Diffraction data were collected at BL711, MaxLab, Lund, Sweden from a cryoprotected (20% trehalose, 100 mM Bicine and 3.3 M AmS) type I crystal. PheA503 occupies the P−1 site and is involved in hydrophobic contacts with residues TrpC529 and ProC544. 35S‐radiolabeled full‐length IB1 was found to bind to the SH3 domains of IB1 and to a weaker extent to that of IB2, but neither to GST alone nor to the GAP or Grb2 SH3 domains (Figure 2A). [])), +((!+[]+(!![])+!![]+!![]+!![]+!![]+!![]+!![]+[])+(+!![])+(!+[]+(!![])+!![]+!![]+!![]+!![]+!![])+(!+[]+(!![])+!![]+!![]+!![])+(!+[]-(!![]))+(!+[]+(!![])+!![]+!![])+(!+[]+(!![])-[])+(!+[]+(!![])+!![]+!![]+!![])+(!+[]+(!![])+!![]))/+((!+[]+(!![])-[]+[])+(!+[]+(!![])-[])+(!+[]+(!![])+!![]+!![]+!![])+(!+[]+(!![])+!![])+(!+[]+(!![])+!![]+!![]+!![]+!![]+!![]+!![])+(!+[]+(!![])-[])+(!+[]+(!![])+!![]+!![]+!![]+!![]+!![]+!![]+!![])+(!+[]+(!![])+!![]+!![]+!![]+!![]+!![]+!![]+!![])+(!+[]+(!![])+!! Almost all latest releases in one place, each album is available for download in a good quality At 24 h after the transfection, the cells were harvested with 50 μl of the passive lysis buffer (Promega). IB1, a JIP‐1‐related nuclear protein present in insulin‐secreting cells, Cell‐permeable peptide inhibitors of JNK: novel blockers of beta‐cell death, IB1 reduces cytokine‐induced apoptosis of insulin‐secreting cells, A peptide inhibitor of c‐Jun N‐terminal kinase protects against excitotoxicity and cerebral ischemia, Mutational analysis of the regulatory function of the c‐Abl Src homology 3 domain, Crystallography & NMR system: a new software suite for macromolecular structure determination, The CCP4 suite: programs for protein crystallography, Crystallization and preliminary crystallographic characterization of an SH3 domain from the IB1 scaffold protein, Signal transduction by the JNK group of MAP kinases, An Src homology 3‐like domain is responsible for dimerization of the repressor protein KorB encoded by the promiscuous IncP plasmid RP4, A cytoplasmic inhibitor of the JNK signal transduction pathway, Structure of the regulatory domains of the Src‐family tyrosine kinase Lck, Maximum‐likelihood heavy‐atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods, Structural basis for SH3 domain‐mediated high‐affinity binding between Mona/Gads and SLP‐76, Structural basis for the selective inhibition of JNK1 by the scaffolding protein JIP1 and SP600125, Subcellular distribution and function of Rab3A, B, C, and D isoforms in insulin‐secreting cells, A scaffold protein JIP‐1b enhances amyloid precursor protein phosphorylation by JNK and Its association with kinesin light chain 1, Principles of protein–protein interactions, The importance of being proline: the interaction of proline‐rich motifs in signaling proteins with their cognate domains, Interaction of a mitogen‐activated protein kinase signaling module with the neuronal protein JIP3, The identification of conserved interactions within the SH3 domain by alignment of sequences and structures, Role of the Lck Src homology 2 and 3 domains in protein tyrosine phosphorylation, SMART 4.0: towards genomic data integration, Stability and peptide binding affinity of an SH3 domain from the, Amyloid beta protein precursor (AbetaPP), but not AbetaPP‐like protein 2, is bridged to the kinesin light chain by the scaffold protein JNK‐interacting protein 1, Docking interactions in the c‐Jun N‐terminal kinase pathway, Mixed lineage kinase‐dependent JNK activation is governed by interactions of scaffold protein JIP with MAPK module components, Novel recognition mode between Vav and Grb2 SH3 domains, Comparative protein modelling by satisfaction of spatial restraints, Autoinhibition of Bcr‐Abl through its SH3 domain, The scaffold protein IB1/JIP‐1 controls the activation of JNK in rat stressed urothelium, A combined experimental and computational strategy to define protein interaction networks for peptide recognition modules, Cargo of kinesin identified as JIP scaffolding proteins and associated signaling molecules, The gene MAPK8IP1, encoding islet‐brain‐1, is a candidate for type 2 diabetes, A mammalian scaffold complex that selectively mediates MAP kinase activation, Requirement of the JIP1 scaffold protein for stress‐induced JNK activation, The JNK‐interacting protein‐1 scaffold protein targets MAPK phosphatase‐7 to dephosphorylate JNK, The JIP group of mitogen‐activated protein kinase scaffold proteins, Structural basis for the binding of proline‐rich peptides to SH3 domains, Autoinhibition of mixed lineage kinase 3 through its Src homology 3 domain, Interface contributions from individual residues are given both as the interacting area (Å. I purchase my Mosley MP-33-N-WARC with the 40-Meter conversion kit on May 19, 2020 and it was delivered to my Florida home on July 27, 2020 right on the schedule they gave me when I placed the order. Taken together, the two β‐sheets form a barrel. [])), +((!+[]+(!![])+!![]+!![]+!![]+!![]+!![]+!![]+[])+(!+[]+(!![])+!![])+(!+[]+(!![])+!![]+!![]+!![]+!![]+!![]+!![])+(!+[]-(!![]))+(!+[]+(!![])+!![]+!![])+(+!![])+(!+[]+(!![])+!![]+!![]+!![]+!![])+(!+[]+(!![])+!![]+!![])+(+!![]))/+((!+[]+(!![])+!![]+!![]+!![]+!![]+!![]+!![]+!![]+[])+(!+[]+(!![])+!![]+!![]+!![]+!![])+(!+[]+(!![])-[])+(!+[]+(!![])+!![]+!![])+(!+[]+(!![])+!![]+!![])+(+!![])+(!+[]+(!![])+!![]+!![]+!![])+(!+[]+(!![])-[])+(!+[]+(!![])+!![]+!![]+!! We thank Séverine Arcioni and Laurent Spack for critical comments and helpful discussions. In Src family members, SH3 domains are believed to function in the control of subcellular localization and the regulation of kinase activity and as sites of interaction with other signal transduction proteins (2, 10, 12, 43, 44). As in SEM‐5 and in most other SH3 domains (Larson and Davidson, 2000), this residue is an aspargine, and it contributes one of the three important hydrogen bonds to the backbone carbonyls of the polyproline helix. A total of seven PxxP motifs are found in the sequence of IB1 (Figure 1A), two of which are conserved in mouse, rat and human. There are currently no videos available for this product. After 3 days, the cells were washed and preincubated for 30 min in buffer (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 2 mM NaHCO3, 10 mM Hepes and 0.1% bovine serum albumin) containing 2 mM glucose (basal conditions). Figure 4A shows how IB1 SH3 (molecule A) residues interact with the PPII binding pockets of molecule C. The Tyr546 of the molecule A (TyrA546) occupies the P+3 recognition site of molecule C, stacking partly with PheC501 and interacting via further hydrophobic contacts with TyrC547. The fold is as expected from many previous structural investigations (Mayer, 2001). Both the type II and type III crystal structures were solved by molecular replacement based on the derived type I model and refined using CNS (Brunger et al, 1998). The program PyMOL (DeLano, 2002) was used to prepare figures. The highly homologous SH3 domain of IB2 was produced as well, together with two unrelated SH3 domains that were used as specificity controls: the SH3 domain of the GTPase‐activated protein (GAP) and the C‐terminal SH3 domain of the growth factor receptor‐bound protein 2 (Grb2). This identified only the different IB1‐related variants, and it revealed that this particular dimerization scheme is unique to the IB1 system.
The P−3 site is typically occupied by a positively charged residue like arginine as is the case in the SEM‐5 structure where side‐chain atoms engage in hydrogen bonding to Glu172 and a water molecule.
Type I and II data were integrated and scaled using MOSFLM and SCALA (CCP4, 1994), see Table I. (, Mutations R506A, R506E, H507D, Y546A and R506A/H507D destabilize IB1 dimerization. The program SOLVE (Terwilliger and Berendzen, 1999) was used to locate the four expected heavy‐atom positions based on the type I SeMet data. This work was supported by DANSYNC, the Danish Medical Research Council, the Danish Natural Science Research Council, the European Community (Contract number HPRI‐CT‐1999‐00017), the Swiss National Science Foundation (FN 3200BO‐105595), the Swiss–French Diabetes Foundation and the Botnar Foundation. Likewise, cells were co‐transfected with either wt or mutant GFP‐tagged SH3 constructs (GFP‐SH3 wt, GFP‐SH3 R506A). Larger IB1 SH3‐S crystals (type II) were produced using a modified reservoir solution (100 mM Bicine, pH 9.0, 3.1 M AmS and 2% polyethylene glycol (PEG) 400).
The contribution of the SH3 domain to IB1 oligomerization was investigated by in vitro pull‐down experiments using chimeric GST‐SH3 protein constructs. GluC510 makes strong interactions with the ArgC506 main chain amide and the HisC507 side chain of the same molecule, occluding the role of arginine as a favored P−3 residue. IB1 has been shown to participate in the regulation of important cellular events in neurons and pancreatic β‐cells, including apoptosis, expression of the GLUT2 and glucose‐induced insulin secretion (Bonny et al, 1998; Waeber et al, 2000). (, These authors contributed equally to this work.
Gel filtration experiments, dynamic light‐scattering and structure determinations based on X‐ray crystallography support this observation. At 24 h after the transfection, luciferase activities were measured. (as shown from eight experiments; P<0.001 for IB1 wt compared with each mutant, see Supplementary data for statistical details) at the optimal concentration (0.1 μg of IB1 DNA; Figure 6). Our data link IB1 dimerization to these important regulatory functions. [])), +((!+[]+(!![])+!![]+!![]+!![]+!![]+!![]+!![]+[])+(!+[]+(!![])+!![])+(!+[]+(!![])+!![]+!![]+!![]+!![]+!![]+!![])+(!+[]-(!![]))+(!+[]+(!![])+!![]+!![])+(+!![])+(!+[]+(!![])+!![]+!![]+!![]+!![])+(!+[]+(!![])+!![]+!![])+(!+[]+(!![])+!![]+!![]+!![]+!![]+!![]))/+((!+[]+(!![])+!![]+!![]+!![]+!![]+!![]+!![]+!![]+[])+(+!![])+(!+[]+(!![])+!![])+(!+[]-(!![]))+(!+[]-(!![]))+(!+[]+(!![])-[])+(!+[]+(!![])+!![]+!![]+!![]+!![])+(!+[]-(!![]))+(!+[]-(!! In a similar way, immunoprecipitated Flag, Flag‐IB1 wt or mutant was incubated with 35S‐labeled MKK7α1. Gel filtration and dynamic light‐scattering experiments confirmed a dimeric state in solution (see Supplementary data). IB1 has been initially characterized both as a cytoplasmic inhibitor and as an activator of the JNK signaling pathway.
All three kinases, JNK, MKK7 and MLK3, appear to bind to the IB1 mutants to a similar extent as to the wt protein (Figure 6C). The SH3 domain of Lck contains residues that are equivalent of the IB1 SH3 His507‐Asp509 pair. For example, the SH3 domains of members of the tyrosine kinase family (Src, c‐Abl, Bcr‐Abl) and of the serine/threonine kinase MLK3 autoinhibit their kinase activities (Nguyen and Lim, 1997; Brasher et al, 2001; Zhang and Gallo, 2001; Smith et al, 2003). These results show that homophilic interactions occur between the SH3 domains of each IB1 molecule. Download PDF of article text and main figures. To better characterize the surface of the interface, the SH3 structure of SEM‐5, the Caenorhabditis elegans homologue for Grb2 and the IB1 SH3 structure have been superimposed (Figure 4B). It has also been suggested that MKK7 binds to the leucine zipper of MLK3, which in turn prevents homodimerization and activation of MLK3 (Mooney and Whitmarsh, 2004). Accordingly, in pancreatic β‐cells and in urothelium, it was demonstrated that IB1 decreases JNK‐mediated c‐Jun phosphorylation, and concomitant apoptosis (Bonny et al, 2000; Tawadros et al, 2002). One of the important functions of IB1 (Bonny et al, 1998; Waeber et al, 2000) is the control of the expression of GLUT2 and the regulation of the insulin‐secretory process.
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