the cytoplasm and at the cell cortex. Immunofluorescence confocal microscopy revealed no colocalization with multimerin or tissue plasminogen activator, i.e., proteins that are released from small granules of ECs by regulated exocytosis. Moreover, the GROalpha/MCP-1-containing granules were Rab27-negative, contrasting the Rab27-positive, WPB. The secretagogues PMA, histamine, and forskolin triggered distinct dose and time-dependent responses of GROalpha release. Furthermore, GROalpha release was more sensitive than IL-8 release to inhibitors and activators of PKA and PKC but not to an activator of Epac, a cAMP-regulated GTPase exchange factor, indicating that GROalpha release is regulated by molecular adaptors different from those regulating exocytosis of the WPB. On the basis of these findings, we designated the GROalpha/MCP-1-containing compartment the type 2 granule of regulated secretion in ECs, considering the WPB the type 1 compartment. In conclusion, we propose that the GROalpha/MCP-1-containing type 2 granule shows preferential responsiveness to important mediators of EC activation, pointing to the existence of selective agonists that would allow differential release of selected chemokines.

Cells, Cultured↗

PubMed2005

The mouse CD1d cytoplasmic tail mediates CD1d trafficking and antigen presentation by adaptor protein 3-dependent and -independent mechanisms.

The short cytoplasmic tail of mouse CD1d (mCD1d) is required for its endosomal localization, for the presentation of some glycolipid Ags, and for the development of Valpha14i NKT cells. This tail has a four-amino acid Tyr-containing motif, Tyr-Gln-Asp-Ile (YQDI), similar to those sequences known to be important for the interaction with adaptor protein complexes (AP) that mediate the endosomal localization of many different proteins. In fact, mCD1d has been shown previously to interact with the AP-3 adaptor complex. In the present study, we mutated each amino acid in the YQDI motif to determine the importance of the entire motif sequence in influencing mCD1d trafficking, its interaction with adaptors, and its intracellular localization. The results indicate that the Y, D, and I amino acids are significant functionally because mutations at each of these positions altered the intracellular distribution of mCD1d and reduced its ability to present glycosphingolipids to NKT cells. However, the three amino acids are not all acting in the same way because they differ with regard to how they influence the intracellular distribution of CD1d, its rate of internalization, and its ability to interact with the mu subunit of AP-3. Our results emphasize that multiple steps, including interactions with the adaptors AP-2 and AP-3, are required for normal trafficking of mCD1d and that these different steps are mediated by only a few cytoplasmic amino acids.

Adaptor Protein Complex 2↗

PubMed2005

The adaptor protein AP-4 as a component of the clathrin coat machinery: a morphological study.

The four members of the AP (adaptor protein) family are heterotetrameric cytosolic complexes that are involved in the intracellular trafficking of cargo proteins between different organelles. They interact with motifs present in the cytoplasmic tails of their specific cargo proteins at different intracellular locations. While AP-1, AP-2 and AP-3 have been investigated extensively, very few studies have focused on the fourth member, AP-4. In the present study, we report on the intracellular localization of AP-4 in the MDCK (Madin-Darby canine kidney) and MelJuSo cell lines after immunogold labelling of ultrathin cryosections. We find that AP-4 is localized mainly in the Golgi complex, as well as on endosomes and transport vesicles. Interestingly, we show for the first time that AP-4 is localized with the clathrin coat machinery in the Golgi complex and in the endocytic pathway. Furthermore, we find that AP-4 is localized with the CI-MPR (cation-independent mannose 6-phosphate receptor), but not with the transferrin receptor, LAMP-2 (lysosomal-associated membrane protein-2) or invariant chain. The difference in morphology between CI-MPR/AP-4-positive vesicles and CI-MPR/AP-1-positive vesicles raises the possibility that AP-4 acts at a location different from that of AP-1 in the intracellular trafficking pathway of CI-MPR.

Adaptor Protein Complex 4↗

PubMed2004

c-Myb associates with PML in nuclear bodies in hematopoietic cells.

The c-Myb transcription factor plays a central role in the regulation of cell growth and differentiation of hematopoietic cells. Being the product of a proto-oncogene, one would expect c-Myb function to be modulated by signal transduction pathways, but our knowledge on such regulation of c-Myb is rather limited. Recently, we and others showed that c-Myb is subjected to sumoylation and that this posttranslational modification has considerable effect on c-Myb's activity. Interestingly, many proteins subjected to SUMO-1 conjugation associate with the promyelocytic leukemia (PML) protein and localize to PML nuclear bodies (PML NBs). Although the precise molecular function of PML NBs still remains to be defined, they seem to play a role in regulation of gene expression and are linked to specific cellular signaling. We show here that c-Myb localizes to PML NBs and that c-Myb interacts with PML as judged by immunofluorescence microcopy and co-immunoprecipitation experiments. Enforced expression of PML IV was shown to enhance c-Myb-dependent reporter activation. Our results imply a role for PML and possibly other components of PML NBs in regulating c-Myb's activity. This novel link between c-Myb and PML, two gene products being implicated in leukemic disorders, suggests that previously unknown mechanisms for regulating c-Myb's activity involving PML may exist.

Animals↗

PubMed2004

Rapid chemokine secretion from endothelial cells originates from 2 distinct compartments.

The neutrophil-attracting chemokine interleukin 8 (IL-8) is stored in the Weibel-Palade body (WPB) of endothelial cells (ECs) from which it can be rapidly released after exposure to the secretagogues histamine or thrombin. In this manner, IL-8 may enable rapid recruitment of leukocytes to inflammatory sites. To explore the possible storage of EC-derived chemokines that may attract other subsets of leukocytes, we examined the intracellular localization and secretagogue responsiveness of growth-related oncogene alpha (GROalpha), monocyte chemoattractant protein-1 (MCP-1), eotaxin-3, interferon-gamma-inducible protein 10 (IP-10), and regulated on activation, normal T-cell expressed and secreted (RANTES). While eotaxin-3, GROalpha, and MCP-1 were rapidly released from ECs, the release of the T-cell attractors RANTES and IP-10 was not sensitive to the secretagogues. Moreover, of the 3 former chemokines, only eotaxin-3 was stored in WPBs. GROalpha and MCP-1 resided mainly in smaller vesicles compatible with sorting to a different, histamine-responsive compartment, which has been described in ECs although not reported to contain chemokines. In conclusion, we propose that rapid release of chemokines is restricted to those primarily recruiting leukocytes of the innate immune system, and that their storage in ECs is not restricted to the WPB compartment.

Carcinogens↗

PubMed2003

The adaptor protein AP-3 is required for CD1d-mediated antigen presentation of glycosphingolipids and development of Valpha14i NKT cells.

Relatively little is known about the pathway leading to the presentation of glycolipids by CD1 molecules. Here we show that the adaptor protein complex 3 (AP-3) is required for the efficient presentation of glycolipid antigens that require internalization and processing. AP-3 interacts with mouse CD1d, and cells from mice deficient for AP-3 have increased cell surface levels of CD1d and decreased expression in late endosomes. Spleen cells from AP-3-deficient mice have a reduced ability to present glycolipids to natural killer T (NKT) cells. Furthermore, AP-3-deficient mice have a significantly reduced NKT cell population, although this is not caused by self-tolerance that might result from increased CD1d surface levels. These data suggest that the generation of the endogenous ligand that selects NKT cells may also be AP-3 dependent. However, the function of MHC class II-reactive CD4+ T lymphocytes is not altered by AP-3 deficiency. Consistent with this divergence from the class II pathway, NKT cell development and antigen presentation by CD1d are not reduced by invariant chain deficiency. These data demonstrate that the AP-3 requirement is a particular attribute of the CD1d pathway in mice and that, although MHC class II molecules and CD1d are both found in late endosomes or lysosomes, different pathways mediate their intracellular trafficking.

Adaptor Protein Complex 3↗

PubMed2003

The cytoplasmic tail of invariant chain modulates antigen processing and presentation.

The MHC class II-associated invariant chain (Ii) has several important functions in antigen presentation. In this study, we have examined the effect of Iip33 expression on endocytic transport and antigen presentation. We find that degradation of both endocytosed antigen and Ii itself is delayed in cells expressing high levels of Ii, whereas a mutant Ii with an altered charge distribution in the cytoplasmic tail was unable to exert this effect. Furthermore, the Ii mutant did not enhance the presentation of an Ii-dependent MHC class II-restricted epitope to the same extent as the wild type. In a parallel study, we investigated the effect of charge in the cytoplasmic tail of Ii. We find that due to exposed negative charges, it promotes endosome fusion events, and we suggest that this causes endosomal retention (Nordeng et al., Mol. Biol. Cell 2002). Together, the data reveal an additional property of the Iip33 cytoplasmic tail that contributes to the modulation of antigen processing and presentation.

Amino Acid Substitution↗

PubMed2003

MHC class II loading of high or low affinity peptides directed by Ii/peptide fusion constructs: implications for T cell activation.

CD4(+) T cells recognize peptides presented on the cell surface of antigen presenting cells in the MHC class II context. The biosynthesis and transport of MHC class II molecules depend on the type II transmembrane invariant chain (Ii) and are tightly regulated processes. Ii is known to bind to the MHC class II peptide-binding groove via its class II-associated Ii peptide (CLIP) region early in the biosynthetic pathway to prevent premature peptide binding. In this study we have genetically exchanged CLIP with peptides of either high or low affinity for the class II peptide binding groove and utilized the properties of Ii to manipulate MHC class II loading. An inducible promoter controlled expression of the Ii/peptide fusion constructs, and presentation at different expression levels was studied. Both peptides were excised from Ii and presented on MHC class II molecules as shown by liquid chromatography-tandem mass spectrometry, but the high affinity peptide was presented more efficiently than the low affinity peptide. Both peptides were efficient in eliciting T cell responses at high Ii/peptide concentration independent of the duration of T cell stimulation. The peptides were also able to elicit an IL-2 response at low expression levels; however, the kinetic differed as the T cells required longer duration of T cell contact to reach a significant T cell response. This probably reflects the number of class II/peptide complexes at the cell surface and is discussed.

Amino Acid Sequence↗

PubMed2002

Structural requirements for interactions between leucine-sorting signals and clathrin-associated adaptor protein complex AP3.

Cytoplasmic tails of LIMPII and the invariant chain contain similar leucine-based sorting signals, but the invariant chain interacts only with AP1 and AP2, whereas LIMPII interacts strongly with AP3. In a series of in vitro experiments, we investigated the effect of residues upstream of the leucine pairs and demonstrated that these residues determine adapter binding, and certain residues favor interactions with AP3. Furthermore, constructs that interacted stronger with AP3 interacted weakly with AP1 and vice versa. Exchanging residues upstream of the leucine-based signal in LIMPII with those of the invariant chain reduced LIMPII binding to AP3 in vitro, and in vivo the corresponding LIMPII mutant was rerouted via the plasma membrane like the invariant chain. These preferential interactions of different leucine signals with different AP complexes may thus be the determining step sorting proteins from the trans-Golgi network to their final destinations. Proteins that interact with AP3 are sorted directly to endosomes/lysosomes, whereas proteins that interact with AP1 are sorted via a different route. At the same time, constructs that exhibited specificity for either AP1 or AP3 might still interact with AP2, suggesting that AP2 may recognize a wider variety of leucine signals. This is consistent with the suggested role of AP2 in internalization of proteins containing general leucine-based signals, including proteins that have been missorted to the plasma membrane.

Adaptor Protein Complex 1↗

PubMed2002

Mechanism of interaction between leucine-based sorting signals from the invariant chain and clathrin-associated adaptor protein complexes AP1 and AP2.

The cytoplasmic tail of the invariant chain contains two leucine-based sorting signals, and each of those seems sufficient to route the invariant chain to its intracellular destination in either normal or polarized cells. It is believed that the intracellular routing of the invariant chain is mediated by its interactions with the clathrin-associated adaptor protein complexes AP1 and AP2. We () have previously demonstrated the in vitro interactions between the cytoplasmic tail of the invariant chain and AP1/AP2 complexes. These interactions were specific and depended on the critical leucine residues in the invariant chain's sorting signals. In the present study, we decided to investigate the molecular mechanism of these interactions. To this end, we constructed a set of glutathione S-transferase fusion proteins that contained the intact cytoplasmic tail of the invariant chain and its various mutants to define residues important for its interactions with AP1 and AP-2. Our results demonstrated the importance of several residues other than the critical leucine residues for such interactions. A strong correlation between in vitro binding of AP2 to the invariant chain and in vivo internalization of the invariant chain was observed, confirming the primary role of AP2 in recognition of endocytic signals. In addition, we demonstrated different requirements for AP1 and AP2 binding to cytoplasmic tail of the invariant chain, which may reflect that the different sorting pathways mediated by AP1 and AP2 involve their recognition of the primary structure of the sorting signal.

Adaptor Protein Complex 1↗

PubMed2002

Uncoating ATPase Hsc70 is recruited by invariant chain and controls the size of endocytic compartments.

Targeting of class II major histocompatibility complex molecules to endocytic compartments is mediated by their association with the invariant chain (Ii). Although the identity of certain sorting signals located in Ii's cytoplasmic tail is known, proteins that interact with Ii's cytoplasmic tail in living cells remain to be identified. Synthesis of a biotinylated trimeric Ii cytoplasmic tail allowed the retrieval of two proteins that interact with this domain. We identify one of them as the 70-kDa heat-shock cognate protein (hsc70), the uncoating ATPase of clathrin-coated vesicles, and the other as its mitochondrial homologue, the glucose-regulated protein grp75. Expression of Ii in COS cells results in the formation of large endocytic compartments. We observe extensive colocalization of hsc70 with Ii in these macrosomes. Expression of a dominant-negative (K71M) green fluorescent protein-tagged version of hsc70 counteracted the ability of Ii to modify the endocytic pathway, demonstrating an interaction in vivo of Ii with hsc70 as part of the machinery responsible for macrosome formation.

Amino Acid Sequence↗

PubMed2002

The cytoplasmic tail of invariant chain regulates endosome fusion and morphology.

The major histocompatibility complex class II associated invariant chain (Ii) has been shown to inhibit endocytic transport and to increase the size of endosomes. We have recently found that this property has a significant impact on antigen processing and presentation. Here, we show in a cell-free endosome fusion assay that expression of Ii can increase fusion after phosphatidylinositol 3-kinase activity is blocked by wortmannin. In live cells wortmannin was also not able to block formation of the Ii-induced enlarged endosomes. The effects of Ii on endosomal transport and morphology depend on elements within the cytoplasmic tail. Data from mutagenesis analysis and nuclear magnetic resonance-based structure calculations of the Ii cytoplasmic tail demonstrate that free negative charges that are not involved in internal salt bridges are essential for both interactions between the tails and for the formation of enlarged endosomes. This correlation indicates that it is interactions between the Ii cytoplasmic tails that are involved in endosome fusion. The combined data from live cells, cell-free assays, and molecular dynamic simulations suggest that Ii molecules on different vesicles can promote endosome docking and fusion and thereby control endosomal traffic of membrane proteins and endosomal content.

Amino Acid Sequence↗