Marc Lewitzky

Essential Role of Ubiquitin-Specific Protease 8 for Receptor Tyrosine Kinase Stability and Endocytic Trafficking In Vivo

ABSTRACT Posttranslational modification by ubiquitin controls multiple cellular functions and is counteracted by the activities of deubiquitinating enzymes. UBPy (USP8) is a growth-regulated ubiquitin isopeptidase that interacts with the HRS-STAM complex. Using Cre-loxP-mediated gene targeting in mice, we show that lack of UBPy results in embryonic lethality, whereas its conditional inactivation in adults causes fatal liver failure. The defect is accompanied by a strong reduction or absence of several growth factor receptor tyrosine kinases (RTKs), like epidermal growth factor receptor, hepatocyte growth factor receptor (c-met), and ERBB3. UBPy-deficient cells exhibit aberrantly enlarged early endosomes colocalizing with enhanced ubiquitination and have reduced levels of HRS and STAM2. Congruently immortalized cells gradually stop proliferation upon induced deletion of UBPy. These results unveil a central and nonredundant role of UBPy in growth regulation, endosomal sorting, and the control of RTKs in vivo.

The C-terminal SH3 domain of the adapter protein Grb2 binds with high affinity to sequences in Gab1 and SLP-76 which lack the SH3-typical P-x-x-P core motif.

The adapter Grb2 is an important mediator of normal cell proliferation and oncogenic signal transduction events. It consists of a central SH2 domain flanked by two SH3 domains. While the binding specificities of the Grb2 SH2 and N-terminal SH3 domain [Grb2 SH3(N)] have been studied in detail, binding properties of the Grb2 SH3(C) domain remained poorly defined. Gab1, a receptor tyrosine kinase substrate which associates with Grb2 and the c-Met receptor, was previously shown to bind Grb2 via a region which lacks a Grb2 SH3(N)-typical motif (P-x-x-P-x-R). Precipitation experiments with the domains of Grb2 show now that Gab1 can bind stably to the Grb2 SH3(C) domain. For further analyses, Gab1 mutants were generated by PCR to test in vivo residues thought to be crucial for Grb2 SH3(C) binding. The Grb2 SH3(C) binding region of Gab1 has significant homology to a region of the adapter protein SLP-76. Peptides corresponding to epitopes SLP-76, Gab1, SoS and other proteins with related sequences, as well as mutant peptides were synthesized and analysed by tryptophan-fluorescence spectrometry and by in vitro competition experiments. These experiments define a 13 amino acid sequence with the unusual consensus motif P-x-x-x-R-x-x-K-P as required for a stable binding to the SH3(C) domain of Grb2. Additional analyses point to a distinct binding specificity of the Grb2-homologous adapter protein Mona (Gads), indicating that the proteins of the Grb2 adapter family may have partially overlapping, yet distinct protein binding properties.

Structural basis for SH3 domain-mediated high-affinity binding between Mona/Gads and SLP-76

SH3 domains are protein recognition modules within many adaptors and enzymes. With more than 500 SH3 domains in the human genome, binding selectivity is a key issue in understanding the molecular basis of SH3 domain interactions. The Grb2‐like adaptor protein Mona/Gads associates stably with the T‐cell receptor signal transducer SLP‐76. The crystal structure of a complex between the C‐terminal SH3 domain (SH3C) of Mona/Gads and a SLP‐76 peptide has now been solved to 1.7 Å. The peptide lacks the canonical SH3 domain binding motif P–x–x–P and does not form a frequently observed poly‐proline type II helix. Instead, it adopts a clamp‐like shape around the circumfence of the SH3C β‐barrel. The central R–x–x–K motif of the peptide forms a 310 helix and inserts into a negatively charged double pocket on the SH3C while several other residues complement binding through hydrophobic interactions, creating a short linear SH3C binding epitope of uniquely high affinity. Interestingly, the SH3C displays ion‐dependent dimerization in the crystal and in solution, suggesting a novel mechanism for the regulation of SH3 domain functions.

Distinct Binding Modes of Two Epitopes in Gab2 that Interact with the SH3C Domain of Grb2

Grb2 and Gab2 form a complex implicated in normal cell signaling and cancer development. Binding of the Grb2SH3C domain to Gab2 is essential for the interaction, but molecular details remained undefined. Using peptide arrays and isothermal titration calorimetry, two Grb2SH3C binding sites in Gab2 (Gab2a and Gab2b) were confirmed and characterized. Gab2a bears similarity to a p27Kip1 epitope that also binds Grb2SH3C. Crystal structures of both Gab2 epitopes complexed with Grb2SH3C reveal that Gab2b contains a 3(10) helix that positions the arginine and lysine of the core-binding motif RxxK in parallel orientation. In contrast, the Gab2a RxxK motif is embedded in a PPII helix with Arg and Lys in staggered orientation. A similar interaction mode is also present in a new complex of Mona/GadsSH3C with an RxxxxK epitope from the putative phosphatase HD-PTP. In summary, our study reveals interaction types of SH3 domains, highlighting their great versatility.

Multiple interactions of the cytosolic polyproline region of the CD95 ligand: hints for the reverse signal transduction capacity of a death factor1

The CD95/Fas/Apo‐1 ligand is expressed on activated lymphocytes, NK cells, platelets, certain immune‐privileged cells and some tumor cells and induces apoptosis through the death receptor CD95/Fas/Apo‐1. In murine T cells, membrane‐bound CD95L (Fas ligand) also acts as a costimulatory receptor to coordinate activation and function in vivo. The molecular basis for this reverse signal transduction is yet unknown. In the present report, we identify individual interaction domains of enzymes and adapter molecules that selectively interact with full‐length CD95L from transfectants and human T cells. These results may help to explain the costimulatory capacity of CD95L.

Mona/Gads SH3C Binding to Hematopoietic Progenitor Kinase 1 (HPK1) Combines an Atypical SH3 Binding Motif, R/KXXK, with a Classical PXXP Motif Embedded in a Polyproline Type II (PPII) Helix

Hematopoietic progenitor kinase 1 (HPK1) is implicated in signaling downstream of the T cell receptor. Its non-catalytic, C-terminal half contains several prolinerich motifs, which have been shown to interact with different SH3 domain-containing adaptor proteins in vitro. One of these, Mona/Gads, was also shown to bind HPK1 in mouse T cells in vivo. The region of HPK1 that binds to the Mona/Gads C-terminal SH3 domain has been mapped and shows only very limited similarity to a recently identified high affinity binding motif in SLP-76, another T-cell adaptor. Using isothermal titration calorimetry and x-ray crystallography, the binding of the HPK1 motif to Mona/Gads SH3C has now been characterized in molecular detail. The results indicate that although charge interactions through an RXXK motif are essential for complex formation, a PXXP motif in HPK1 strongly complements binding. This unexpected binding mode therefore differs considerably from the previously described interaction of Mona/Gads SH3C with SLP-76. The crystal structure of the complex highlights the great versatility of SH3 domains, which allows interactions with very different proteins. This currently limits our ability to categorize SH3 binding properties by simple rules.

Potential disease targets for drugs that disrupt protein-- protein interactions of Grb2 and Crk family adaptors.

This review summarises some of the knowledge we have about Crk and Grb2 family adaptor protein signalling in health and disease and outlines the current status and the challenges still remaining in the development of efficient and selective inhibitors of their protein - protein interactions. It also highlights briefly some recent successes and problems of inhibitors for proteins that functionally interact with Crk and Grb2 family adaptors, as well as opportunities, which may arise from combination therapies. Grb2 and Crk family adaptors regulate signalling pathways linked to human diseases. They are mainly composed of Src homology 2 (SH2) and Src homology 3 (SH3) domains, which serve as docking sites for signalling proteins, including various receptors, cytoplasmic kinases and GTPase regulators. Considerable insight into the biological functions and mechanisms of action of small SH2/SH3 domain adaptors has been gained in the last years from experimental approaches as diverse as targeted gene disruption and structural studies at the atomic level. This has already indicated several strategies to utilise SH2 and SH3 domain interaction inhibitors in human disease therapy. Additional molecular targets for Crk and Grb2 domain interaction blockers are expected to surface as further protein-protein interactions are discovered. Examples include newly found DOCK family proteins (DOCK3, DOCK4, and DOCK5) which are known or suspected effectors of Crk proteins and the interaction of Grb2 with the cell cycle regulator p27Kip1.

Role of the Kaposi's Sarcoma-Associated Herpesvirus K15 SH3 Binding Site in Inflammatory Signaling and B-Cell Activation

ABSTRACT The Kaposis sarcoma-associated herpesvirus (KSHV) contains several open reading frames (ORFs) that encode proteins capable of initiating and modulating cellular signaling pathways. Among them is ORF K15, encoding a 12-transmembrane-spanning protein with a cytoplasmic C-terminal domain. Through conserved binding motifs, such as Src homology 2 (SH2) and SH3 binding sites, K15 interacts with cellular proteins, activates the NF-κB, MEK/Erk, and Jun N-terminal protein kinase (JNK) pathways, and induces the expression of several inflammatory and angiogenic genes. In this study, we investigated the role of an SH3 domain binding site centered on a PPLP motif in K15. We screened libraries of cellular SH3 domains to identify signaling molecules interacting with the KSHV PPLP motif. We found its affinities for two Src kinase family members, Lyn and Hck, to exceed those of other viral proteins. While the SH2 binding motif YEEV is essential for the inflammatory response induced by KSHV K15, recruitment of Lyn and Hck to the K15 PPLP motif seems to be dispensable for this inflammatory response. However, the PPLP motif is essential for the decrease in B-cell receptor-mediated signaling induced by K15, as measured by calcium mobilization assays.

Conformational Recognition of an Intrinsically Disordered Protein

There is a growing interest in understanding the properties of intrinsically disordered proteins (IDPs); however, the characterization of these states remains an open challenge. IDPs appear to have functional roles that diverge from those of folded proteins and revolve around their ability to act as hubs for protein-protein interactions. To gain a better understanding of the modes of binding of IDPs, we combined statistical mechanics, calorimetry, and NMR spectroscopy to investigate the recognition and binding of a fragment from the disordered protein Gab2 by the growth factor receptor-bound protein 2 (Grb2), a key interaction for normal cell signaling and cancer development. Structural ensemble refinement by NMR chemical shifts, thermodynamics measurements, and analysis of point mutations indicated that the population of preexisting bound conformations in the free-state ensemble of Gab2 is an essential determinant for recognition and binding by Grb2. A key role was found for transient polyproline II (PPII) structures and extended conformations. Our findings are likely to have very general implications for the biological behavior of IDPs in light of the evidence that a large fraction of these proteins possess a specific propensity to form PPII and to adopt conformations that are more extended than the typical random-coil states.

Beyond 'furballs' and 'dumpling soups' - towards a molecular architecture of signaling complexes and networks.

The molecular architectures of intracellular signaling networks are largely unknown. Understanding their design principles and mechanisms of processing information is essential to grasp the molecular basis of virtually all biological processes. This is particularly challenging for human pathologies like cancers, as essentially each tumor is a unique disease with vastly deranged signaling networks. However, even in normal cells we know almost nothing. A few ‘signalosomes’, like the COP9 and the TCR signaling complexes have been described, but detailed structural information on their architectures is largely lacking. Similarly, many growth factor receptors, for example EGF receptor, insulin receptor and c‐Met, signal via huge protein complexes built on large platform proteins (Gab, Irs/Dok, p130Cas[BCAR1], Frs families etc.), which are structurally not well understood. Subsequent higher order processing events remain even more enigmatic. We discuss here methods that can be employed to study signaling architectures, and the importance of too often neglected features like macromolecular crowding, intrinsic disorder in proteins and the sophisticated cellular infrastructures, which need to be carefully considered in order to develop a more mature understanding of cellular signal processing.