Spatial proteomics reveal that the protein phosphatase PTP1B interacts with and may modify tyrosine phosphorylation of the rhomboid protease RHBDL4 (original) (raw)
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Functional Proteomics Identifies Protein-tyrosine Phosphatase 1B as a Target of RhoA Signaling
Molecular & Cellular Proteomics, 2006
Rho GTPases are signal transduction effectors that control cell motility, cell attachment, and cell shape by the control of actin polymerization and tyrosine phosphorylation. To identify cellular targets regulated by Rho GT-Pases, we screened global protein responses to Rac1, Cdc42, and RhoA activation by two-dimensional gel electrophoresis and mass spectrometry. A total of 22 targets were identified of which 19 had never been previously linked to Rho GTPase pathways, providing novel insight into pathway function. One novel target of RhoA was protein-tyrosine phosphatase 1B (PTP1B), which catalyzes dephosphorylation of key signaling molecules in response to activation of diverse pathways. Subsequent analysis demonstrated that RhoA enhances post-translational modification of PTP1B, inactivates phosphotyrosine phosphatase activity, and up-regulates tyrosine phosphorylation of p130Cas, a key mediator of focal adhesion turnover and cell migration. Thus, protein profiling reveals a novel role for PTP1B as a mediator of RhoA-dependent phosphorylation of p130Cas.
PLoS ONE, 2012
PTP1B is an endoplasmic reticulum (ER) anchored enzyme whose access to substrates is partly dependent on the ER distribution and dynamics. One of these substrates, the protein tyrosine kinase Src, has been found in the cytosol, endosomes, and plasma membrane. Here we analyzed where PTP1B and Src physically interact in intact cells, by bimolecular fluorescence complementation (BiFC) in combination with temporal and high resolution microscopy. We also determined the structural basis of this interaction. We found that BiFC signal is displayed as puncta scattered throughout the ER network, a feature that was enhanced when the substrate trapping mutant PTP1B-D181A was used. Time-lapse and colocalization analyses revealed that BiFC puncta did not correspond to vesicular carriers; instead they localized at the tip of dynamic ER tubules. BiFC puncta were retained in ventral membrane preparations after cell unroofing and were also detected within the evanescent field of total internal reflection fluorescent microscopy (TIRFM) associated to the ventral membranes of whole cells. Furthermore, BiFC puncta often colocalized with dark spots seen by surface reflection interference contrast (SRIC). Removal of Src myristoylation and polybasic motifs abolished BiFC. In addition, PTP1B active site and negative regulatory tyrosine 529 on Src were primary determinants of BiFC occurrence, although the SH3 binding motif on PTP1B also played a role. Our results suggest that ER-bound PTP1B dynamically interacts with the negative regulatory site at the C-terminus of Src at random puncta in the plasma membrane/substrate interface, likely leading to Src activation and recruitment to adhesion complexes. We postulate that this functional ER/plasma membrane crosstalk could apply to a wide array of protein partners, opening an exciting field of research.
Biochemical Journal, 2002
It has been postulated that PtdIns(3,4)P 2 , one of the immediate breakdown products of PtdIns(3,4,5)P 3 , functions as a signalling molecule in insulin-and growth-factor-stimulated pathways. To date, the tandem-PH-domain-containing protein-1 (TAPP1) and related TAPP2 are still the only known PH-domain-containing proteins that interact strongly and specifically with PtdIns(3,4)P 2 . In this study we demonstrate that endogenously expressed TAPP1, is constitutively associated with the protein-tyrosine-phosphataselike protein-1 (PTPL1 also known as FAP-1). We show that PTPL1 binds to TAPP1 and TAPP2, principally though its first PDZ domain [where PDZ is postsynaptic density protein (PSD-95)/Drosophila disc large tumour suppressor (dlg)/tight junction protein (ZO1)] and show that this renders PTPL1 capable of associating with PtdIns(3,4)P 2 in vitro. Our data suggest that the binding of TAPP1 to PTPL1 does not influence PTPL1 phosphatase activity, but instead functions to maintain PTPL1 in the cytoplasm. Following stimulation of cells with hydrogen peroxide to induce PtdIns(3,4)P 2 production, PTPL1, complexed to TAPP1, translocates to the plasma membrane. This study provides the first evidence that TAPP1 and PtdIns(3,4)P 2 could function to regulate the membrane localization of PTPL1. We speculate that if PTPL1 was recruited to the plasma membrane by increasing levels of PtdIns(3,4)P 2 , it could trigger a negative feedback loop in which phosphoinositide-3-kinase-dependent or other signalling pathways could be switched off by the phosphatase-catalysed dephosphorylation of receptor tyrosine kinases or tyrosine phosphorylated adaptor proteins such as IRS1 or IRS2. Consistent with this notion we observed RNA-interference-mediated knock-down of TAPP1 in HEK-293 cells, enhanced activation and phosphorylation of PKB following IGF1 stimulation.
Biochemistry, 2003
The receptor protein tyrosine phosphatase R (RPTPR) is a transmembrane receptor with two intracellular protein tyrosine phosphatase domains, a catalytically active membrane proximal domain (D1) and a membrane distal phosphatase domain with minimal catalytic activity (D2). Here we elucidate the crystal structure of RPTPR's D2 domain. Unlike D1, D2 exists as a monomer and lacks the N-terminal inhibitory wedge motif. The N-terminal portion of D2 is disordered, and this region linking D1 to D2 is proteolytically labile in solution whether part of D2 alone or tethered to D1, indicating that the polypeptide backbone of this part of D2 is highly flexible, and therefore accessible to proteases under native conditions. Furthermore, we have crystallized the SH2 domain of the protein tyrosine kinase c-Src, a RPTPR substrate, with a phosphopeptide encompassing the C-terminal phosphorylation site of D2 (pTyr789). The SH2 domain of Src binds RPTPR in an extended conformation. The structural and functional data support a D1-D2 arrangement with significant flexibility between phosphatase domains of RPTPR that is likely to be important for dynamic alterations in intra-and/or intermolecular interactions that are critical for RPTPR function.
Journal of Biological Chemistry, 2015
Background: The AAA-ATPase VCP/p97 and the deubiquitinase YOD1 are required in the endoplasmic reticulum-associated degradation (ERAD) of misfolded proteins. Results: Three ERAD substrates (NHK-␣1〈⌻, NS1-kLC, and Tetherin) become cytosolically exposed independently of p97 and YOD1, whereas MHC-I␣-and CD4-induced retro-translocation requires them. Conclusion: VCP/p97 and YOD1 have distinct substrate-dependent activities in ERAD. Significance: We demonstrate two different levels of p97 and YOD1 requirements in ERAD. Endoplasmic reticulum-associated degradation (ERAD) is an essential quality control mechanism of the folding state of proteins in the secretory pathway that targets unfolded/misfolded polypeptides for proteasomal degradation. The cytosolic p97/ valosin-containing protein is an essential ATPase for degradation of ERAD substrates. It has been considered necessary during retro-translocation to extract proteins from the endoplasmic reticulum that are otherwise supposed to accumulate in the endoplasmic reticulum lumen. The activity of the p97-associated deubiquitinylase YOD1 is also required for substrate disposal. We used the in vivo biotinylation retro-translocation assay in mammalian cells under conditions of impaired p97 or YOD1 activity to directly discriminate their requirements and diverse functions in ERAD. Using different ERAD substrates, we found that both proteins participate in two distinct retro-translocation steps. For CD4 and MHC-I␣, which are induced to degradation by the HIV-1 protein Vpu and by the CMV immunoevasins US2 and US11, respectively, p97 and YOD1 have a retro-translocation-triggering role. In contrast, for three other spontaneous ERAD model substrates (NS1, NHK-␣1AT, and BST-2/Tetherin), p97 and YOD1 are required in the downstream events of substrate deglycosylation and proteasomal degradation. Valosin-containing protein, p97 (VCP/p97, Cdc48 in yeast) is an abundant and conserved ATPase belonging to the type II ATPases family, associated with diverse cellular activities (1). p97 is organized into a homohexameric ring-shaped complex. Each protomer contains a flexible N-terminal domain and two ATPase domains (2). The N-terminal portion is involved in interactions with a large number of partners having distinct domains (i.e. UBX/UBX-like (ubiquitin regulatory X), UBD (ubiquitin D), PUB (PNGase/ubiquitin-associated), SHP box, PUL (PLAP (phospholipase A2-activating protein), Ufd3p, and Lub1p), VIM (VCP-interacting motif), VBM (VCP-binding motif)) (3). Many of the various p97 functions are connected to the ubiquitin pathway (4-12). Endoplasmic reticulum-associated degradation (ERAD) 5 represents the main mechanism by which cells control the folding state of molecules within the secretory pathway. Several ER-resident proteins, including chaperones and lectins, participate in the recognition of misfolded or terminally unfolded molecules that are then targeted for proteasomal degradation (13, 14). A crucial step in ERAD, still poorly understood, is the retro-translocation from the ER lumen to the cytosol (15-21). Cytosolic p97 is a key player of ERAD in complex with the heterodimeric co-factor formed by ubiquitin fusion-degradating protein 1 (Ufd1) and nuclear protein localization protein 4 homolog (Npl4) (22, 23). The common view is that the p97-Ufd1-Npl4 complex is recruited to the ER membrane, where several different membrane-embedded ERAD protein components having p97-binding motifs reside (6, 24, 25). The precise mechanism and function of the p97 complex is not very clear. It is well established, however, that loss of p97 ATPase activity blocks the proteasomal degradation of several different ERAD substrates (26-29). These results have been generally interpreted as a stringent requirement of p97 activity in the retrotranslocation step, therefore concluding that stabilization of the substrate protein occurs in the ER lumen or in partially * The authors declare that they have no conflicts of interest with the contents of this article.
Journal of Biological Chemistry, 2010
There is growing evidence that tyrosine phosphatases display an intrinsic enzymatic preference for the sequence context flanking the target phosphotyrosines. On the other hand, substrate selection in vivo is decisively guided by the enzymesubstrate connectivity in the protein interaction network. We describe here a system wide strategy to infer physiological substrates of protein-tyrosine phosphatases. Here we integrate, by a Bayesian model, proteome wide evidence about in vitro substrate preference, as determined by a novel high-density peptide chip technology, and "closeness" in the protein interaction network. This allows to rank candidate substrates of the human PTP1B phosphatase. Ultimately a variety of in vitro and in vivo approaches were used to verify the prediction that the tyrosine phosphorylation levels of five high-ranking substrates, PLC-␥1, Gab1, SHP2, EGFR, and SHP1, are indeed specifically modulated by PTP1B. In addition, we demonstrate that the PTP1B-mediated dephosphorylation of Gab1 negatively affects its EGF-induced association with the phosphatase SHP2. The dissociation of this signaling complex is accompanied by a decrease of ERK MAP kinase phosphorylation and activation. Protein-tyrosine phosphatases (PTPs) 4 in concert with tyrosine kinases contribute to the maintenance of regulated levels of tyrosine phosphorylation in multicellular organisms (1, 2). Evidence accumulated over the past decade has now led to the recognition that PTPs play specific, active and even dominant roles in setting the levels of tyrosine phosphorylation in cells thus participating in the regulation of many physiological processes including cell growth, tissue differentiation, and intercellular communication (3-5). Disruption of PTPs activity or its dys-* This work was supported by grants from the Italian Association for Cancer Research (AIRC), Telethon, and the European Network of Excellence ENFIN.
Herp coordinates compartmentalization and recruitment of HRD1 and misfolded proteins for ERAD
Molecular Biology of the Cell, 2014
A functional unfolded protein response (UPR) is essential for endoplasmic reticulum (ER)-associated degradation (ERAD) of misfolded secretory proteins, reflecting the fact that some level of UPR activation must exist under normal physiological conditions. A coordinator of the UPR and ERAD processes has long been sought. We previously showed that the PKR-like, ER-localized eukaryotic translation initiation factor 2α kinase branch of the UPR is required for the recruitment of misfolded proteins and the ubiquitin ligase HRD1 to the ERderived quality control compartment (ERQC), a staging ground for ERAD. Here we show that homocysteine-induced ER protein (Herp), a protein highly upregulated by this UPR branch, is responsible for this compartmentalization. Herp localizes to the ERQC, and our results suggest that it recruits HRD1, which targets to ERAD the substrate presented by the OS-9 lectin at the ERQC. Predicted overall structural similarity of Herp to the ubiquitin-proteasome shuttle hHR23, but including a transmembrane hairpin, suggests that Herp may function as a hub for membrane association of ERAD machinery components, a key organizer of the ERAD complex.
Biochemical Journal, 2003
It has been postulated that PtdIns(3,4)P 2 , one of the immediate breakdown products of PtdIns(3,4,5)P 3 , functions as a signalling molecule in insulin-and growth-factor-stimulated pathways. To date, the tandem-PH-domain-containing protein-1 (TAPP1) and related TAPP2 are still the only known PH-domain-containing proteins that interact strongly and specifically with PtdIns(3,4)P 2. In this study we demonstrate that endogenously expressed TAPP1, is constitutively associated with the protein-tyrosine-phosphataselike protein-1 (PTPL1 also known as FAP-1). We show that PTPL1 binds to TAPP1 and TAPP2, principally though its first PDZ domain [where PDZ is postsynaptic density protein (PSD-95)/Drosophila disc large tumour suppressor (dlg)/tight junction protein (ZO1)] and show that this renders PTPL1 capable of associating with PtdIns(3,4)P 2 in vitro. Our data suggest that the binding of TAPP1 to PTPL1 does not influence PTPL1 phosphatase activity, but instead functions to maintain PTPL1 in the cytoplasm. Following stimulation of cells with hydrogen peroxide to induce PtdIns(3,4)P 2 production, PTPL1, complexed to TAPP1, translocates to the plasma membrane. This study provides the first evidence that TAPP1 and PtdIns(3,4)P 2 could function to regulate the membrane localization of PTPL1. We speculate that if PTPL1 was recruited to the plasma membrane by increasing levels of PtdIns(3,4)P 2 , it could trigger a negative feedback loop in which phosphoinositide-3-kinase-dependent or other signalling pathways could be switched off by the phosphatase-catalysed dephosphorylation of receptor tyrosine kinases or tyrosine phosphorylated adaptor proteins such as IRS1 or IRS2. Consistent with this notion we observed RNA-interference-mediated knock-down of TAPP1 in HEK-293 cells, enhanced activation and phosphorylation of PKB following IGF1 stimulation.
A subset of RAB proteins modulates PP2A phosphatase activity
Scientific Reports, 2016
Protein phosphatase 2A (PP2A) is one of the most abundant serine-threonine phosphatases in mammalian cells. PP2A is a hetero-trimeric holoenzyme participating in a variety of physiological processes whose deregulation is often associated to cancer. The specificity and activity of this phosphatase is tightly modulated by a family of regulatory B subunits that dock the catalytic subunit to the substrates. Here we characterize a novel and unconventional molecular mechanism controlling the activity of the tumor suppressor PP2A. By applying a mass spectrometry-based interactomics approach, we identified novel PP2A interacting proteins. Unexpectedly we found that a significant number of RAB proteins associate with the PP2A scaffold subunit (PPP2R1A), but not with the catalytic subunit (PPP2CA). Such interactions occur in vitro and in vivo in specific subcellular compartments. Notably we demonstrated that one of these RAB proteins, RAB9, competes with the catalytic subunit PPP2CA in binding to PPP2R1A. This competitive association has an important role in controlling the PP2A catalytic activity, which is compromised in several solid tumors and leukemias. Protein phosphatases act in concert with kinases to fine-tune signaling events by modulating the level of phosphorylated serine, threonine and tyrosine residues 1,2. Protein phosphatase 2A is the most abundant serine/threonine phosphatase in mammals 3 , controlling key physiological processes, including proliferation, apoptosis, differentiation and cell migration 4. Such broad functional specificity is mediated by the array of subunits that associate in a combinatorial fashion to form the functional PP2A holoenzyme 5. The core enzyme is a heterodimer, formed by a catalytic subunit C (encoded by two genes, PPP2CA and PPP2CB) and a scaffold subunit A (encoded by PPP2R1A and PPP2R1B genes) 6. The enzyme core can interact with at least 25 different regulatory subunits, resulting in more than 70 distinct trimeric complexes, differing for their subcellular localization, substrate specificity and enzyme activity 5. Given the importance of protein-protein interactions in defining the function of PP2A, we have recently exploited an immunoprecipitation assay combined with mass spectrometry (MS)-based proteomic analysis to investigate the PP2A interactome 7. Besides recapitulating most of the known PP2A interactors, we found that only the scaffold subunit, and not the catalytic nor the regulatory ones, interacts with a significant number of RAB family members. RAB GTPases (Ras-related in brain) belong to the RAS superfamily of small GTPases and play a prominent role in controlling vesicle trafficking from the donor compartments to the acceptor ones 8. Similarly to other GTPases, the RAB family members can switch from the active GTP-bound conformation, which interacts with downstream effectors proteins, to the inactive GDP-bound form 9. Here we report that RAB8 and RAB9 proteins interact with the PP2A scaffold subunit, PPP2R1A, in a GTP independent manner. This interaction impairs the assembly of the PP2A holoenzyme, which consequently is inactivated. Our results are consistent with a model whereby some specific members of the RAB family play a crucial role in selectively inhibiting the PP2A tumor suppressor in specific subcellular compartments. Results The PP2A holoenzyme protein interaction network. Protein-protein interactions play a pivotal role in defining the function of PP2A, one of the most abundant serine/threonine phosphatase implicated in cancer development. In order to investigate the PP2A interactome, we have recently exploited an immunoprecipitation assay combined with mass spectrometry (MS)-based proteomic analysis to investigate the PP2A interactome in