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Biomedical subjects

H Stenmark

Publications and source records attributed to H Stenmark.

At least 19 recordsLinked to original sources
PubMed2004

Protein targeting to endosomes and phagosomes via FYVE and PX domains.

Phosphatidylinositol 3-phosphate (PI3P) is generated on early endosomal and phagosomal membranes by PI 3-kinases. This lipid serves important regulatory functions in phagocytosis, endocytic traffic, receptor signalling and microbial killing through the recruitment and activation of a number of effector proteins. Almost all of these effectors contain FYVE or PX domains, functional protein modules which are conserved from yeast to mammals. Structural information is available regarding the binding of FYVE and PX domains to PI3P. The two domains are highly different, but they have in common that clusters of basic residues mediate ligand binding through interactions with the phosphate groups of PI3P. Most proteins that contain FYVE or PX domains serve as regulators of endocytic membrane trafficking, whereas others function as regulators of phagosome maturation, signal transduction, microbial killing and other cellular activities of relevance for the immune system.

Animals↗
PubMed2003

PI3P signaling regulates receptor sorting but not transport in the endosomal pathway.

While evidence is accumulating that phosphoinositide signaling plays a crucial role in growth factor and hormone receptor down-regulation, this signaling pathway has also been proposed to regulate endosomal membrane transport and multivesicular endosome biogenesis. Here, we have followed the fate of the down-regulated EGF receptor (EGFR) and bulk transport (fluid phase) markers in the endosomal pathway in vivo and in vitro. We find that bulk transport from early to late endosomes is not affected after inhibition of the phosphatidylinositol-3-phosphate (PI3P) signaling pathway, but that the EGFR then remains trapped in early endosomes. Similarly, we find that hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is not directly involved in bulk solute transport, but is required for EGFR sorting. These observations thus show that transport and sorting can be uncoupled in the endosomal pathway. They also show that PI3P signaling does not regulate the core machinery of endosome biogenesis and transport, but controls the sorting of down-regulated receptor molecules in early endosomes via Hrs.

Animals↗
PubMed2001

A PtdIns(3)P-specific probe cycles on and off host cell membranes during Salmonella invasion of mammalian cells.

Salmonella invade nonphagocytic cells by eliciting their own internalization; upon contact with the host cell, the bacteria induce membrane ruffles highly localized to the point of contact between the invading bacterium and the host cell. The bacterium is then internalized into an unusual cytosolic organelle, the Salmonella-containing vacuole (SCV). Early endosomal markers (including EEA1) have recently been shown to be associated with the SCV shortly after invasion. EEA1, a protein involved in early endosome fusion, is recruited to early endosomal membranes in part by the interaction between its FYVE finger and phosphatidylinositol 3-phosphate [PtdIns(3)P], a characteristic lipid of early endosomes. This suggests a possible role for PtdIns(3)P during Salmonella infection. To investigate this, we generated a highly specific probe for PtdIns(3)P that was used to follow invasion of Salmonella in nonphagocytic cells. Here, we show that PtdIns(3)P is present on the membranes of SCVs shortly after invasion and also that it is present on the membrane ruffles produced immediately prior to invasion. We also show that this specific probe cycles on and off the membranes of nascent SCVs even when PtdIns 3-kinase activity is inhibited, demonstrating that invading Salmonella influence the composition of the membranes that envelop them during invasion.

Androstadienes↗
PubMed2001

Phosphoinositides and phagocytosis.

Phosphoinositide 3 kinases (PI3Ks)*Abbreviation used in this paper: PI3K, phosphoinositide 3 kinase. are known as regulators of phagocytosis. Recent results demonstrate that class I and III PI3Ks act consecutively in phagosome formation and maturation, and that their respective products, phosphatidylinositol 3,4,5-trisphosphate (PI[3,4,5]P(3)) and phosphatidylinositol 3-phosphate (PI[3]P), accumulate transiently at different stages. Phagosomes containing Mycobacterium tuberculosis do not acquire the PI(3)P-binding protein EEA1, which is required for phagosome maturation. This suggests a possible mechanism of how this microorganism evades degradation in phagolysosomes.

Humans↗
PubMed2001

Hrs recruits clathrin to early endosomes.

The hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, has been implicated in intracellular trafficking and signal transduction. Hrs contains a phosphatidylinositol 3-phosphate-binding FYVE domain that contributes to its endosomal targeting. Here we show that Hrs and EEA1, a FYVE domain protein involved in endocytic membrane fusion, are localized to different regions of early endosomes. We demonstrate that Hrs co-localizes with clathrin, and that the C-terminus of Hrs contains a functional clathrin box motif that interacts directly with the terminal beta-propeller domain of clathrin heavy chain. A massive recruitment of clathrin to early endosomes was observed in cells transfected with Hrs, but not with Hrs lacking the C-terminus. Furthermore, the phosphatidylinositol 3-kinase inhibitor wortmannin caused the dissociation of both Hrs and clathrin from endosomes. While overexpression of Hrs did not affect endocytosis and recycling of transferrin, endocytosed epidermal growth factor and dextran were retained in early endosomes. These results provide a molecular mechanism for the recruitment of clathrin onto early endosomes and suggest a function for Hrs in trafficking from early to late endosomes.

Amino Acid Sequence↗
PubMed2001

The Rab GTPase family.

SUMMARY: The Rab family is part of the Ras superfamily of small GTPases. There are at least 60 Rab genes in the human genome, and a number of Rab GTPases are conserved from yeast to humans. The different Rab GTPases are localized to the cytosolic face of specific intracellular membranes, where they function as regulators of distinct steps in membrane traffic pathways. In the GTP-bound form, the Rab GTPases recruit specific sets of effector proteins onto membranes. Through their effectors, Rab GTPases regulate vesicle formation, actin- and tubulin-dependent vesicle movement, and membrane fusion.

Amino Acid Sequence↗
PubMed2001

Cellular functions of phosphatidylinositol 3-phosphate and FYVE domain proteins.

PtdIns3P is a phosphoinositide 3-kinase product that has been strongly implicated in regulating membrane trafficking in both mammalian and yeast cells. PtdIns3P has been shown to be specifically located on membranes associated with the endocytic pathway. Proteins that contain FYVE zinc-finger domains are recruited to PtdIns3P-containing membranes. Structural information is now available concerning the interaction between FYVE domains and PtdIns3P. A number of proteins have been identified which contain a FYVE domain, and in this review we discuss the functions of PtdIns3P and its FYVE-domain-containing effector proteins in membrane trafficking, cytoskeletal regulation and receptor signalling.

Amino Acid Sequence↗
PubMed2001

Intracellular trafficking and turnover of phosphatidylinositol 3-phosphate.

Phosphatidylinositol 3-kinases (PI 3-kinases) regulate cellular functions through the 3'-phosphorylation of phosphatidylinositol (PI) and its derivatives. The PI 3-kinase product phosphatidylinositol 3-phosphate [PI(3)P] functions to recruit and activate effector proteins containing FYVE zinc finger domains. These proteins have various functions in endocytic membrane trafficking, cytoskeletal regulation and signal transduction. In order to understand the function of FYVE proteins, it is essential to study the formation, localisation, trafficking and turnover of PI(3)P. Here we review recent evidence that PI(3)P is formed on early endosomes through the activity of a PI 3-kinase which is recruited by the GTPase Rab5, and that the PI(3)P is subsequently internalised into intralumenal vesicles of multivesicular endosomes for turnover.

Animals↗
PubMed2001

The role of phosphoinositides in membrane transport.

Phosphoinositides serve as intrinsic membrane signals that regulate intracellular membrane trafficking. Recently, phosphoinositides have been found to direct the localization and activity of effector proteins containing consensus sequence motifs such as FYVE, PH and ENTH domains. In addition, recent results show that regulated synthesis and turnover of phosphoinositides by membrane-associated phosphoinoside kinases and phosphatases spatially restrict the location of effectors critical for cellular transport processes, such as clathrin-mediated endocytosis, autophagy, phagocytosis, macropinocytosis and biosynthetic trafficking.

Animals↗
PubMed2001

Function of Hrs in endocytic trafficking and signalling.

The hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, becomes tyrosine-phosphorylated upon the binding of various growth factors and cytokines to their receptors. This protein is essential for ventral folding morphogenesis, and it shares structural similarity with Vps27p, which is involved in vacuolar protein sorting in yeast. Since Hrs is localized to endosomes and has been implicated in the regulation of signal transduction as well as membrane trafficking, it has been regarded as a potential co-ordinator of endosomal receptor sorting and signalling. Here we discuss the possible functions of Hrs in light of its interactions with phosphatidylinositol 3-phosphate and multiple proteins.

Amino Acid Sequence↗
PubMed2001

Trafficking of phosphatidylinositol 3-phosphate from the trans-Golgi network to the lumen of the central vacuole in plant cells.

Very limited information is available on the role of phosphatidylinositol 3-phosphate (PI[3]P) in vesicle trafficking in plant cells. To investigate the role of PI(3)P during the vesicle trafficking in plant cells, we exploited the PI(3)P-specific binding property of the endosome binding domain (EBD) (amino acids 1257 to 1411) of human early endosome antigen 1, which is involved in endosome fusion. When expressed transiently in Arabidopsis protoplasts, a green fluorescent protein (GFP):EBD fusion protein exhibited PI(3)P-dependent localization to various compartments--such as the trans-Golgi network, the prevacuolar compartment, the tonoplasts, and the vesicles in the vacuolar lumen--that varied with time. The internalized GFP:EBD eventually disappeared from the lumen. Deletion experiments revealed that the PI(3)P-dependent localization required the Rab5 binding motif in addition to the zinc finger motif. Overexpression of GFP:EBD inhibited vacuolar trafficking of sporamin but not trafficking of H(+)-ATPase to the plasma membrane. On the basis of these results, we propose that the trafficking of GFP:EBD reflects that of PI(3)P and that PI(3)P synthesized at the trans-Golgi network is transported to the vacuole through the prevacuolar compartment for degradation in plant cells.

Arabidopsis↗
PubMed2001

FYVE and coiled-coil domains determine the specific localisation of Hrs to early endosomes.

Hrs, an essential tyrosine kinase substrate, has been implicated in intracellular trafficking and signal transduction pathways. The protein contains several distinctive domains, including an N-terminal VHS domain, a phosphatidylinositol 3-phosphate (PtdIns(3)P)-binding FYVE domain and two coiled-coil domains. Here we have investigated the roles of these domains in the subcellular localisation of Hrs. Hrs was found to colocalise extensively with EEA1, an established marker of early endosomes. While the membrane association of EEA1 was abolished in the presence of a dominant negative mutant of the endosomal GTPase Rab5, the localisation of Hrs to early endosomes was Rab5 independent. The VHS-domain was nonessential for the subcellular targeting of Hrs. In contrast, the FYVE domain as well as the second coiled-coil domain, which has been shown to bind to SNAP-25, were required for targeting of Hrs to early endosomes. A small construct consisting of only these two domains was correctly localised to early endosomes, whereas a point mutation (R183A) in the PtdIns(3)P-binding pocket of the FYVE domain inhibited the membrane targeting of Hrs. Thus, like EEA1, the endosomal targeting of Hrs is mediated by a PtdIns(3)P-binding FYVE domain in cooperation with an additional domain. We speculate that binding to PtdIns(3)P and a SNAP-25-related molecule may target Hrs specifically to early endosomes.

Amino Acid Motifs↗
PubMed2000

Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells.

Phosphatidylinositol 3-kinase (PI3K) regulates several vital cellular processes, including signal transduction and membrane trafficking. In order to study the intracellular localization of the PI3K product, phosphatidylinositol 3-phosphate [PI(3)P], we constructed a probe consisting of two PI(3)P-binding FYVE domains. The probe was found to bind specifically, and with high affinity, to PI(3)P both in vitro and in vivo. When expressed in fibroblasts, a tagged probe localized to endosomes, as detected by fluorescence microscopy. Electron microscopy of untransfected fibroblasts showed that PI(3)P is highly enriched on early endosomes and in the internal vesicles of multivesicular endosomes. While yeast cells deficient in PI3K activity (vps15 and vps34 mutants) were not labelled, PI(3)P was found on intralumenal vesicles of endosomes and vacuoles of wild-type yeast. vps27Delta yeast cells, which have impaired endosome to vacuole trafficking, showed a decreased vacuolar labelling and increased endosome labelling. Thus PI(3)P follows a conserved intralumenal degradation pathway, and its generation, accessibility and turnover are likely to play a crucial role in defining the early endosome and the subsequent steps leading to multivesicular endosome formation.

Animals↗
PubMed2000

Interaction of the EEA1 FYVE finger with phosphatidylinositol 3-phosphate and early endosomes. Role of conserved residues.

FYVE zinc finger domains, which are conserved in multiple proteins from yeast to man, interact specifically with the membrane lipid phosphatidylinositol 3-phosphate (PtdIns(3)P). Here we have investigated the structural requirements for the interaction of the FYVE finger of the early endosome antigen EEA1 with PtdIns(3)P and early endosomes. The binding of the FYVE finger to PtdIns(3)P is Zn(2+)-dependent, and Zn(2+) could not be replaced by any other bivalent cations tested. By surface plasmon resonance, the wild-type FYVE finger was found to bind to PtdIns(3)P with an apparent K(D) of about 50 nm and a 1:1 stoichiometry. Mutagenesis of cysteines involved in Zn(2+) coordination, basic residues thought to be directly involved in ligand binding and other conserved residues, resulted in a 6- to >100-fold decreased affinity for PtdIns(3)P. A mutation in the putative PtdIns(3)P-binding pocket, R1375A, may prove particularly informative, because it led to a strongly decreased affinity for PtdIns(3)P without affecting the FYVE three-dimensional structure, as measured by fluorescence spectroscopy. Whereas the C terminus of EEA1 localizes to early endosomes when expressed in mammalian cells, all the FYVE mutants with reduced affinity for PtdIns(3)P were found to be largely cytosolic. Furthermore, whereas expression of the wild-type EEA1 C terminus interferes with early endosome morphology, the point mutants were without detectable effect. These results support recently proposed models for the ligand binding of the FYVE domain and indicate that PtdIns(3)P binding is crucial for the localization and function of EEA1.

Amino Acid Sequence↗
PubMed2000

Cycling lipids.

When synaptic vesicles fuse with the presynaptic plasma membrane, their membrane constituents are recycled by internalisation into clathrin-coated vesicles. To form new synaptic vesicles, the clathrin coat must be shed and recent studies reveal that a lipid phosphatase, synaptojanin, plays a central role in this process.

Calcium-Binding Proteins↗