Effects of deficiencies of STAMs and Hrs,mammalian class E Vps proteins,on receptor downregulation

The STAM family proteins, STAM1 and STAM2/EAST/Hbp, are phosphotyrosine proteins that contain SH3 domains and ubiquitin-interacting motifs. Their yeast homologue, Hse1, and its binding protein, Vps27, are involved in the vacuolar membrane transport machinery. Here we show that STAM1 and STAM2 are localized to the endosomal membrane. Some of these complexes contain Eps15, an endocytic protein, which accumulates in clumps upon expression of a dominant-negative form of Vps4-A, an AAA-type ATPase, that is required for normal endosome function. These results support the idea that the STAMs are mammalian vacuolar protein sorting (Vps) proteins. We also demonstrate that ligand-mediated epidermal growth factor receptor (EGFR) degradation is partially but not completely impaired in both Hrs(-/-) and STAM1(-/-)STAM2(-/-) mouse embryonic fibroblasts. Furthermore, endosome swelling is seen in both Hrs(-/-) and STAM1(-/-)STAM2(-/-) cells. These results suggest that the STAMs and Hrs play important roles in the mammalian endosomal/vacuolar protein sorting pathway.     

A deubiquitinating enzyme UBPY interacts with the Src homology 3 domain of Hrs-binding protein via a novel binding motif PX(V/I)(D/N)RXXKP

Hrs-binding protein (Hbp) is a Src homology 3 (SH3) domain-containing protein that tightly associates with Hrs. Hbp together with Hrs is thought to play a regulatory role in endocytic trafficking of growth factor-receptor complexes through early endosomes. Association of Hbp with a binding partner(s) via the SH3 domain seems to be essential for Hbp to exert its function. In this study, we searched for Hbp-binding proteins by a far Western screening and isolated a mouse cDNA clone encoding a deubiquitinating enzyme mUBPY as an Hbp SH3-binding protein. mUBPY has two Hbp-SH3 domain binding sites. Mutagenic analysis identified a consensus sequence PX(V/I)(D/N)RXXKP as the Hbp-SH3 domain binding motif. It is a novel SH3-binding motif and does not contain the canonical proline-rich consensus binding motif, PXXP. Ubiquitination of growth factor receptors is thought to regulate their intracellular degradation. Thus, UBPY may play a regulatory role in the degradation by interaction with the SH3 domain of Hbp via the novel SH3-binding motif.     

STAM proteins bind ubiquitinated proteins on the early endosome via the VHS domain and ubiquitin-interacting motif

Conjugation with ubiquitin acts as a sorting signal for proteins in the endocytic and biosynthetic pathways at the endosome. Signal-transducing adaptor molecule (STAM) proteins, STAM1 and STAM2, are associated with hepatocyte growth factor-regulated substrate (Hrs) but their function remains unknown. Herein, we show that STAM proteins bind ubiquitin and ubiquitinated proteins and that the tandemly located VHS (Vps27/Hrs/STAM) domain and ubiquitin-interacting motif serve as the binding site(s). STAM proteins colocalize with Hrs on the early endosome. Overexpression of STAM proteins, but not their mutants lacking the ubiquitin-binding activity, causes the accumulation of ubiquitinated proteins and ligand-activated epidermal growth factor receptor on the early endosome. These results suggest that through interaction with ubiquitinated cargo proteins on the early endosome via the VHS domain and ubiquitin-interacting motif, STAM proteins participate in the sorting of cargo proteins for trafficking to the lysosome.     

Structural insight into modest binding of a non-PXXP ligand to the signal transducing adaptor molecule-2 Src homology 3 domain

Although some exceptional motifs have been identified, it is well known that the PXXP motif is the motif of ligand proteins generally recognized by the Src homology 3 (SH3) domain. SH3-ligand interactions are usually weak, with ordinary KD approximately 10 microM. The structural basis for a tight and specific association (KD = 0.24 microm) between Gads SH3 and a novel motif, PX(V/I)(D/N)RXXKP, was revealed in a previous structural analysis of the complex formed between them. In this paper, we report the crystal structure of the signal transducing adaptor molecule-2 (STAM2) SH3 domain in complex with a peptide with a novel motif derived from a ligand protein, UBPY. The derived KD value for this complex is 27 microM. The notable difference in affinity for these parallel complexes may be explained because the STAM2 SH3 structure does not provide a specificity pocket for binding, whereas the Gads SH3 structure does. Instead, the structure of STAM2 SH3 is analogous to that of Grb2 SH3 which, in addition to normal PXXP ligands, has also been shown to moderately recognize the novel motif discussed herein. Thus, the extremely tight interaction observed between Gads SH3 and the novel motif is caused not by an innate ability of the novel motif but rather by an evolutionary change in the Gads SH3 domain. Instead, SH3 domains of STAM2 and Grb2 retain the moderate characteristics of recognizing their ligand proteins like other SH3 domains for appropriate transient interactions between signaling molecules.     

Possible involvement of a novel STAM-associated molecule "AMSH" in intracellular signal transduction mediated by cytokines.

STAM containing an SH3 (Src homology 3) domain and an immunoreceptor tyrosine-based activation motif was previously revealed to be implicated in signaling pathways immediately downstream of Jak2 and Jak3 tyrosine kinases associated with cytokine receptors. We molecularly cloned a novel molecule interacting with the SH3 domain of STAM, which was named AMSH (associated molecule with the SH3 domain of STAM). AMSH contains a putative bipartite nuclear localization signal and a homologous region of a c-Jun activation domain-binding protein 1 (JAB1) subdomain in addition to a binding site for the SH3 domain of STAM. AMSH mutant deleted of the C-terminal half conferred dominant negative effects on signaling for DNA synthesis and c-myc induction mediated by interleukin 2 and granulocyte macrophage-colony-stimulating factor. These results suggest that AMSH plays a critical role in the cytokine-mediated intracellular signal transduction downstream of the Jak2/Jak3.STAM complex.     

Solution structure of UIM and interaction of tandem ubiquitin binding domains in STAM1 with ubiquitin

STAM1 and Hrs are the components of ESCRT-0 complex for lysosomal degradation of membrane proteins is composed of STAM1 Hrs and has multiple ubiquitin binding domains. Here, the solution structure of STAM1 UIM, one of the ubiquitin binding motif, was determined by NMR spectroscopy. The structure of UIM adopts an α-helix with amphipathic nature. The central hydrophobic residues in UIM provides the binding surface for ubiquitin binding and are flanked with positively and negatively charged residues on both sides. The docking model of STAM1 UIM-ubiquitin complex is suggested. In NMR and ITC experiments with the specifically designed mutant proteins, we investigated the ubiquitin interaction of tandem ubiquitin binding domains from STAM1. The ubiquitin binding affinity of the VHS domain and UIM in STAM1 was 52.4 and 94.9 μM, and 1.5 and 2.2 fold increased, respectively, than the value obtained from the isolated domain or peptide. The binding affinities here would be more physiologically relevant and provide more precise understanding in ESCRT pathway of lysosomal degradation.     

Identification of AMSH-LP containing a Jab1/MPN domain metalloenzyme motif

We have isolated a cDNA clone encoding a new AMSH (associated molecule with the SH3 domain of STAM) family protein, termed AMSH-like protein (AMSH-LP). AMSH-LP has similar characteristics to AMSH; both AMSH-LP and AMSH are expressed ubiquitously in various human tissues, contain a putative nuclear localization signal (NLS), an Mpr/Pad1/N-terminal (MPN) domain, and a Jab1/MPN domain metalloenzyme (JAMM) motif in their structures, and are excluded from the nucleus when lacking either the NLS or MPN domain. Moreover, we observed an enhancement of interleukin 2 (IL-2)-mediated c-myc induction in AMSH-LP-transfected cells similar to that seen in AMSH-transfected cells, suggesting a functional similarity between AMSH-LP and AMSH. However, the present study demonstrated that AMSH-LP, unlike AMSH, fails to bind to the SH3 domains of STAM1 (signal transducing adaptor molecule 1) and Grb2. These results suggest that AMSH-LP and AMSH may have different functions.     

Interaction of the deafness-dystonia protein DDP/TIMM8a with the signal transduction adaptor molecule STAM1

The Mohr-Tranebjaerg-Jensen deafness-dystonia-optic atrophy protein DDP/TIMM8a is translated on cytoplasmic ribosomes but targeted ultimately to the mitochondrial intermembrane space, where it is involved in mitochondrial protein import. STAM1 is a cytoplasmic signal-transducing adaptor molecule implicated in cytokine signaling. We report here a direct interaction between DDP and STAM1, identified by yeast two-hybrid screening and confirmed by co-immunoprecipitation, fusion protein "pull downs," and nuclear redistribution assays. DDP coordinates Zn(2+), and Zn(2+) was found to stimulate the DDP-STAM1 interaction in vitro. Endogenous STAM1 localizes predominantly to early endosomes, and we found no evidence that STAM1 is imported into mitochondria in vitro. Thus, the DDP-STAM1 interaction likely occurs in the cytoplasm or at the mitochondrial outer membrane. The DDP-STAM1 interaction requires a coiled-coil region in STAM1 that overlaps with the immunoreceptor tyrosine-based activation motif (ITAM), a region previously shown to be important for interaction with Jak2/3 and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs). Thus, DDP binding may alter the interactions of STAM1 with several cytoplasmic proteins involved in cell signaling and endosomal trafficking.     

Novel Mechanism of Hemin Capture by Hbp2, the Hemoglobin-binding Hemophore from Listeria monocytogenes

Iron is an essential nutrient that is required for the growth of the bacterial pathogen Listeria monocytogenes. In cell cultures, this microbe secretes hemin/hemoglobin-binding protein 2 (Hbp2; Lmo2185) protein, which has been proposed to function as a hemophore that scavenges heme from the environment. Based on its primary sequence, Hbp2 contains three NEAr transporter (NEAT) domains of unknown function. Here we show that each of these domains mediates high affinity binding to ferric heme (hemin) and that its N- and C-terminal domains interact with hemoglobin (Hb). The results of hemin transfer experiments are consistent with Hbp2 functioning as an Hb-binding hemophore that delivers hemin to other Hbp2 proteins that are attached to the cell wall. Surprisingly, our work reveals that the central NEAT domain in Hbp2 binds hemin even though its primary sequence lacks a highly conserved YXXXY motif that is used by all other previously characterized NEAT domains to coordinate iron in the hemin molecule. To elucidate the mechanism of hemin binding by Hbp2, we determined crystal structures of its central NEAT domain (Hbp2^N2; residues 183–303) in its free and hemin-bound states. The structures reveal an unprecedented mechanism of hemin binding in which Hbp2^N2 undergoes a major conformational rearrangement that facilitates metal coordination by a non-canonical tyrosine residue. These studies highlight previously unrecognized plasticity in the hemin binding mechanism of NEAT domains and provide insight into how L. monocytogenes captures heme iron.     

The death-domain fold of the ASC PYRIN domain, presenting a basis for PYRIN/PYRIN recognition

The PYRIN domain is a conserved sequence motif identified in more than 20 human proteins with putative functions in apoptotic and inflammatory signalling pathways. The three-dimensional structure of the PYRIN domain from human ASC was determined by NMR spectroscopy. The structure determination reveals close structural similarity to death domains, death effector domains, and caspase activation and recruitment domains, although the structural alignment with these other members of the death-domain superfamily differs from previously predicted amino acid sequence alignments. Two highly positively and negatively charged surfaces in the PYRIN domain of ASC result in a strong electrostatic dipole moment that is predicted to be present also in related PYRIN domains. These results suggest that electrostatic interactions play an important role for the binding between PYRIN domains. Consequently, the previously reported binding between the PYRIN domains of ASC and ASC2/POP1 or between the zebrafish PYRIN domains of zAsc and Caspy is proposed to involve interactions between helices 2 and 3 of one PYRIN domain with helices 1 and 4 of the other PYRIN domain, in analogy to previously reported homophilic interactions between caspase activation and recruitment domains.     

Backbone 1H, 13C, and 15N assignments for the tandem ubiquitin binding domains of signal transducing adapter molecule 1

Signal transducing adapter molecule (STAM) forms the endosomal sorting complex required for transport-0 (ESCRT-0) complex with hepatocyte growth factor-regulated substrate (Hrs) to sort the ubiquitinated cargo proteins from the early endosomes to the ESCRT-1 complex. ESCRT-0 complex, STAM and Hrs, contains multiple ubiquitin binding domains, in which STAM has two ubiquitin binding domains, Vps27/Hrs/Stam (VHS) and ubiquitin interacting motif (UIM) at its N-terminus. By the cooperation of the multiple ubiquitin binding domains, the ESCRT-0 complex recognizes poly-ubiquitin, especially Lys63-linked ubiquitin. Here, we report the backbone resonance assignments and the secondary structure of the N-terminal 191 amino acids of the human STAM1 which includes the VHS domain and UIM. The {¹H}-¹⁵N heteronuclear NOE experiments revealed that an unstructured and flexible loop region connects the VHS domain and UIM. Our work provides the basic information for the further NMR investigation of the interaction between STAM1 and poly-ubiquitin.     

The MIT domain of UBPY constitutes a CHMP binding and endosomal localization signal required for efficient epidermal growth factor receptor degradation

We have identified and characterized a Microtubule Interacting and Transport (MIT) domain at the N terminus of the deubiquitinating enzyme UBPY/USP8. In common with other MIT-containing proteins such as AMSH and VPS4, UBPY can interact with CHMP proteins, which are known to regulate endosomal sorting of ubiquitinated receptors. Comparison of binding preferences for the 11 members of the human CHMP family between the UBPY MIT domain and another ubiquitin isopeptidase, AMSH, reveals common interactions with CHMP1A and CHMP1B but a distinct selectivity of AMSH for CHMP3/VPS24, a core subunit of the ESCRT-III complex, and UBPY for CHMP7. We also show that in common with AMSH, UBPY deubiquitinating enzyme activity can be stimulated by STAM but is unresponsive to its cognate CHMPs. The UBPY MIT domain is dispensable for its catalytic activity but is essential for its localization to endosomes. This is functionally significant as an MIT-deleted UBPY mutant is unable to rescue its binding partner STAM from proteasomal degradation or reverse a block to epidermal growth factor receptor degradation imposed by small interfering RNA-mediated depletion of UBPY.     

STAM and Hrs are subunits of a multivalent ubiquitin-binding complex on early endosomes

STAM1 and STAM2, which have been identified as regulators of receptor signaling and trafficking, interact directly with Hrs, which mediates the endocytic sorting of ubiquitinated membrane proteins. The STAM proteins interact with the same coiled-coil domain that is involved in the targeting of Hrs to endosomes. In this work, we show that STAM1 and STAM2, as well as an endocytic regulator protein, Eps15, can be co-immunoprecipitated with Hrs both from membrane and cytosolic fractions and that recombinant Hrs, STAM1/STAM2, and Eps15 form a ternary complex. We find that overexpression of Hrs causes a strong recruitment of STAM2 to endosome membranes. Moreover, STAM2, like Hrs and Eps15, binds ubiquitin, and Hrs, STAM2, and Eps15 colocalize with ubiquitinated proteins in clathrin-containing endosomal microdomains. The localization of Hrs, STAM2, Eps15, and clathrin to endosome membranes is controlled by the AAA ATPase mVps4, which has been implicated in multivesicular body formation. Depletion of cellular Hrs by small interfering RNA results in a strongly reduced recruitment of STAM2 to endosome membranes and an impaired degradation of endocytosed epidermal growth factor receptors. We propose that Hrs, Eps15, and STAM proteins function in a multivalent complex that sorts ubiquitinated proteins into the multivesicular body pathway.     

Loss of hippocampal CA3 pyramidal neurons in mice lacking STAM1

STAM1, a member of the STAM (signal transducing adapter molecule) family, has a unique structure containing a Src homology 3 domain and ITAM (immunoreceptor tyrosine-based activation motif). STAM1 was previously shown to be associated with the Jak2 and Jak3 tyrosine kinases and to be involved in the regulation of intracellular signal transduction mediated by interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. Here we generated mice lacking STAM1 by using homologous recombination with embryonic stem cells. STAM1(-/-) mice were morphologically indistinguishable from their littermates at birth. However, growth retardation in the third week after birth was observed for the STAM1(-/-) mice. Unexpectedly, despite the absence of STAM1, hematopoietic cells, including T- and B-lymphocyte and other hematopoietic cell populations, developed normally and responded well to several cytokines, including IL-2 and GM-CSF. However, histological analyses revealed the disappearance of hippocampal CA3 pyramidal neurons in STAM1(-/-) mice. Furthermore, we observed that primary hippocampal neurons derived from STAM1(-/-) mice are vulnerable to cell death induced by excitotoxic amino acids or an NO donor. These data suggest that STAM1 is dispensable for cytokine-mediated signaling in lymphocytes but may be involved in the survival of hippocampal CA3 pyramidal neurons.     

Binding of EMSY to HP1beta: implications for recruitment of HP1beta and BS69

EMSY is a large nuclear protein that binds to the transactivation domain of BRCA2. EMSY contains an approximately 100-residue segment at the amino terminus called the ENT (EMSY N-terminal) domain. Plant proteins containing ENT domains also contain members of the royal family of chromatin-remodelling domains. It has been proposed that EMSY may have a role in chromatin-related processes. This is supported by the observation that a number of chromatin-regulator proteins, including HP1beta and BS69, bind directly to EMSY by means of a conserved motif adjacent to the ENT domain. Here, we report the crystal structure of residues 1-108 of EMSY at 2.0 A resolution. The structure contains both the ENT domain and the HP1beta/BS69-binding motif. This binding motif forms an extended peptide-like conformation that adopts distinct orientations in each subunit of the dimer. Biophysical and nuclear magnetic resonance analyses show that the main complex formed by EMSY and the chromoshadow domain of HP1 (HP1-CSD) consists of one EMSY dimer sandwiched between two HP1-CSD dimers. The HP1beta-binding motif is necessary and sufficient for EMSY to bind to the chromoshadow domain of HP1beta.     

The deubiquitinating enzyme DUB2A enhances CSF3 signalling by attenuating lysosomal routing of the CSF3 receptor

Ubiquitination of the CSF3R [CSF3 (colony-stimulating factor 3) receptor] occurs after activated CSF3Rs are internalized and reside in early endosomes. CSF3R ubiquitination is crucial for lysosomal routing and degradation. The E3 ligase SOCS3 (suppressor of cytokine signalling 3) has been shown to play a major role in this process. Deubiquitinating enzymes remove ubiquitin moieties from target proteins by proteolytic cleavage. Two of these enzymes, AMSH [associated molecule with the SH3 domain of STAM (signal transducing adaptor molecule)] and UBPY (ubiquitin isopeptidase Y), interact with the general endosomal sorting machinery. Whether deubiquitinating enzymes control CSF3R trafficking from early towards late endosomes is unknown. In the present study, we asked whether AMSH, UBPY or a murine family of deubiquitinating enzymes could fulfil such a role. This DUB family (deubiquitin enzyme family) comprises four members (DUB1, DUB1A, DUB2 and DUB2A), which were originally described as being haematopoietic-specific and cytokine-inducible, but their function in cytokine receptor routing and signalling has remained largely unknown. We show that DUB2A expression is induced by CSF3 in myeloid 32D cells and that DUB2 decreases ubiquitination and lysosomal degradation of the CSF3R, leading to prolonged signalling. These results support a model in which CSF3R ubiquitination is dynamically controlled at the early endosome by feedback mechanisms involving CSF3-induced E3 ligase (SOCS3) and deubiquitinase (DUB2A) activities.