Structural basis for ubiquitin recognition by SH3 domains

The SH3 domain is a protein-protein interaction module commonly found in intracellular signaling and adaptor proteins. The SH3 domains of multiple endocytic proteins have been recently implicated in binding ubiquitin, which serves as a signal for diverse cellular processes including gene regulation, endosomal sorting, and protein destruction. Here we describe the solution NMR structure of ubiquitin in complex with an SH3 domain belonging to the yeast endocytic protein Sla1. The ubiquitin binding surface of the Sla1 SH3 domain overlaps substantially with the canonical binding surface for proline-rich ligands. Like many other ubiquitin-binding motifs, the SH3 domain engages the Ile44 hydrophobic patch of ubiquitin. A phenylalanine residue located at the heart of the ubiquitin-binding surface of the SH3 domain serves as a key specificity determinant. The structure of the SH3-ubiquitin complex explains how a subset of SH3 domains has acquired this non-traditional function.     

Distinct interactions between ubiquitin and the SH3 domains involved in immune signaling

The SH3 domain is a versatile protein interaction motif that generally recognizes proline rich sequences (PRS). Recently, it has been shown that some SH3 domains in the endocytotic pathway can bind to ubiquitin. Moreover, Phe73 in the SH3 domain has been proposed to be an important determinant of the interaction, as the SH3 domains having Tyr73, either naturally or by mutation, failed to bind. Since SH3 domains are also important in immune receptor signaling, we investigated the interactions between immunologically relevant SH3 domains and ubiquitin. We observed that some of these SH3 domains can also bind to ubiquitin. Interestingly, we found that Nck2-SH3-3 bound to ubiquitin despite its Tyr at residue 73 (Tyr56 in our actual construct), but that CD2BP1-SH3 failed to bind, even though it has Phe at an equivalent position. Through detailed NMR binding studies on SH3 domains with Phes and Tyrs at the 73 position, we found that the two types of SH3 domains exhibit mechanistic differences in ubiquitin binding. We showed that the relative contribution of each binding sub-region in both SH3 domains and ubiquitin is quite different in the two binding modes. Such results raise the possibility that the mechanistic variety of these immunologically relevant SH3 domains might contribute to their functional diversity.     

Proteins recruited by SH3 domains of Ruk/CIN85 adaptor identified by LC-MS/MS

Background  

Ruk/CIN85 is a mammalian adaptor molecule with three SH3 domains. Using its SH3 domains Ruk/CIN85 can cluster multiple proteins and protein complexes, and, consequently, facilitates organisation of elaborate protein interaction networks with diverse regulatory roles. Previous research linked Ruk/CIN85 with the regulation of vesicle-mediated transport and cancer cell invasiveness. Despite the recent findings, precise molecular functions of Ruk/CIN85 in these processes remain largely elusive and further research is hampered by a lack of complete lists of its partner proteins.     

Atypical recognition consensus of CIN85/SETA/Ruk SH3 domains revealed by target-assisted iterative screening

Target-assisted iterative screening applied to random peptide libraries unveiled a novel and atypical recognition consensus shared by CIN85/SETA/Ruk SH3 domains, PX(P/A)XXR. Confirmed by mutagenesis and in vitro binding experiments, the novel consensus allowed for the accurate mapping of CIN85 SH3 binding sites within known CIN85 interactors, c-Cbl, BLNK, Cbl-b, AIP1/Alix, SB1, and CD2 proteins, as well as the prediction of CIN85 novel-interacting partners in protein databases. Synaptojanin 1, PAK2, ZO-2, and TAFII70, which contain CIN85 SH3 recognition consensus sites, were selectively precipitated from mouse brain lysates by CIN85 SH3 domains in glutathione S-transferase pull-down experiments. A direct interaction of synaptojanin 1 and PAK2 with CIN85 SH3 domains was confirmed by Far Western blotting.     

CIN85 regulates ubiquitination and degradative endosomal sorting of the EGF receptor

CIN85 has been demonstrated to interact with a number of proteins involved in endocytosis and intracellular sorting. However, the exact functional role of CIN85 in endocytosis remains unclear. We have investigated whether CIN85 plays a role in EGF-induced EGF receptor (EGFR) internalization, as previously suggested, or whether CIN85 is rather involved in endosomal sorting of the EGFR. When over-expressing a dominant negative interfering CIN85 mutant consisting of three SH3 domains only, we found that internalization of EGF was inhibited. However, when knocking down CIN85 by RNAi, the EGF–EGFR uptake appeared similar to in control cells. Furthermore, in CIN85 depleted cells, EGF-induced ubiquitination of the EGFR was decreased, and degradation of EGF–EGFR complexes was delayed. Our data further demonstrated that depletion of CIN85 increased the recycling of EGF, suggesting that CIN85 plays a role in endosomal sorting of the ubiquitinated EGFR. Our data also demonstrated that CIN85 was constitutively associated with Hrs, and this strengthens the hypothesis of a functional role of CIN85 in endosomal EGFR sorting.     

CIN85 phosphorylation is essential for EGFR ubiquitination and sorting into multivesicular bodies

Ubiquitination of the epidermal growth factor receptor (EGFR) by cbl and its cognate adaptor cbl-interacting protein of 85 kDa (CIN85) is known to play an essential role in directing this receptor to the lysosome for degradation. The mechanisms by which this ubiquitin modification is regulated are not fully defined, nor is it clear where this process occurs. In this study we show that EGFR activation leads to a pronounced src-mediated tyrosine phosphorylation of CIN85 that subsequently influences EGFR ubiquitination. Of importance, phospho-CIN85 interacts with the Rab5-positive endosome, where it mediates the sequestration of the ubiquitinated receptor into multivesicular bodies (MVBs) for subsequent degradation. These findings provide novel insights into how src- kinase-based regulation of a cbl adaptor regulates the fate of the EGFR.     

Interactions of polyomavirus middle T with the SH2 domains of the pp85 subunit of phosphatidylinositol-3-kinase.

The binding of phosphatidylinositol-3-kinase to the polyomavirus middle T antigen is facilitated by tyrosine phosphorylation of middle T on residue 315. The pp85 subunit of phosphatidylinositol-3-kinase contains two SH2 domains, one in the middle of the molecule and one at the C terminus. When assayed by blotting with phosphorylated middle T, the more N-terminal SH2 domain is responsible for binding to middle T. When assayed in solution with glutathione S transferase fusions, both SH2s are capable of binding phosphorylated middle T. While both SH2 fusions can compete with intact pp85 for binding to middle T, the C-terminal SH2 is the more efficient of the two. Interaction between pp85 or its SH2 domains and middle T can be blocked by a synthetic peptide comprising the tyrosine phosphorylation sequence around middle T residue 315. Despite the fact that middle T can interact with both SH2s, these domains are not equivalent. Only the C-terminal SH2-middle T interaction was blocked by anti-SH2 antibody; the two SH2 fusions also interact with different cellular proteins.     

Signalling through SH2 and SH3 domains

In 1986, Pawson's group recognized a region of homology between two oncogenic tyrosine kinases that lay outside the catalytic domain. They termed this the Src homology 2, or SH2, domain. In the ensuing years, SH2 domains have been found in an impressive variety of proteins, as has a second region of homology, inevitably termed SH3. These domains appear to mediate controlled protein-protein interactions. Many proteins that contain SH2 and SH3 domains are involved in signal transduction, suggesting a new paradigm for regulation of intracellular signalling pathways.     

Inhibition of CIN85-Mediated Invasion by a Novel SH3 Domain Binding Motif in the Lysyl Oxidase Propeptide

The lysyl oxidase gene inhibits Ras signaling in transformed fibroblasts and breast cancer cells. Its activity was mapped to the 162 amino acid propeptide domain (LOX-PP) of the lysyl oxidase precursor protein. LOX-PP inhibited the Her-2/Ras signaling axis in breast cancer cells, and reduced the Her-2-driven breast tumor burden in a xenograft model. Since its mechanism of action is largely unknown, co-affinity-purification/mass spectrometry was performed and the “Cbl-interacting protein of 85-kDa” (CIN85) identified as an associating protein. CIN85 is an SH3-containing adapter protein that is overexpressed in invasive breast cancers. The CIN85 SH3 domains interact with c-Cbl, an E3 ubiquitin ligase, via an unconventional PxxxPR ligand sequence, with the highest affinity displayed by the SH3-B domain. Interaction with CIN85 recruits c-Cbl to the AMAP1 complex where its ubiquitination activity is necessary for cancer cells to develop an invasive phenotype and to degrade the matrix. Direct interaction of LOX-PP with CIN85 was confirmed using co-immunoprecipitation analysis of lysates from breast cancer cells and of purified expressed proteins. CIN85 interaction with c-Cbl was reduced by LOX-PP. Domain specific CIN85 regions and deletion mutants of LOX-PP were prepared and used to map the sites of interaction to the SH3-B domain of CIN85 and to an epitope encompassing amino acids 111 to 116 of LOX-PP. Specific LOX-PP point mutant proteins P111A and R116A failed to interact with CIN85 or to compete for CIN85 binding with c-Cbl. Structural modeling identified a new atypical PxpxxRh SH3-binding motif in this region of LOX-PP. The LOX-PP interaction with CIN85 was shown to reduce the invasive phenotype of breast cancer cells, including their ability to degrade the surrounding extracellular matrix and for Matrigel outgrowth. Thus, LOX-PP interacts with CIN85 via a novel SH3-binding motif and this association reduces CIN85-promoted invasion by breast cancer cells.     

Transitional forms between the three domains of life and evolutionary implications

The question as to the origin and relationship between the three domains of life is lodged in a phylogenetic impasse. The dominant paradigm is to see the three domains as separated. However, the recently characterized bacterial species have suggested continuity between the three domains. Here, we review the evidence in support of this hypothesis and evaluate the implications for and against the models of the origin of the three domains of life. The existence of intermediate steps between the three domains discards the need for fusion to explain eukaryogenesis and suggests that the last universal common ancestor was complex. We propose a scenario in which the ancestor of the current bacterial Planctomycetes, Verrucomicrobiae and Chlamydiae superphylum was related to the last archaeal and eukaryotic common ancestor, thus providing a way out of the phylogenetic impasse.     

Making ends meet: the importance of the N- and C-termini for the structure, stability, and function of the third SH3 domain of CIN85

SH3 domains are common structure, interaction, and regulation modules found in more than 200 human proteins. In this report, we studied the third SH3 domain from the human CIN85 adaptor protein, which plays an important role in both receptor tyrosine kinase downregulation and phosphatidylinositol 3 kinase inhibition. The structure of this domain includes an additional 90° kink after the last canonical β-strand and features unusual interactions between the termini well outside the boundaries of the standard SH3 domain definition. The extended portions of the domain are well-structured and held together entirely by side chain-side chain interactions. Extensive expression screening showed that these additional contacts provide significantly increased stability to the domain. A similar 90° kink is found in only one other SH3 domain structure, while side chain contacts linking the termini have never been described before. As a result of the increased size of CIN85 SH3 domain C, the proximal proline rich region is positioned such that a possible intramolecular interaction is structurally inhibited. Using the key interactions of the termini as the basis for sequence analysis allowed the identification of several SH3 domains with flanking sequences that could adopt similar structures. This work illustrates the importance of careful experimental analysis of domain boundaries even for a well-characterized fold such as the SH3 domain.     

Novel Insights into the Mechanisms of CIN85 SH3 Domains Binding to Cbl Proteins: Solution-Based Investigations and In Vivo Implications

CIN85 is a multifunctional protein that plays key roles in endocytic down-regulation of receptor tyrosine kinases, apoptosis, cell adhesion, and cytoskeleton rearrangement. Its three SH3 domains (CIN85A, CIN85B, and CIN85C) allow it to recruit multiple binding partners. To understand the manifold interactions of CIN85, we present a detailed high-resolution solution structural study of CIN85A and CIN85B binding to proline-arginine peptides derived from the cognate ligands Cbl and Cbl-b. We report the structure of CIN85B and provide evidence that both CIN85A and CIN85B, in isolation or when linked, form heterodimeric complexes with the peptides. We report unusual curved chemical shift changes for several residues of CIN85A when titrated with Cbl-b peptide, indicating the existence of more than one complex form. Here we demonstrate that CIN85A and CIN85B use different mechanisms for peptide binding.     

Interactions between the quality control ubiquitin ligase CHIP and ubiquitin conjugating enzymes

Background  

Ubiquitin (E3) ligases interact with specific ubiquitin conjugating (E2) enzymes to ubiquitinate particular substrate proteins. As the combination of E2 and E3 dictates the type and biological consequence of ubiquitination, it is important to understand the basis of specificity in E2:E3 interactions. The E3 ligase CHIP interacts with Hsp70 and Hsp90 and ubiquitinates client proteins that are chaperoned by these heat shock proteins. CHIP interacts with two types of E2 enzymes, UbcH5 and Ubc13-Uev1a. It is unclear, however, why CHIP binds these E2 enzymes rather than others, and whether CHIP interacts preferentially with UbcH5 or Ubc13-Uev1a, which form different types of polyubiquitin chains.     

Mutagenic analysis of the v-crk oncogene: requirement for SH2 and SH3 domains and correlation between increased cellular phosphotyrosine and transformation.

We have constructed a series of mutants with deletion, linker insertion, and point mutations in the v-crk oncogene of avian sarcoma virus CT10. The v-crk gene contains no apparent catalytic domain, but does contain two blocks of homology to putative regulatory domains, termed SH2 and SH3, found in a variety of proteins implicated in signal transduction. Infection with CT10 causes a dramatic increase in the level of tyrosine phosphorylation of several cellular proteins. We found that mutation of either the SH2 or SH3 domain of v-crk reduced or eliminated transforming activity, whereas mutation of regions outside the conserved domains had no effect. Deletion of amino-terminal gag sequences caused a partial loss of transforming activity and a change in subcellular distribution of the crk protein. In all cases, there was an absolute correlation between increased cellular phosphotyrosine and transformation.     

Specificity of the interaction between ubiquitin-associated domains and ubiquitin

Ubiquitin-associated (UBA) domains are found in a large number of proteins with diverse functions involved in ubiquitination, DNA repair, and signaling pathways. Recent studies have shown that several UBA domain proteins interact with ubiquitin (Ub), specifically p62, the phosphotyrosine-independent ligand of the SH2 domain of p56(lck); HHR23A, a human nucleotide excision repair protein; and DDI1, another damage-inducible protein. NMR chemical shift mapping reveals that Ub binds specifically but weakly to a conserved hydrophobic epitope on HHR23A UBA(1) and UBA(2) and that the UBA domains bind on the hydrophobic patch on the surface of the five-stranded beta-sheet of Ub. Models of the UBA(1)-Ub and UBA(2)-Ub complexes obtained from de novo docking reveal different orientations of the UBA domains on the Ub surface compared with those obtained by homology modeling with the related CUE domains, which also bind Ub. Our results suggest that UBA domains may interact with Ub as well as other proteins in more than one way while utilizing the same binding surface.