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Biomedical subjects

Harald Stenmark

Publications and source records attributed to Harald Stenmark.

At least 37 records · Page 2Linked to original sources
PubMed2005

p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death.

Autophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it is not known how the autophagic machinery recognizes such aggregates. In this study, we report that polymerization of the polyubiquitin-binding protein p62/SQSTM1 yields protein bodies that either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomal structures. Inhibition of autophagy led to an increase in the size and number of p62 bodies and p62 protein levels. The autophagic marker light chain 3 (LC3) colocalized with p62 bodies and co-immunoprecipitated with p62, suggesting that these two proteins participate in the same complexes. The depletion of p62 inhibited recruitment of LC3 to autophagosomes under starvation conditions. Strikingly, p62 and LC3 formed a shell surrounding aggregates of mutant huntingtin. Reduction of p62 protein levels or interference with p62 function significantly increased cell death that was induced by the expression of mutant huntingtin. We suggest that p62 may, via LC3, be involved in linking polyubiquitinated protein aggregates to the autophagy machinery.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Alix regulates cortical actin and the spatial distribution of endosomes.

Alix/AIP1 is a proline-rich protein that has been implicated in apoptosis, endocytic membrane trafficking and viral budding. To further elucidate the functions of Alix, we used RNA interference to specifically suppress its expression. Depletion of Alix caused a striking redistribution of early endosomes from a peripheral to a perinuclear location. The redistribution of endosomes did not affect transferrin recycling or degradation of endocytosed epidermal growth factor receptors, although the uptake of transferrin was mildly reduced when Alix was downregulated. Quantitative immunoelectron microscopy showed that multivesicular endosomes of Alix-depleted cells contained normal amounts of CD63, whereas their levels of lysobisphosphatidic acid were reduced. Alix depletion also caused an accumulation of unusual actin structures that contained clathrin and cortactin, a protein that couples membrane dynamics to the cortical actin cytoskeleton. Our results suggest that Alix functions in the actin-dependent intracellular positioning of endosomes, but that it is not essential for endocytic recycling or for trafficking of membrane proteins between early and late endosomes in non-polarised cells.

Actins↗
PubMed2005

Modulation of receptor recycling and degradation by the endosomal kinesin KIF16B.

Different classes of endosomes exhibit a characteristic intracellular steady-state distribution governed by interactions with the cytoskeleton. We found a kinesin-3, KIF16B, that transports early endosomes to the plus end of microtubules in a process regulated by the small GTPase Rab5 and its effector, the phosphatidylinositol-3-OH kinase hVPS34. In vivo, KIF16B overexpression relocated early endosomes to the cell periphery and inhibited transport to the degradative pathway. Conversely, expression of dominant-negative mutants or ablation of KIF16B by RNAi caused the clustering of early endosomes to the perinuclear region, delayed receptor recycling to the plasma membrane, and accelerated degradation. These results suggest that KIF16B, by regulating the plus end motility of early endosomes, modulates the intracellular localization of early endosomes and the balance between receptor recycling and degradation. We propose that this mechanism could have important implications for signaling.

Amino Acid Sequence↗
PubMed2005

Eap45 in mammalian ESCRT-II binds ubiquitin via a phosphoinositide-interacting GLUE domain.

Ubiquitination serves as a key sorting signal in the lysosomal degradation of endocytosed receptors through the ability of ubiquitinated membrane proteins to be recognized and sorted by ubiquitin-binding proteins along the endocytic route. The ESCRT-II complex in yeast contains one such protein, Vps36, which harbors a ubiquitin-binding NZF domain and is required for vacuolar sorting of ubiquitinated membrane proteins. Surprisingly, the presumptive mammalian ortholog Eap45 lacks the ubiquitin-binding module of Vps36, and it is thus not clear whether mammalian ESCRT-II functions to bind ubiquitinated cargo. In this paper, we provide evidence that Eap45 contains a novel ubiquitin-binding domain, GLUE (GRAM-like ubiquitin-binding in Eap45), which binds ubiquitin with similar affinity and specificity as other ubiquitin-binding domains. The GLUE domain shares similarities in its primary and predicted secondary structures to phosphoinositide-binding GRAM and PH domains. Accordingly, we find that Eap45 binds to a subset of 3-phosphoinositides, suggesting that ubiquitin recognition could be coordinated with phosphoinositide binding. Furthermore, we show that Eap45 colocalizes with ubiquitinated proteins on late endosomes. These results are consistent with a role for Eap45 in endosomal sorting of ubiquitinated cargo.

Amino Acid Sequence↗
PubMed2005

Actin-based motility of endosomes is linked to the polar tip growth of root hairs.

Plant tip growth has been recognized as an actin-based cellular process requiring targeted exocytosis and compensatory endocytosis to occur at the growth cone. However, the identity of subcellular compartments involved in polarized membrane trafficking pathways remains enigmatic in plants. Here we characterize endosomal compartments in tip-growing root hair cells. We demonstrate their presence at the growing tip and differential distribution upon cessation of tip growth. We also show that both the presence of endosomes as well as their rapid movements within the tip region depends on an intact actin cytoskeleton and involves actin polymerization. In conclusion, actin-propelled endosomal motility is tightly linked to the polar tip growth of root hairs.

Actins↗
PubMed2005

The Rab5 effector Rabaptin-5 and its isoform Rabaptin-5delta differ in their ability to interact with the small GTPase Rab4.

Rabaptin-5 is an effector for the small GTPase Rab5, a regulator of the early steps in endocytosis. In addition, Rabaptin-5 interacts with the small GTPase Rab4 that has been implicated in recycling from early endosomes to the cell surface. Recently we have identified a ubiquitous transcript encoding the Rabaptin-5 isoform, Rabaptin-5delta. To evaluate the interaction properties of Rabaptin-5delta with the small GTPases Rab4 and Rab5, we have applied protein interaction assays using the yeast two-hybrid system and a glutathione S-transferase pull-down assay. We found that unlike Rabaptin-5, that interacts with both GTPases in GTP-bound conformations, Rabaptin-5delta interacts only with GTP-bound Rab5, and does not interact with Rab4, presumably due to a disrupted Rab4 binding site. Immunofluorescence microscopy analysis carried out to address the localization of Rabaptin-5delta relative to GTP-bound Rab4 and Rab5 in BHK-21 cells supported these data. Our data suggests that while Rabaptin-5 was proposed to act as a molecular linker between Rab5 and Rab4, to coordinate endocytic and recycling traffic, Rabaptin-5delta is involved only in the Rab5-driven events.

Animals↗
PubMed2004

Alfy, a novel FYVE-domain-containing protein associated with protein granules and autophagic membranes.

Phosphatidylinositol-3-phosphate [PtdIns(3)P] regulates endocytic and autophagic membrane traffic. In order to understand the downstream effects of PtdIns(3)P in these processes, it is important to identify PtdIns(3)P-binding proteins, many of which contain FYVE zinc-finger domains. Here, we describe a novel giant FYVE-domain-containing protein, named autophagy-linked FYVE protein (Alfy). Alfy is ubiquitously expressed, shares sequence similarity with the Chediak-Higashi-syndrome protein and has putative homologues in flies, nematodes and fission yeast. Alfy binds PtdIns(3)P in vitro and partially colocalizes with PtdIns(3)P in vivo. Unlike most other FYVE-domain proteins, Alfy is not found on endosomes but instead localizes mainly to the nuclear envelope. When HeLa cells are starved or treated with a proteasome inhibitor, Alfy relocalizes to characteristic filamentous cytoplasmic structures located close to autophagic membranes and ubiquitin-containing protein aggregates. By electron microscopy, similar structures can be found within autophagosomes. We propose that Alfy might target cytosolic protein aggregates for autophagic degradation.

Adaptor Proteins, Signal Transducing↗
PubMed2004

The growth-regulatory protein HCRP1/hVps37A is a subunit of mammalian ESCRT-I and mediates receptor down-regulation.

The biogenesis of multivesicular bodies and endosomal sorting of membrane cargo are driven forward by the endosomal sorting complexes required for transport, ESCRT-I, -II, and -III. ESCRT-I is characterized in yeast as a complex consisting of Vps23, Vps28, and Vps37. Whereas mammalian homologues of Vps23 and Vps28 (named Tsg101 and hVps28, respectively) have been identified and characterized, a mammalian counterpart of Vps37 has not yet been identified. Here, we show that a regulator of proliferation, hepatocellular carcinoma related protein 1 (HCRP1), interacts with Tsg101, hVps28, and their upstream regulator Hrs. The ability of HCRP1 (which we assign the alternative name hVps37A) to interact with Tsg101 is conferred by its mod(r) domain and is shared with hVps37B and hVps37C, two other mod(r) domain-containing proteins. HCRP1 cofractionates with Tsg101 and hVps28 by size exclusion chromatography and colocalizes with hVps28 on LAMP1-positive endosomes. Whereas depletion of Tsg101 by siRNA reduces cellular levels of both hVps28 and HCRP1, depletion of HCRP1 has no effect on Tsg101 or hVps28. Nevertheless, HCRP1 depletion strongly retards epidermal growth factor (EGF) receptor degradation. Together, these results indicate that HCRP1 is a subunit of mammalian ESCRT-I and that its function is essential for lysosomal sorting of EGF receptors.

Amino Acid Sequence↗
PubMed2004

Defective downregulation of receptor tyrosine kinases in cancer.

Most growth factors control cellular functions by activating specific receptor tyrosine kinases (RTKs). While overactivation of RTK signalling pathways is strongly associated with carcinogenesis, it is becoming increasingly clear that impaired deactivation of RTKs may also be a mechanism in cancer. A major deactivation pathway, receptor downregulation, involves ligand-induced endocytosis of the RTK and subsequent degradation in lysosomes. A complex molecular machinery that uses the small protein ubiquitin as a key regulator assures proper endocytosis and degradation of RTKs. Here we discuss evidence that implicates deregulation of this machinery in cancer.

Animals↗
PubMed2004

Cbl-dependent ubiquitination is required for progression of EGF receptors into clathrin-coated pits.

Ligand binding causes the EGF receptor (EGFR) to become ubiquitinated by Cbl upon association with the adaptor protein Grb2. We have investigated the role of ubiquitin and Grb2 in ligand-induced endocytosis of the EGFR. Incubation of cells with EGF on ice caused translocation of Grb2 and Cbl from the cytosol to the rim of coated pits. Grb2 with point mutations in both SH3 domains inhibited recruitment of the EGFR to clathrin-coated pits, in a Ras-independent manner. On overexpression of the Cbl-binding protein Sprouty, ubiquitination of the EGFR was inhibited, the EGFR was recruited only to the rim of coated pits, and endocytosis of the EGFR was inhibited. Conjugation-defective ubiquitin similarly inhibited recruitment of EGF-EGFR to clathrin-coated pits. Even though this does not prove that cargo must be ubiquitinated, this indicates the importance of interaction of ubiquitinated protein(s) with proteins harboring ubiquitin-interacting domains. We propose that Grb2 mediates transient anchoring of the EGFR to an Eps15-containing molecular complex at the rim of coated pits and that Cbl-induced ubiquitination of the EGFR allows relocation of EGFR from the rim to the center of clathrin-coated pits.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Misfolding diverts CFTR from recycling to degradation: quality control at early endosomes.

To investigate the degradation mechanism of misfolded membrane proteins from the cell surface, we used mutant cystic fibrosis transmembrane conductance regulators (CFTRs) exhibiting conformational defects in post-Golgi compartments. Here, we show that the folding state of CFTR determines the post-endocytic trafficking of the channel. Although native CFTR recycled from early endosomes back to the cell surface, misfolding prevented recycling and facilitated lysosomal targeting by promoting the ubiquitination of the channel. Rescuing the folding defect or down-regulating the E1 ubiquitin (Ub)-activating enzyme stabilized the mutant CFTR without interfering with its internalization. These observations with the preferential association of mutant CFTRs with Hrs, STAM-2, TSG101, hVps25, and hVps32, components of the Ub-dependent endosomal sorting machinery, establish a functional link between Ub modification and lysosomal degradation of misfolded CFTR from the cell surface. Our data provide evidence for a novel cellular mechanism of CF pathogenesis and suggest a paradigm for the quality control of plasma membrane proteins involving the coordinated function of ubiquitination and the Ub-dependent endosomal sorting machinery.

Animals↗
PubMed2004

Programmed autophagy in the Drosophila fat body is induced by ecdysone through regulation of the PI3K pathway.

Eukaryotic cells catabolize their own cytoplasm by autophagy in response to amino acid starvation and inductive signals during programmed tissue remodeling and cell death. The Tor and PI3K signaling pathways have been shown to negatively control autophagy in eukaryotes, but the mechanisms that link these effectors to overall animal development and nutritional status in multicellular organisms remain poorly understood. Here, we reveal a complex regulation of programmed and starvation-induced autophagy in the Drosophila fat body. Gain-of-function genetic analysis indicated that ecdysone receptor signaling induces programmed autophagy whereas PI3K signaling represses programmed autophagy. Genetic interaction studies showed that ecdysone signaling downregulates PI3K signaling and that this represents the effector mechanism for induction of programmed autophagy. Hence, these studies link hormonal induction of autophagy to the regulatory function of the PI3K signaling pathway in vivo.

Amino Acids↗
PubMed2004

The structure of an endosomal protein sorter.

Three "endosomal sorting complexes required for transport," ESCRT-I, -II, and -III, mediate sorting of ubiquitinated membrane proteins into intraluminal endosomal vesicles that are destined for degradation in lysosomes. Two recent reports, one in Nature and one in this issue of Developmental Cell, reveal the crystal structure of the yeast form of ESCRT-II.

Amino Acid Sequence↗
PubMed2004

Acquisition of Hrs, an essential component of phagosomal maturation, is impaired by mycobacteria.

Pathogenic mycobacteria survive within macrophages by precluding the fusion of phagosomes with late endosomes or lysosomes. Because the molecular determinants of normal phagolysosome formation are poorly understood, the sites targeted by mycobacteria remain unidentified. We found that Hrs, an adaptor molecule involved in protein sorting, associates with phagosomes prior to their fusion with late endosomes or lysosomes. Recruitment of Hrs required the interaction of its FYVE domain with phagosomal phosphatidylinositol 3-phosphate, but two other attachment sites were additionally involved. Depletion of Hrs by use of small interfering RNA impaired phagosomal maturation, preventing the acquisition of lysobisphosphatidic acid and reducing luminal acidification. As a result, the maturation of phagosomes formed in Hrs-depleted cells was arrested at an early stage, characterized by the acquisition and retention of sorting endosomal markers. This phenotype is strikingly similar to that reported to occur in phagosomes of cells infected by mycobacteria. We therefore tested whether Hrs is recruited to phagosomes containing mycobacteria. Hrs associated readily with phagosomes containing inert particles but poorly with mycobacterial phagosomes. Moreover, Hrs was found more frequently in phagosomes containing avirulent Mycobacterium smegmatis than in phagosomes with the more virulent Mycobacterium marinum. These findings suggest that the inability to recruit Hrs contributes to the arrest of phagosomal maturation induced by pathogenic mycobacteria.

Androstadienes↗
PubMed2003

Endocytosis and signaling: a relationship under development.

The ability to internalize macromolecules by endocytosis is a property of all eukaryotic cells. Frontline research on endocytosis has been presented in a successful series of biannual meetings in Europe. This year's meeting on "Membrane Dynamics in Endocytosis" was held September 13-18 in Acquafredda di Maratea, on the coast of southern Italy. Four key questions were addressed: What are the molecular mechanisms of endocytic membrane trafficking? How does endocytosis modulate receptor signaling and vice versa? What is the importance of endocytosis during development? How do endocytic organelles contribute to immunity or susceptibility to pathogens?

Adaptor Protein Complex 2↗
PubMed2003

Phosphatidylinositol 3-phosphate is found in microdomains of early endosomes.

Phosphatidylinositol 3-phosphate [PI(3)P] is a phosphatidylinositol 3-kinase product whose localisation is restricted to the limiting membranes of early endosomes and to the internal vesicles of multivesicular bodies. In this study the intracellular distribution of PI(3)P was compared with those of another phosphoinositide and a number of endosomal proteins. Using a 2xFYVE probe specific for PI(3)P we found that PI(3)P is present in microdomains within the endosome membrane, whereas a phosphoinositide required for clathrin-mediated endocytosis, PI(4,5)P2, was only detected at the plasma membrane. The small GTPase Rab5 as well as the PI(3)P-binding proteins EEA1, SARA and CISK were found to be abundant within PI(3)P-containing endosomal microdomains. In contrast, another PI(3)P-binding protein, Hrs, was found concentrated in clathrin-coated endosomal microdomains with low levels of PI(3)P. While PI(3)P-containing microdomains could be readily distinguished on enlarged endosomes in cells transfected with a constitutively active Rab5 mutant, such domains could also be detected in endosomes of non-transfected cells. We conclude that the membranes of early endosomes consist of microdomains in which PI(3)P and specific proteins are concentrated. These microdomains may be necessary for the assembly of distinct multimolecular complexes that specify organelle identity, membrane trafficking and receptor signalling.

Endosomes↗
PubMed2003

Peroxisomal targeting as a tool for assaying potein-protein interactions in the living cell: cytokine-independent survival kinase (CISK) binds PDK-1 in vivo in a phosphorylation-dependent manner.

Current methods to detect protein-protein interactions are either laborious to implement or not adaptable for mammalian systems or in vitro methods. By adding a peroxisomal targeting signal (PTS) onto one protein, binding partners lacking a targeting signal were co-transported into the peroxisomes in a "piggy-back" fashion, as visualized by confocal and electron microscopy. A fragment of colicin E2 and its tightly interacting immunity protein, ImmE2, were both expressed in the cytosol. When either one contained a PTS tag, both proteins were co-localized in the peroxisomes. The cytokine-independent survival kinase (CISK) containing a PTS tag was not efficiently targeted to the peroxisomes unless the Phox homology (PX) domain, attaching the protein to endosomal membranes, was removed. However, PTS-tagged CISK with deleted PX domain was able to direct 3-phosphoinositide-dependent protein kinase-1 (PDK-1) into the peroxisomes. This demonstrates that the two proteins interact in vivo. Mutating Ser486, which is phosphorylated in activated CISK, to Ala prevented the interaction, indicating that CISK and PDK-1 interact in a phosphorylation-dependent manner. The method therefore allows assessment of protein-protein interactions that depend on post-translational modifications that are cell-specific or dependent on the physiological state of the cell.

3-Phosphoinositide-Dependent Protein Kinases↗