共查询到20条相似文献,搜索用时 15 毫秒
1.
Andrew Severin 《Journal of molecular biology》2009,387(3):726-9615
We report here the NMR-derived structure of the binary complex formed by the interleukin-2 tyrosine kinase (Itk) Src homology 3 (SH3) and Src homology 2 (SH2) domains. The interaction is independent of both a phosphotyrosine motif and a proline-rich sequence, the classical targets of the SH2 and SH3 domains, respectively. The Itk SH3/SH2 structure reveals the molecular details of this nonclassical interaction and provides a clear picture for how the previously described prolyl cis/trans isomerization present in the Itk SH2 domain mediates SH3 binding. The higher-affinity cis SH2 conformer is preorganized to form a hydrophobic interface with the SH3 domain. The structure also provides insight into how autophosphorylation in the Itk SH3 domain might increase the affinity of the intermolecular SH3/SH2 interaction. Finally, we can compare this Itk complex with other examples of SH3 and SH2 domains engaging their ligands in a nonclassical manner. These small binding domains exhibit a surprising level of diversity in their binding repertoires. 相似文献
2.
3.
Mills JE Whitford PC Shaffer J Onuchic JN Adams JA Jennings PA 《Journal of molecular biology》2007,365(5):1460-1468
The SH2 domain of the C-terminal Src kinase [Csk] contains a unique disulfide bond that is not present in other known SH2 domains. To investigate whether this unusual disulfide bond serves a novel function, the effects of disulfide bond formation on catalytic activity of the full-length protein and on the structure of the SH2 domain were investigated. The kinase activity of full-length Csk decreases by an order of magnitude upon formation of the disulfide bond in the distal SH2 domain. NMR spectra of the fully oxidized and fully reduced SH2 domains exhibit similar chemical shift patterns and are indicative of similar, well-defined tertiary structures. The solvent-accessible disulfide bond in the isolated SH2 domain is highly stable and far from the small lobe of the kinase domain. However, reduction of this bond results in chemical shift changes of resonances that map to a cluster of residues that extend from the disulfide bond across the molecule to a surface that is in direct contact with the small lobe of the kinase domain in the intact molecule. Normal mode analyses and molecular dynamics calculations suggest that disulfide bond formation has large effects on residues within the kinase domain, most notably within the active-site cleft. Overall, the data indicate that reversible cross-linking of two cysteine residues in the SH2 domain greatly impacts catalytic function and interdomain communication in Csk. 相似文献
4.
The very C-terminus of c-Src is a ligand for PDZ domains. In a screen for PDZ domains that interact with c-Src, we identified one of the PDZ domains of the Ligand-of-Numb protein X1 (LNX1), a multiple PDZ domain scaffold and RING type E3 ubiquitin ligase. We demonstrate that the interaction of c-Src with LNX1 depends on the C-terminal PDZ ligand of c-Src. Furthermore, we show that c-Src phosphorylates LNX1. Moreover, c-Src itself is ubiquitinated by LNX1, suggesting an interdependent regulation of c-Src and LNX1. 相似文献
5.
Nandan D Tran T Trinh E Silverman JM Lopez M 《Biochemical and biophysical research communications》2007,364(3):601-607
The macrophage protein tyrosine phosphatase-1 SHP-1 has been implicated in the pathogenesis of infection with leishmania. To identify the factors that may interact with SHP-1, Leishmania donovani promastigote lysates were added to a GST-SHP-1 affinity matrix. A 44 kDa specifically bound protein was identified as leishmania fructose-1,6-bisphosphate aldolase (aldolase). Purified leishmania aldolase bound to SHP-1 indicating that the interaction was direct. In contrast, purified mammalian aldolase did not bind to SHP-1. Consistent with this, leishmania aldolase activated SHP-1 in vitro, whereas mammalian aldolase did not. The presence of leishmania aldolase in the cytosolic fractions prepared from infected macrophages indicated that leishmania aldolase is exported from phagolysosomes in infected cells where it can target host cytosolic proteins. In fact, co-immunoprecipitation showed association of leishmania aldolase with SHP-1. Moreover, leishmania aldolase-expressing macrophages showed the deactivated phenotype of leishmania infected cells as judged by much reduced inability to induce expression of nitric-oxide synthase in response to interferon-γ treatment. Collectively, these data show that leishmania aldolase is a novel SHP-1 binding and activating protein that contributes to macrophage dysfunction. 相似文献
6.
7.
The neuronal protein FE65 functions in brain development and amyloid precursor protein (APP) signaling through its interaction with the mammalian enabled (Mena) protein and APP, respectively. The recognition of short polyproline sequences in Mena by the FE65 WW domain has a central role in axon guidance and neuronal positioning in the developing brain. We have determined the crystal structures of the human FE65 WW domain (residues 253-289) in the apo form and bound to the peptides PPPPPPLPP and PPPPPPPPPL, which correspond to human Mena residues 313-321 and 347-356, respectively. The FE65 WW domain contains two parallel ligand-binding grooves, XP (formed by residues Y269 and W280) and XP2 (formed by Y269 and W271). Both Mena peptides adopt a polyproline helical II conformation and bind to the WW domain in a forward (N-C) orientation through selection of the PPPPP motif by the XP and XP2 grooves. This mode of ligand recognition is strikingly similar to polyproline interaction with SH3 domains. Importantly, comparison of the FE65 WW structures in the apo and liganded forms shows that the XP2 groove is formed by an induced-fit mechanism that involves movements of the W271 and Y269 side-chains upon ligand binding. These structures elucidate the molecular determinants underlying polyproline ligand selection by the FE65 WW domain and provide a framework for the design of small molecules that would interfere with FE65 WW-ligand interaction and modulate neuronal development and APP signaling. 相似文献
8.
9.
Huntingtin interacting protein 1 (HIP1) is a member of a family of proteins whose interaction with Huntingtin is critical to prevent cells from initiating apoptosis. HIP1, and related protein HIP12/1R, can also bind to clathrin and membrane phospholipids, and HIP12/1R links the CCV to the actin cytoskeleton. HIP1 and HIP12/1R interact with the clathrin light chain EED regulatory site and stimulate clathrin lattice assembly. Here, we report the X-ray structure of the coiled-coil domain of HIP1 (residues 482-586) that includes residues crucial for binding clathrin light chain. The dimeric HIP1 crystal structure is partially splayed open. The comparison of the HIP1 model with coiled-coil predictions revealed the heptad repeat in the dimeric trunk (S2 path) is offset relative to the register of the heptad repeat from the N-terminal portion (S1 path) of the molecule. Furthermore, surface analysis showed there is a third hydrophobic path (S3) running parallel with S1 and S2. We present structural evidence supporting a role for the S3 path as an interaction surface for clathrin light chain. Finally, comparative analysis suggests the mode of binding between sla2p and clathrin light chain may be different in yeast. 相似文献
10.
Al-Jassar C Knowles T Jeeves M Kami K Behr E Bikker H Overduin M Chidgey M 《Journal of molecular biology》2011,411(5):1049-1061
Desmoplakin is a cytoplasmic desmosomal protein that plays a vital role in normal intercellular adhesion. Mutations in desmoplakin can result in devastating skin blistering diseases and arrhythmogenic right ventricular cardiomyopathy, a heart muscle disorder associated with ventricular arrhythmias, heart failure, and sudden death. The desmoplakin N-terminal region is a 1056-amino-acid sequence of unknown structure. It mediates interactions with other desmosomal proteins, is found in a variety of plakin proteins, and spans what has been termed the “plakin domain,” which includes residues 180-1022 and consists of six spectrin repeats (SRs) and an Src homology 3 domain. Herein we elucidate the architecture of desmoplakin's plakin domain, as well as its constituent tandem SRs. Small-angle X-ray scattering analysis shows that the entire plakin domain has an “L” shape, with a long arm and a short arm held at a perpendicular angle. The long arm is 24.0 nm long and accommodates four stably folded SRs arranged in tandem. In contrast, the short arm is 17.9 nm in length and accommodates two independently folded repeats and an extended C-terminus. We show that mutations linked to arrhythmogenic right ventricular cardiomyopathy (K470E and R808C) cause local conformational alterations, while the overall folded structure is maintained. This provides the first structural and mechanistic insights into an entire plakin domain and provides a basis for understanding the critical role of desmoplakin in desmosome function. 相似文献
11.
Hom RA Vora M Regner M Subach OM Cho W Verkhusha VV Stahelin RV Kutateladze TG 《Journal of molecular biology》2007,373(2):412-423
Epsin and AP180 are essential components of the endocytotic machinery, which controls internalization of protein receptors and other macromolecules at the cell surface. Epsin and AP180 are recruited to the plasma membrane by their structurally and functionally related N-terminal ENTH and ANTH domains that specifically recognize PtdIns(4,5)P2. Here, we show that membrane anchoring of the ENTH and ANTH domains is regulated by the acidic environment. Lowering the pH enhances PtdIns(4,5)P2 affinity of the ENTH and ANTH domains reinforcing their association with lipid vesicles and monolayers. The pH dependency is due to the conserved histidine residues of the ENTH and ANTH domains, protonation of which is necessary for the strong PtdIns(4,5)P2 recognition, as revealed by liposome binding, surface plasmon resonance, NMR, monolayer surface tension and mutagenesis experiments. The pH sensitivity of the ENTH and ANTH domains is reminiscent to the pH dependency of the FYVE domain suggesting a common regulatory mechanism of membrane anchoring by a subset of the PI-binding domains. 相似文献
12.
Lemeer S Bluwstein A Wu Z Leberfinger J Müller K Kramer K Kuster B 《Journal of Proteomics》2012,75(12):3465-3477
The receptor tyrosine kinase DDR1 has been implicated in multiple human cancers and fibrosis and is targeted by the leukemia drug Gleevec. This suggests that DDR1 might be a new therapeutic target. However, further insight into the DDR1 signaling pathway is required in order to support its further development. Here, we investigated DDR1 proximal signaling by the analysis of protein-protein interactions using proteomic approaches. All known interactors of DDR1 were identified and localized to specific phosphotyrosine residues on the receptor. In addition, we identified numerous signaling proteins as new putative phosphotyrosine mediated interactors including RasGAP, SHIP1, SHIP2, STATs, PI3K and the SRC family kinases. Most of the new proteins contain SH2 and PTB domains and for all interactors we could directly point the site of interaction to specific phosphotyrosine residues on the receptor. The identified proteins have roles in the early steps of the signaling cascade, propagating the signal from the DDR1 receptor into the cell. The map of phosphotyrosine mediated interactors of DDR1 created in this study will serve as a starting point for functional investigations which will enhance our knowledge on the role of the DDR1 receptor in health and disease. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry. 相似文献
13.
14.
RASSF5 is a member of the Ras association domain family, which is known to be involved in cell growth regulation. Expression of RASSF5 is extinguished selectively by epigenetic mechanism(s) in different cancers and cell lines, and reexpression usually suppresses cell proliferation and tumorigenicity. To date, the mechanism regulating RASSF5 nuclear transport and its role in cell growth regulation remains unclear. Using heterokaryon assay, we have demonstrated that RASSF5 shuttles between the nucleus and the cytoplasm, and its export from the nucleus is sensitive to leptomycin B, suggesting that RASSF5 is exported from the nucleus by a CRM-1-dependent export pathway. We further demonstrate that RASSF5 contains a hydrophobic-rich nuclear export signal (NES) towards the C-terminus and two nuclear localization signals—one each at the N-terminus and the C-terminus. Combination of mutational and immunofluorescence analyses suggests that the functional NES residing between amino acids 260 and 300 in the C-terminus is necessary for the efficient export of RASSF5 from the nucleus. In addition, substitution of conserved hydrophobic residues within the minimal NES impaired RASSF5 export from the nucleus. Furthermore, exchange of proline residues within the putative Src homology 3 binding motifs altered the export of RASSF5 from the nucleus despite the presence of functional NES, suggesting that multiple domains independently modulate the nucleocytoplasmic transport of RASSF5. Interestingly, the present investigation provided evidence that RASSF5 interacts with the tyrosine kinase Lck through its C-terminal Src homology 2 binding motif and showed that Lck-mediated phosphorylation is critical for the efficient translocation of RASSF5 into the nuclear compartment. Interestingly, our data demonstrate that wild type and nuclear export defective (ΔNES) mutant of RASSF5 but not the import defective mutant of accumulate the cells at G1/S phase and induce apoptosis. Furthermore, the Lck-interaction-defective mutant of RASSF5 induces apoptosis without altering cell cycle progression, suggesting that RASSF5 induces apoptosis independent of cell cycle arrest. Together, our data demonstrate that interaction with Lck is critical for RASSF5 phosphorylation, which in turn regulates the cell growth control activity of RASSF5. Finally, we have shown that RASSF5 encodes four splice variants and is translocated to the nucleus by the classical nuclear import pathway. One of the splice variants, RASSF5C, was found to be localized in the cytoplasm and translocated into the nucleus upon leptomycin B treatment despite the absence of N-terminal nuclear localization signal, suggesting that distribution of RASSF5 variants in different cellular compartments may be critical for Ras-dependent cell growth regulation. Collectively, the present investigation provided evidence that Lck-mediated phosphorylation regulates the nucleocytoplasmic shuttling and cell growth control activities of RASSF5. 相似文献
15.
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. 相似文献
16.
Zhang J Li X Yao B Shen W Sun H Xu C Wu J Shi Y 《Biochemical and biophysical research communications》2007,357(4):931-937
Solution structure of the first Src homology (SH) 3 domain of human vinexin (V_SH3_1) was determined using nuclear magnetic resonance (NMR) method and revealed that it was a canonical SH3 domain, which has a typical beta-beta-beta-beta-alpha-beta fold. Using chemical shift perturbation and surface plasmon resonance experiments, we studied the binding properties of the SH3 domain with two different peptides from vinculin hinge regions: P856 and P868. The observations illustrated slightly different affinities of the two peptides binding to V_SH3_1. The interaction between P868 and V_SH3_1 belonged to intermediate exchange with a modest binding affinity, while the interaction between P856 and V_SH3_1 had a low binding affinity. The structure and ligand-binding interface of V_SH3_1 provide a structural basis for the further functional study of this important molecule. 相似文献
17.
Ca(2+)/calmodulin-dependent protein kinase kinase alpha (CaMKKalpha) plays critical roles in the modulation of neuronal cell survival as well as many other cellular activities. Here we show that 14-3-3 proteins directly regulate CaMKKalpha when the enzyme is phosphorylated by protein kinase A on either Ser74 or Ser475. Mutational analysis revealed that these two serines are both functional: the CaMKKalpha mutant with a mutation at either of these residues, but not the double mutant, was inhibited significantly by 14-3-3. The mode of regulation described herein differs the recently described mode of 14-3-3 regulation of CaMKKalpha. 相似文献
18.
Functional implications of the conformational switch in AICD peptide upon binding to Grb2-SH2 domain
It has been hypothesized previously that synergistic effect of both amyloid precursor protein intracellular C-terminal domain (AICD) and Aβ aggregation could contribute to Alzheimer's disease pathogenesis. Structural studies of AICD have found no stable globular fold over a broad range of pH. Present work is based on the premises that a conformational switch involving the flipping of C-terminal helix of AICD would be essential for effective binding with the Src homology 2 (SH2) domain of growth factor receptor binding protein-2 (Grb2) and subsequent initiation of Grb2-mediated endo-lysosomal pathway. High-resolution crystal structures of Grb2-SH2 domain bound to AICD peptides reveal a unique mode of binding where the peptides assume a noncanonical conformation that is unlike other structures of AICD peptides bound to protein-tyrosine-binding domains or that of its free state; rather, a flipping of the C-terminal helix of AICD is evident. The involvement of different AICD residues in Grb2-SH2 interaction is further elucidated through fluorescence-based assays. Our results reveal the significance of a specific interaction of the two molecules to optimize the rapid transport of AICD inside endosomal vesicles presumably to reduce the cytotoxic load. 相似文献
19.
Activation of extracellular signal-regulated kinase (ERK) is known to be regulated by cell adhesion, namely "anchorage dependence". Most studies on the anchorage-dependent regulation have focused on the upstream activating components. We previously reported that the focal adhesion protein vinexin beta can induce the anchorage-independent activation of ERK2. We show here that vinexin beta-induced anchorage-independent activation of ERK2 involves prevention of the dephosphorylation of ERK2, but not the promotion of MEK1 or Raf1 activity. Furthermore, knockdown of vinexin beta resulted in a faster dephosphorylation of ERK2 in A549 cells. Moreover, the coexpression of MKP3/rVH6, an ERK2 specific phosphatase, suppressed the anchorage-independent activation of ERK2 induced by vinexin beta. These results suggest that vinexin beta can prevent the dephosphorylation of ERK2 stimulated by cell detachment, leading to the anchorage-independent activation of ERK2. Furthermore, we found that phosphatase activity directed against activated ERK2 was higher in suspended cells than in adherent cells. In addition, orthovanadate efficiently induces anchorage-independent activation of ERK2 without marked activation of MEK1 in NIH3T3 cells. These observations suggest that the anchorage dependence of ERK1/2 activation is regulated not only by upstream kinases, Raf1 and MEK, but also by phosphatases acting against ERK1/2 and that vinexin beta can induce anchorage-independent activation of ERK by preventing the inactivation of ERK1/2. 相似文献
20.
The protein kinase KIS is made by the juxtaposition of a unique kinase domain and a C-terminal domain with a U2AF homology motif (UHM), a sequence motif for protein interaction initially identified in the heterodimeric pre-mRNA splicing factor U2AF. This domain of KIS is closely related to the C-terminal UHM domain of the U2AF large subunit, U2AF65. KIS phosphorylates the splicing factor SF1, which in turn enhances SF1 binding to U2AF65 and the 3′ splice site, an event known to take place at an early step of spliceosome assembly. Here, the analysis of the subcellular localization of mutated forms of KIS indicates that the kinase domain of KIS is the necessary domain for its nuclear localization. As in the case of U2AF65, the UHM-containing C-terminal domain of KIS is required for binding to the splicing factors SF1 and SF3b155. The efficiency of KIS binding to SF1 and SF3b155 is similar to that of U2AF65 in pull-down assays. These results further support the functional link of KIS with splicing factors. Interestingly, when compared to other UHM-containing proteins, KIS presents a different specificity for the UHM docking sites that are present in the N-terminal region of SF3b155, thus providing a new insight into the variety of interactions mediated by UHM domains. 相似文献