SH3 Domains Of CIN85 Research Articles

Biochemical and Structural Studies of the Interaction between ARAP1 and CIN85.

Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 1 (ARAP1), Cbl-interacting protein of 85 kDa (CIN85), and casitas B-lineage lymphoma (Cbl) play important roles in epidermal growth factor receptor (EGFR) internalization and recycling. In previous studies, ARAP1 was found to interact with CIN85, and their interaction attenuated the ubiquitination of EGFR. However, the molecular mechanism was still unclear. In this study, we first biochemically and structurally characterized the interaction between ARAP1 and CIN85, and found that the CIN85 SH3B domain bound to the ARAP1 PXPXXRX (except P) XXR/H/K motif with high affinity and specificity. Based on this binding model, we further predicted other potential CIN85 binding partners and tested their interactions biochemically. Moreover, our swapping data and structure alignment analysis suggested that the β2-β3 loops of the CIN85 SH3 domains and the H87ARAP1/E132CIN85 interaction were critical for ARAP1 binding specificity. Finally, our competitive analytical gel-filtration chromatography and isothermal titration calorimetry (ITC) results showed that ARAP1 could compete with Cbl for CIN85 binding, which provides a biochemical basis for the regulatory roles of ARAP1 in the CIN85-mediated EGFR internalizing process.

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.

SH3KBP1‐binding protein 1 prevents epidermal growth factor receptor degradation by the interruption of c‐Cbl‐CIN85 complex

The binding of Cbl-interacting protein of 85 kDa (CIN85) to c-Cbl is important to endocytosis and degradation of epidermal growth factor receptor (EGFR). The proline-arginine motif PXXXPR in c-Cbl and SH3 domains of CIN85 are essential to this interaction. Here, we demonstrated that SH3KBP1-binding protein 1 (SHKBP1), which also contains two PXXXPR motifs, constitutively bound to SH3 domains of CIN85. Importantly, the binding of SHKBP1 prevented the interaction of CIN85 with c-Cbl and inhibited the translocation of CIN85 to EGFR-containing vesicles, thus reducing EGFR degradation and enhancing EGF-induced serum response element transcription activity. Therefore, our results indicated that SHKBP1 could promote EGFR signaling pathway by interrupting c-Cbl-CIN85 complex and inhibiting EGFR degradation.

Phosphorylation of Human Tristetraprolin in Response to Its Interaction with the Cbl Interacting Protein CIN85

BackgroundTristetraprolin (TTP) is the prototype member of a family of CCCH tandem zinc finger proteins and is considered to be an anti-inflammatory protein in mammals. TTP plays a critical role in the decay of tumor necrosis factor alpha (TNF) mRNA, among others, by binding AU-rich RNA elements in the 3′-untranslated regions of this transcript and promoting its deadenylation and degradation.Methodology/Principal FindingsWe used yeast two-hybrid analysis to identify potential protein binding partners for human TTP (hTTP). Various regions of hTTP recovered 31 proteins that fell into 12 categories based on sequence similarities. Among these, the interactions between hTTP and CIN85, cytoplasmic poly (A) binding protein (PABP), nucleolin and heat shock protein 70 were confirmed by co-immunoprecipitation experiments. CIN85 and hTTP co-localized in the cytoplasm of cells as determined by confocal microscopy. CIN85 contains three SH3 domains that specifically bind a unique proline-arginine motif (PXXXPR) found in several CIN85 effectors. We found that the SH3 domains of CIN85 bound to a PXXXPR motif located near the C-terminus of hTTP. Co-expression of CIN85 with hTTP resulted in the increased phosphorylation of hTTP at serine residues in positions 66 and 93, possibly due in part to the demonstrated association of mitogen-activated protein kinase kinase kinase 4 (MEKK4) to both proteins. The presence of CIN85 did not appear to alter hTTP's binding to RNA probes or its stimulated breakdown of TNF mRNA.Conclusions/SignificanceThese studies describe interactions between hTTP and nucleolin, cytoplasmic PABP, heat shock protein 70 and CIN85; these interactions were initially discovered by two-hybrid analysis, and confirmed by co-immunoprecipitation. We found that CIN85 binding to a C-terminal motif within hTTP led to the increased phosphorylation of hTTP, possibly through enhanced association with MEKK4. The functional consequences to each of the members of this putative complex remain to be determined.

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 Three CIN85 SH3 Domains and Ubiquitin: Implications for CIN85 Ubiquitination

CIN85 is an adaptor protein linking the ubiquitin ligase Cbl and clathrin-binding proteins in clathrin-mediated receptor endocytosis. The SH3 domains of CIN85 bind to a proline-rich region of Cbl. Here we show that all three SH3 domains of CIN85 bind to ubiquitin. We also present a data-based structural model of the CIN85 SH3-C domain in complex with ubiquitin. In this complex, ubiquitin binds to the canonical interaction surface of the SH3 domain for proline-rich ligands and mimics the PPII helix, and we provide evidence that ubiquitin competes with these ligands for binding. We demonstrate that disruption of ubiquitin binding results in constitutive ubiquitination of CIN85 and an increased level of ubiquitination of EGFR in the absence of EGF stimulation. These results suggest that competition between Cbl and ubiquitin binding to CIN85 regulates Cbl function and EGFR endocytosis.

Regulation of the interaction of Disabled‐1 with CIN85 by phosphorylation with Cyclin‐dependent kinase 5

Disabled-1 (Dab1) is an adaptor protein mediating Reelin signaling in neuronal migration during brain development. Cyclin-dependent kinase 5 (Cdk5)-p35 is a proline-directed Ser/Thr kinase also involved in neuronal migration. The interaction between Dab1 and Cdk5 is in need of investigation. Dab1 was phosphorylated at Ser400 and Ser491 by Cdk5 in vivo. We search for proteins that interact with Dab1 in a phosphorylation-dependent manner at these sites, and identified CIN85, an SH3-containing adaptor protein involved in endocytosis, and CPalpha/CPbeta, which are subunits of barbed end F-actin-capping proteins (CP), as proteins bound to unphosphorylated Dab1 by mass spectrometric analysis. It was shown that the PTPAPR sequence of Dab1, conforming to the PxxxPR atypical SH3-binding motif, was the binding site for SH3 domains of CIN85. The results that phosphorylation at Ser491 close to the PTPAPR sequence inhibited association with CIN85 may provide a mechanism regulating the interaction between the PxxxPR motif proteins and SH3 domains of CIN85 family proteins. Together with previous results that CIN85 regulates actin assembly, present results raise the possibility that Cdk5 modulates actin dynamics through regulation of CIN85-Dab1 interaction by the Dab1 phosphorylation.

Abl-SH3 binding protein 2, 3BP2, interacts with CIN85 and HIP-55

The adapter 3BP2 is involved in leukocyte signaling downstream Src/Syk-kinases coupled immunoreceptors. Here, we show that 3BP2 directly interacts with the endocytic scaffold protein CIN85 and the actin-binding protein HIP-55. 3BP2 co-localized with CIN85 and HIP-55 in T cell rafts and at the T cell/APC synapse, an active zone of receptors and proteins recycling. A binding region of CIN85 SH3 domains on 3BP2 was mapped to a PVPTPR motif in the first proline-rich region of 3BP2, whereas the C-terminal SH3 domain of HIP-55 bound a more distal proline-rich domain of 3BP2. Together, our data suggest an unexpected role of 3BP2 in endocytic and cytoskeletal regulation through its interaction with CIN85 and HIP-55.

CIN85 regulates the ability of MEKK4 to activate the p38 MAP kinase pathway

CIN85 is a multi-adaptor protein involved in different cellular functions including the down-regulation of activated receptor tyrosine kinases and survival of neuronal cells. CIN85 contains three SH3 domains that specifically bind a unique proline-arginine motif (PxxxPR) found in several CIN85 effectors. In this report, we show that the MAP kinase kinase kinase MEKK4 is a new CIN85-interacting partner. This interaction is mediated by the engagement of the SH3 domains of CIN85 to three PxxxPR motifs located within MEKK4 sequence. By disrupting this interaction we demonstrated that CIN85 binding to MEKK4 enhances the activation of MKK6 and of the downstream p38 MAP kinase following oxidative stress and growth factor stimulation. CIN85 was also shown to regulate the activation of MEKK4 by GADD45 proteins and promote multi-ubiquitination of MEKK4. Taken together, these results indicate a novel role for CIN85 in the regulation of cellular stress response via the MAPK pathways.

Cbl promotes clustering of endocytic adaptor proteins

The ubiquitin ligases c-Cbl and Cbl-b play a crucial role in receptor downregulation by mediating multiple monoubiquitination of receptors and promoting their sorting for lysosomal degradation. Their function is modulated through interactions with regulatory proteins including CIN85 and PIX, which recognize a proline-arginine motif in Cbl and thus promote or inhibit receptor endocytosis. We report the structures of SH3 domains of CIN85 and beta-PIX in complex with a proline-arginine peptide from Cbl-b. Both structures reveal a heterotrimeric complex containing two SH3 domains held together by a single peptide. Trimerization also occurs in solution and is facilitated by the pseudo-symmetrical peptide sequence. Moreover, ternary complexes of CIN85 and Cbl are formed in vivo and are important for the ability of Cbl to promote epidermal growth factor receptor (EGFR) downregulation. These results provide molecular explanations for a novel mechanism by which Cbl controls receptor downregulation.

Sprouty2 acts at the Cbl/CIN85 interface to inhibit epidermal growth factor receptor downregulation

The ubiquitin ligase Cbl mediates ubiquitination of activated receptor tyrosine kinases (RTKs) and interacts with endocytic scaffold complexes, including CIN85/endophilins, to facilitate RTK endocytosis and degradation. Several mechanisms regulate the functions of Cbl to ensure the fine-tuning of RTK signalling and cellular homeostasis. One regulatory mechanism involves the binding of Cbl to Sprouty2, which sequesters Cbl away from activated epidermal growth factor receptors (EGFRs). Here, we show that Sprouty2 associates with CIN85 and acts at the interface between Cbl and CIN85 to inhibit EGFR downregulation. The CIN85 SH3 domains A and C bind specifically to proline-arginine motifs present in Sprouty2. Intact association between Sprouty2, Cbl and CIN85 is required for inhibition of EGFR endocytosis as well as EGF-induced differentiation of PC12 cells. Moreover, Sprouty4, which lacks CIN85-binding sites, does not inhibit EGFR downregulation, providing a molecular explanation for functional differences between Sprouty isoforms. Sprouty2 therefore acts as an inducible inhibitor of EGFR downregulation by targeting both the Cbl and CIN85 pathways.

CIN85 associates with TNF receptor 1 via Src and modulates TNF-α-induced apoptosis

CIN85 is a multidomain protein that associates with receptors carrying tyrosine kinase domains. Here we report that it is also a component of the signaling complex associated with tumor necrosis factor receptor 1 (TNFR1), which lacks a tyrosine kinase domain. This was established by showing that CIN85 was co-precipitated with TNFR1, TRADD, cIAP-1 and TARF1/2, but not with FADD, RIP, caspase-8 or TRAF6. However, CIN85 did not bind directly to the cytoplasmic domain of TNFR1 (TNFR1-CYT) but to Src family kinases, Cbl and the p85α subunit of phosphatidylinositol 3-kinase (PI3-K p85α). Src bound directly to TNFR1-CYT, but Cbl and PI3-K p85α did not. A human cell line ectopically expressing CIN85 was 10 times more susceptible to TNF-α-induced apoptosis than control cells, which expressed identical levels of TNFR1 on their surface. However, the susceptibility of these two cell lines to CD95-induced apoptosis was the same. The three SH3 domains of CIN85 were essential for this increased susceptibility to apoptosis and its proline-rich regions were also required for maximal effect. TNF-α treatment recruited CIN85 to the TNFR1 signaling complex. Taken together, these results indicate that CIN85 associates with TNFR1 via Src and modulates TNF-α-induced apoptosis.

Dab2 links CIN85 with clathrin-mediated receptor internalization

CIN85 is a multidomain scaffold protein involved in downregulation of receptor tyrosine kinases. Here we show that disabled-2 (Dab2), an endocytic adaptor molecule implicated in clathrin-coat assembly, associates with CIN85 in mammalian cells. All three SH3 domains of CIN85 were able to bind to the PKPAPR peptide in the carboxyl-terminal part of Dab2, possibly enabling CIN85 to simultaneously interact with multiple Dab2 molecules. CIN85 association with Dab2 is essential for its recruitment to clathrin coat and appears to be modulated by growth factor stimulation. Dab2 and clathrin dissociated from CIN85 following growth factor treatment, enabling other molecules, such as Cbl, to bind to CIN85. Taken together, our data indicate a dynamic interplay between CIN85 and its effectors during endocytosis of receptor tyrosine kinases.

Identification of a Novel Proline-Arginine Motif Involved in CIN85-dependent Clustering of Cbl and Down-regulation of Epidermal Growth Factor Receptors

CIN85 is a multidomain adaptor protein implicated in Cbl-mediated down-regulation of receptor tyrosine kinases. CIN85 binding to Cbl is increased after growth factor stimulation and is critical for targeting receptor tyrosine kinases to clathrin-mediated endocytosis. Here we report the identification of a novel polyproline-arginine motif (PXXXPR), specifically recognized by the SH3 domains of CIN85 and its homologue CMS/CD2AP. This motif was indispensable for CIN85 binding to Cbl/Cbl-b, to other CIN85 SH3 domains' effectors, and for mediating an intramolecular interaction between the SH3-A domain and the proline-rich region of CIN85. Individual SH3 domains of CIN85 bound to PXXXPR peptides of Cbl/Cbl-b with micromolar affinities, whereas an extended structure of two or three SH3 domains bound with higher stoichiometry and increased affinity to the same peptides. This enabled full size CIN85 to simultaneously interact with multiple Cbl molecules, promoting their clustering in mammalian cells. The ability of CIN85 to cluster Cbl was important for ligand-induced stabilization of CIN85.Cbl.epidermal growth factor receptor complexes, as well as for epidermal growth factor receptor degradation in the lysosome. Thus, specific interactions of CIN85 SH3 domains with the PXXXPR motif in Cbl play multiple roles in down-regulation of receptor tyrosine kinases.

New SH3 Recognition Motif

Proteins of the CIN85 and CMS adaptor family contain SH3 domains, which are proline recognition sequences mediating protein interactions. However, the binding sites within known CIN85 target proteins do not appear to be canonical P-X-X-P motifs. Kurakin et al . used target-assisted iterative screening (TAIS) to identify sequences preferred by the first, second, and third SH3 domains of CIN85. Although there were variations in residues tolerated in the "X" positions, each of the CIN85 SH3 domains recognized the sequence P-X-(P or A)-X-X-R. Homology searches for the CIN85 SH3 recognition motif showed many potential new partners and confirmed the presence of this motif in known CIN85 partners. Four of the newly identified potential partners were investigated further to confirm an interaction with CIN85. Interaction with two of these, synaptojanin 1 and the kinase PAK2, was detected in mouse brain lysates by glutathione S -transferase (GST) fusion protein pull-down assay and by Far Western analysis; Z0-2 and TAF II 70 were only detected in a GST pull-down assay. This approach and the results demonstrate (i) that TAIS is an effective method for identifying binding motifs, which can then be used to identify new partners, (ii) that the CIN85 SH3 domain recognizes a noncanonical proline sequence, and (iii) that CIN85 may also interact with synaptojanin 1, PAK2, ZO-2, and TAF II 70, which suggests additional functions. A. V. Kurakin, S. Wu, D. E. Bredesen, Atypical recognition consensus of CIN85/SETA/Ruk SH3 domains revealed by target-assisted iterative screening. J. Biol. Chem. 278 , 34102-34109 (2003). [Abstract] [Full Text]

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.

Characterization of the CIN85 Adaptor Protein and Identification of Components Involved in CIN85 Complexes

CIN85 is an 85-kDa adaptor protein whose functions in signaling pathways are presently unknown. Using the yeast two-hybrid screen, the B cell linker protein (BLNK) was identified as a binding partner of CIN85. Coimmunoprecipitation experiments using mammalian cells revealed that CIN85 directly bound to BLNK through its SH3 domains. Immunostaining analysis showed that CIN85 and BLNK were colocalized in the cytoplasm. These results indicate a potential role of CIN85 in the B cell receptor-mediated signaling pathway. It was also found that Crk-I, Crk-II, p130Cas, p85-PI3K, Grb2, and Sos1 were components of CIN85 complexes. CIN85 interacted with itself through its coiled-coil region, resulting in formation of a tetramer. Both the coiled-coil region and SH3 domains of CIN85 were responsible for its subcellular localization. Our data suggest that CIN85 may serve for regulation of various signaling events through formation of its diverse complexes.