共查询到20条相似文献,搜索用时 15 毫秒
1.
S Arold R O'Brien P Franken MP Strub F Hoh C Dumas JE Ladbury 《Canadian Metallurgical Quarterly》1998,37(42):14683-14691
Understanding the issue of specificity imposed in the interactions of SH3 domains has largely been addressed in studies investigating the interaction of proline-rich amino acid sequences derived from potential ligands for these domains. Although the interaction with this motif forms an essential platform in the binding of SH3 domains, in many cases little specificity is observed and the difference in affinity for so-called specific and nonspecific proline-rich sequences is not great. Furthermore, the binding interface between an SH3 domain and a protein ligand appears to encompass more interactions than are represented by that involving the proline-rich motif. Here we investigate the issue of specificity from the opposite point of view; namely, how does a ligand recognize different SH3 domains? We present the crystal structure of the unbound SH3 domain from hemopoietic cell kinase (Hck) which is a member of the Src family of tyrosine kinases. This structure reveals that, unlike the structures of other Src kinase SH3 domains, the RT loop region is highly mobile and lacks a network of hydrogen bonds that is elsewhere apparent. The RT loop has been shown to form a major part of the binding interface between SH3 domains and HIV-1 Nef. Thermodynamic data, derived from isothermal titration calorimetry, for the binding of Hck SH3 to HIV-1 Nef show that the binding of Hck (KD = 1.5 microM) is approximately an order of magnitude tighter than those of other Src family kinases that were investigated (Fyn, Lck, and Src). This increase in affinity is attributed to, among other effects, the inherent flexibility in the RT loop which does not require breaking the network of hydrogen bonds to adopt the conformation required for binding. 相似文献
2.
CJ Morton DJ Pugh EL Brown JD Kahmann DA Renzoni ID Campbell 《Canadian Metallurgical Quarterly》1996,4(6):705-714
BACKGROUND: The Src family of tyrosine kinases is involved in the propagation of intracellular signals from many transmembrane receptors. Each member of the family contains two domains that regulate interactions with other molecules, one of which is the Src homology 3 (SH3) domain. Although structures have previously been determined for SH3 domains, and ideas about peptide-binding modes have been proposed, their physiological role is still unclear. RESULTS: We have determined the solution structure of the SH3 domain from the Src family tyrosine kinase Fyn in two forms: unbound and complexed with a peptide corresponding to a putative ligand sequence from phosphatidylinositol 3' kinase. Fyn SH3 shows the typical SH3 topology of two perpendicular three-stranded beta sheets and a single turn of 3(10) helix. The interaction of SH3 with three potential ligand peptides was investigated, demonstrating that they all bind to the same site on the molecule. A previous model for ligand binding to SH3 domains predicts binding in one of two orientations (class I or II), each characterized by a consensus sequence. The ligand with the closest match to the class I consensus sequence bound with highest affinity and in the predicted orientation. CONCLUSIONS: The Fyn SH3 domain has a well-defined structure in solution. The relative binding affinities of the three ligand peptides and their orientation within the Fyn SH3 complex were consistent with recently proposed models for the binding of 'consensus' polyproline sequences. Although the affinities of consensus and non-consensus peptides are different, the degree of difference is not very large, suggesting that SH3 domains bind to polyproline peptides in a promiscuous manner. 相似文献
3.
A dileucine motif in HIV-1 Nef is essential for sorting into clathrin-coated pits and for downregulation of CD4 总被引:1,自引:0,他引:1
M Greenberg L DeTulleo I Rapoport J Skowronski T Kirchhausen 《Canadian Metallurgical Quarterly》1998,8(22):1239-1242
Nef, a approximately 200 residue multifunctional regulatory protein of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), interacts with components of host cell signal transduction and clathrin-dependent protein sorting pathways. The downregulation of surface CD4 molecules and major histocompatibility complex (MHC) class I antigens by Nef is believed to be important in AIDS pathogenesis [1-7]. Nef contains a globular core domain and two disordered segments--a myristylated arm at the amino terminus and a carboxy-terminal loop projecting from the globular core [8,9]. Here, we aimed to determine the sorting signals in HIV-1 Nef that were responsible for its involvement in the clathrin-mediated pathway. We found that a sequence in the carboxy-terminal disordered loop of Nef is essential for downregulation of CD4. This sequence resembles the dileucine motif, one of two well-characterized sorting signals that target membrane proteins to clathrin-coated vesicles. The dileucine-motif-containing segment of Nef bound directly and specifically to the beta-adaptin subunit of the clathrin adaptor complexes AP-1 and AP-2, which are responsible for recruiting sorted proteins into coated pits. Unlike wild-type Nef, a mutant form of Nef that lacked the dileucine motif did not localize to clathrin-coated pits and did not downregulate CD4 expression, although it could downregulate MHC class I surface expression. Thus, the dileucine motif in HIV-1 is required for CD4 downregulation and for interaction with clathrin adaptor complexes. 相似文献
4.
To identify novel proteins capable of associating with the Raf-1 serine/threonine kinase, we investigated whether Raf-1 could interact with the Src homology 2 (SH2) domains of various signal-transducing molecules. In this report, we demonstrate that Raf-1 associated with the SH2 domain of Fyn (a member of the Src tyrosine kinase family) but not with the SH2 domains of phospholipase C-gamma 1, the p85 alpha subunit of phosphatidylinositol 3-kinase, and SH2-containing protein tyrosine phosphatase 2. Unlike most SH2 domain interactions that require tyrosine-phosphorylated residues, the Raf-1/Fyn SH2 domain association was dependent on the serine phosphorylation of Raf-1. Our results also demonstrate that Raf-1 interacted with the SH2 domain of Src and that this interaction was destabilized by mutation of Arg175 found within the conserved SH2 domain FLVRES sequence. In addition, we show that inclusion of additional Src sequences containing the SH3 domain increased the association of Raf-1 with the Src SH2 domain. Finally, using the baculovirus/Sf9 cell system, we show that coexpression of Raf-1 with full-length Fyn/Src resulted in the coimmunoprecipitation of Raf-1 with Fyn/Src, the tyrosine phosphorylation of Raf-1, and the stimulation of Raf-1 kinase activity. These results suggest that Raf-1 may form a functional complex with Fyn/Src mediated in part by SH2 domains and the serine phosphorylation of Raf-1. 相似文献
5.
6.
Primate lentiviruses encode for an unique nef gene with an essential function in both viral replication and pathogenicity in the host. The molecular basis for this function remains however poorly defined. Several Nef-binding cellular proteins are thought to be instrumental in its function. Indeed, Nef contains a proline-rich motif implicated in the binding to the Src-like tyrosine kinase Hck and also to a Ser/Thr kinase of molecular weight 62 kDa. The disruption of this motif affects the binding to both these kinases as well as viral replication. Whereas Hck is expressed in the myeloid lineage and hence may account for the nef function in infected monocytes, we and others have reported previously that Nef also interacts with the T-lymphocyte Src-kinase Lck, leading to specific cell signaling impairment. This interaction occurs through the binding of Nef to both Lck SH2 and SH3 domains. Both the proline motif and phosphorylation of Nef on tyrosine residue were proposed to account for these interactions. Here, we investigate the mechanism of Lck SH2 binding by HIV-1 Nef. Using recombinant fusion proteins to precipitate lysates, we show that although SH2 binding is dependent on phosphorylation events, it occurs in a tyrosine independent manner because it requires neither tyrosine residues in Nef nor the phosphotyrosine binding pocket from the Lck SH2 domain, hence suggesting a role for a phosphoserine or a phosphothreonine residue. Further, we show that Hck SH2 does not interact with Nef, indicating that Hck SH3 binding is sufficient for Nef binding, whereas Lck SH2 cooperate together with SH3 to allow Nef binding to a level similar to Hck SH3. Together, our results establish different mechanisms for Hck and Lck binding by HIV-1 Nef protein, and identify a novel mechanism for Src-like tyrosine kinase targeting by a viral protein. 相似文献
7.
SH2 domains are small protein domains of approximately 100 amino acids that bind to phosphotyrosine (pY) in the context of a specific sequence surrounding the target pY. In general, the residues C-terminal to the pY of the binding target are considered most important for defining the binding specificity, and in particular the pY + 1 and pY + 3 residues (i.e., the first and third amino acids C-terminal to the pY). However, our previous studies with the SH2 domains of the protein tyrosine phosphatase SHP-2 [Huyer, G., Li, Z. M., Adam, M., Huckle, W. R., and Ramachandran, C. (1995) Biochemistry 34, 1040-1049] indicated important interactions with the pY - 2 residue as well. In the SH2 domains of SHP-2, the highly conserved alphaA2 Arg is replaced by Gly. A comparison of the published crystal structures of the Src SH2 domain and the N-terminal SH2 domain of SHP-2 complexed with high-affinity peptides suggested that the alphaA2 Gly of SHP-2 creates a gap which is filled by the side chain of the pY - 2 residue of the bound peptide. It was predicted that replacing this Gly with Arg would alter or eliminate the involvement of the pY - 2 residue in binding. The alphaA2 Gly --> Arg mutant was constructed, and indeed, this mutant no longer required residues N-terminal to the target pY for high-affinity binding, making its specificity more like that of other SH2 domains. The alphaA2 Gly is clearly involved in directing the unusual requirement for the pY - 2 residue in the binding sequence of this SH2 domain, which has important implications for its in vivo targeting and specificity. 相似文献
8.
NJ Chalupny A Aruffo JM Esselstyn PY Chan J Bajorath J Blake LK Gilliland JA Ledbetter MA Tepper 《Canadian Metallurgical Quarterly》1995,25(10):2978-2984
CD19 is a B cell surface protein capable of forming non-covalent molecular complexes with a number of other B cell surface proteins including the CD21/CD81/Leu-13 complex as well as with surface immunoglobulin. CD19 tyrosine phosphorylation increases after B cell activation, and is proposed to play a role in signal transduction through its cytoplasmic domain, which contains nine tyrosine residues. Several second messenger proteins have been shown to immunoprecipitate with CD19, including p59 Fyn (Fyn), p59 Lyn (Lyn) and phosphatidylinositol-3 kinase (PI-3 kinase). These associations are predicted to occur via the src-homology 2 (SH2) domains of the second messenger proteins. Two of the cytoplasmic tyrosines in the CD19 cytoplasmic region contain the consensus binding sequence for the PI-3 kinase SH2 domain (YPO4-X-X-M). However, the reported consensus binding sequence for the Fyn and Lyn SH2 domains (YPO4-X-X-I/L) is not found in CD19. We investigated the capacity of CD19 cytoplasmic tyrosines to bind both Fyn and PI-3 kinase SH2-domain fusion proteins. In activated B cells, both Fyn and PI-3 kinase SH2-domain fusion proteins precipitate CD19. Using synthetic tyrosine-phosphorylated peptides comprising each of the CD19 cytoplasmic tyrosines and surrounding amino acids, we investigated the ability of the Fyn SH2 and PI-3 kinase SH2 fusion proteins to bind to the different CD19 cytoplasmic phosphotyrosine peptides. ELISA revealed that the two CD19 cytoplasmic tyrosine residues contained within the Y-X-X-M sequences (Y484 and Y515) bound preferentially to the PI-3 kinase SH2-domain fusion proteins. Two different tyrosines (Y405 and Y445) bound preferentially to the Fyn SH2-domain fusion protein via a novel sequence, Y-E-N-D/E, different from that previously reported for the Fyn SH2 domain. In precipitation studies, peptide Y484 was able to compete with tyrosine phosphorylated CD19 specifically for binding to the PI-3 kinase SH2 domain fusion proteins, while peptides Y405 and Y445 were able to compete specifically for binding to the Fyn SH2 domain fusion proteins. These results indicate that CD19 may be capable of binding both Fyn and PI-3 kinase concurrently, suggesting a mechanism for CD19 signal transduction, in which binding of PI-3 kinase to the Fyn SH3 domain results in activation of PI-3 kinase. 相似文献
9.
F Hajós K Zilles A Zsarnovszky P Sótonyi K Gallatz A Schleicher 《Canadian Metallurgical Quarterly》1998,85(1):45-52
The Nef gene of the human and simian immunodeficiency viruses HIV and SIV has been implicated in pathogenicity; however, the mechanism by which Nef induces disease is still unknown. An impact on signal transduction in cells has been suggested by the interaction of Nef from an HIV-1 strain and tyrosine kinases like HCK and LCK as well as serine/threonine kinases. We have confirmed the binding of HCK to HIV-1 subtype B Nef and demonstrated an equally strong interaction with a subtype E Nef protein but weaker binding to Nef of HIV-2 subtype A (HIV-2D194). No binding, however, was observed to HIV-2 subtype B Nef (HIV-2D205). Instead, this protein bound to a novel cellular protein, Nefin 1, with characteristics of an adaptor protein and strong expression in all human hematopoietic tissues. Nefin 1 binds through an amino-terminal domain, which is related to SH3 domains. For interaction of Nef with Nefin 1, the PxxP motif and the three-dimensional conformation of the molecule appear necessary. In conclusion, this study demonstrates that Nef proteins of divergent strains of HIV-1 and HIV-2 may use different elements of signal transduction pathways for the induction of pathogenicity in vivo. 相似文献
10.
A Mangasarian V Piguet JK Wang YL Chen D Trono 《Canadian Metallurgical Quarterly》1999,73(3):1964-1973
The Nef protein of primate lentiviruses triggers the accelerated endocytosis of CD4 and of class I major histocompatibility complex (MHC-I), thereby down-modulating the cell surface expression of these receptors. Nef acts as a connector between the CD4 cytoplasmic tail and intracellular sorting pathways both in the Golgi and at the plasma membrane, triggering the de novo formation of CD4-specific clathrin-coated pits (CCP). The downstream partners of Nef in this event are the adapter protein complex (AP) of CCP and possibly a subunit of the vacuolar ATPase. Whether Nef-induced MHC-I down-regulation stems from a similar mechanism is unknown. By comparing human immunodeficiency virus type 1 (HIV-1) Nef mutants for their ability to affect either CD4 or MHC-I expression, both in transient-transfection assays and in the context of HIV-1 infection, it was determined that Nef-induced CD4 and MHC-I down-regulation constitute genetically and functionally separate properties. Mutations affecting only CD4 regulation mapped to residues previously shown to mediate the binding of Nef to this receptor, such as W57 and L58, as well as to an AP-recruiting dileucine motif and to an acidic dipeptide in the C-terminal region of the protein. In contrast, mutation of residues in an alpha-helical region in the proximal portion of Nef and amino acid substitutions in a proline-based SH3 domain-binding motif selectively affected MHC-I down-modulation. Although both the N-terminal alpha-helix and the proline-rich region of Nef have been implicated in recruiting Src family protein kinases, the inhibitor herbimycin A did not block MHC-I down-regulation, suggesting that the latter process is not mediated through an activation of this family of tyrosine kinases. 相似文献
11.
The actin filament-associated protein AFAP-110 forms a stable complex with activated variants of Src in chick embryo fibroblast cells. Stable complex formation requires the integrity of the Src SH2 and SH3 domains. In addition, AFAP-110 encodes two adjacent SH3 binding motifs and six candidate SH2 binding motifs. These data indicate that both SH2 and SH3 domains may work cooperatively to facilitate Src/AFAP-110 stable complex formation. As a test for this hypothesis, we sought to understand whether one or both SH3 binding motifs in AFAP-110 modulate interactions with the Src SH3 domain and if this interaction was required to present AFAP-110 for tyrosine phosphorylation by, and stable complex formation with, Src. A proline to alanine site-directed mutation in the amino terminal SH3 binding motif (SH3bm I) was sufficient to abrogate absorption of AFAP-110 with GST-SH3STC. Co-expression of activated Src (pp60(527F)) with AFAP-110 in Cos-1 cells permit tyrosine phosphorylation of AFAP-110 and stable complex formation with pp60(527F). However, co-expression of the SH3 null-binding mutant (AFAP71A) with pp60(527F) revealed a 2.7 fold decrease in steady-state levels of tyrosine phosphorylation, compared to AFAP-110. Although a lower but detectable level of AFAP71A was phosphorylated on tyrosine, AFAP71A could not be detected in stable complex with pp60(527F), unlike AFAP-110. These data indicate that SH3 interactions facilitate presentation of AFAP-110 for tyrosine phosphorylation and are also required for stable complex formation with pp60(527F). 相似文献
12.
Requirement for c-Src catalytic activity and the SH3 domain in platelet-derived growth factor BB and epidermal growth factor mitogenic signaling 总被引:1,自引:0,他引:1
The Src family protein-tyrosine kinases are required for mitogenic signaling from the platelet-derived growth factor (PDGF), colony stimulating factor-1, and epidermal growth factor (EGF) receptor protein-tyrosine kinases (RPTK) (Twamley-Stein, G. M., Pepperkok, R., Ansorge, W., and Courtneidge, S. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7696-7700; Roche, S., Koegl, M., Barone, M. V., Roussel, M. F., and Courtneidge, S. A.(1995) Mol. Cell. Biol. 15, 1102-1109). In NIH3T3 fibroblasts, c-Src, Fyn, and c-Yes associate with the activated PDGF receptor, are substrates for receptor phosphorylation, and are themselves activated. Src family catalytic function is required for RPTK mitogenic signaling as evidenced by the SH2-dependent dominant negative phenotype exhibited by kinase-inactive Src and Fyn mutants (Twamley-Stein, G. M., Pepperkok, R., Ansorge, W., and Courtneidge, S. A.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7696-7700). Here, we have generated clonal Src- murine fibroblast cell lines overexpressing various murine c-Src mutants and studied the effect of these mutant Src proteins on PDGF- and EGF-induced mitogenesis. Two c-Src SH3 domain mutants, Y133F and Y138F, each inhibited PDGF BB- and EGF-induced DNA synthesis in quiescent cells. This demonstrates an involvement of the Src SH3 domain in PDGFbeta and EGF receptor mitogenic signaling. Since both Tyr-133 and Tyr-138 are located on the ligand binding surface of the SH3 domain, these results suggest that the c-Src SH3 domain is required for PDGF and EGF mitogenic signaling. The dominant negative effect of either single mutant on PDGF receptor signaling was reversed by a second SH2-inactivating mutation. We conclude that the c-Src SH3 domain function requires the SH2 domain in the case of the PDGF receptor, presumably because binding of c-Src to the receptor via its SH2 domain is a prerequisite for the SH3 domain function. In contrast, SH2 function is apparently not essential for the SH3 function in EGF receptor signaling. 相似文献
13.
CD28 provides a costimulatory signal that results in optimal activation of T cells. The signal transduction pathways necessary for CD28-mediated costimulation are presently unknown. Engagement of CD28 leads to its tyrosine phosphorylation and subsequent binding to Src homology 2 (SH2)-containing proteins including the p85 subunit of phosphatidylinositol 3'-kinase (PI3K); however, the contribution of PI3K to CD28-dependent costimulation remains controversial. Here we show that CD28 is capable of binding the Src homology 3 (SH3) domains of several proteins, including Grb2. The interaction between Grb2 and CD28 is mediated by the binding of Grb2-SH3 domains to the C-terminal diproline motif present in the cytoplasmic domain of CD28. While the affinity of the C-terminal SH3 domain of Grb2 for CD28 is greater than that of the N-terminal SH3 domain, optimal binding requires both SH3 domains. Ligation of CD28, but not tyrosine-phosphorylation, is required for the SH3-mediated binding of Grb2 to CD28. We propose a model whereby the association of Grb2 with CD28 occurs via an inducible SH3-mediated interaction and leads to the recruitment of tyrosine-phosphorylated proteins such as p52(shc) bound to the SH2 domain of Grb2. The inducible interaction of Grb2 to the C-terminal region of CD28 may form the basis for PI3K-independent signaling through CD28. 相似文献
14.
B Gay P Furet C García-Echeverría J Rahuel P Chène H Fretz J Schoepfer G Caravatti 《Canadian Metallurgical Quarterly》1997,36(19):5712-5718
SH2 domains mediate protein-protein interactions and are involved in a wide range of intracellular signaling events. SH2 domains are 100-amino acid stretches of protein that bind to other proteins containing phosphotyrosine residues. A current major research goal is formulation of the structural principles which govern peptide-binding specificity in SH2 domains. Several structures (both X-ray and NMR) of SH2 domains have now been determined. Short peptide fragments on the carboxyl-terminal side of the phosphotyrosine residue carry the sequence specific information for SH2 recognition. The bound peptides are held in an extended conformation. However, for the GRB2 SH2 domain, the peptide adopts a beta-turn as the motif for recognition [Rahuel, J., et al. (1996) Nat. Struct. Biol. 3, 586-589]. Our SAR data and molecular modeling studies suggest that many SH2 domains, such as the SH2 domains of Lck, Src, and p85, can interact with high affinity with short peptide sequences at least in two ways which are sequence-dependent. The peptide forms either an extended chain across the D-strand of SH2 domains with anchors at pY and pY+3 or, as in the case of GRB2 SH2, a beta-turn with anchors at pY and pY+2. Due to a bulky tryptophan in its EF1 loop, GRB2 SH2 cannot bind peptide conformations such as the extended chain and thus has a unique specificity. 相似文献
15.
DA Renzoni DJ Pugh G Siligardi P Das CJ Morton C Rossi MD Waterfield ID Campbell JE Ladbury 《Canadian Metallurgical Quarterly》1996,35(49):15646-15653
The interaction of the Fyn SH3 domain with the p85 subunit of PI3-kinase is investigated using structural detail and thermodynamic data. The solution structure complex of the SH3 domain with a proline-rich peptide mimic of the binding site on the p85 subunit is described. This indicates that the peptide binds as a poly(L-proline) type II helix. Circular dichroism spectroscopic studies reveal that in the unbound state the peptide exhibits no structure. Thermodynamic data for the binding of this peptide to the SH3 domain suggest that the weak binding (approximately 31 microM) of this interaction is, in part, due to the entropically unfavorable effect of helix formation (delta S0 = -78 J.mol-1.K-1). Binding of the SH3 domain to the intact p85 subunit (minus its own SH3 domain) is tighter, and the entropic and enthalpic contributions are very different from those given by the peptide interaction (delta S0 = +252 J.mol-1.K-1; delta H0 = +44 kJ.mol-1). From these dramatically different thermodynamic measurements we are able to conclude that the interaction of the proline-rich peptide does not effectively mimic the interaction of the intact p85 subunit with the SH3 domain and suggest that other interactions could be important. 相似文献
16.
Paired immunoglobulin-like receptor (PIR)-A and PIR-B possess similar ectodomains with six immunoglobulin-like loops, but have distinct transmembrane and cytoplasmic domains. PIR-B bears immunoreceptor tyrosine-based inhibitory motif (ITIM) sequences in its cytoplasmic domain that recruit Src homology (SH)2 domain-containing tyrosine phosphatases SHP-1 and SHP-2, leading to inhibition of B and mast cell activation. In contrast, the PIR-A protein has a charged Arg residue in its transmembrane region and a short cytoplasmic domain that lacks ITIM sequences. Here we show that Fc receptor gamma chain, containing an immunoreceptor tyrosine-based activation motif (ITAM), associates with PIR-A. Cross-linking of this PIR-A complex results in mast cell activation such as calcium mobilization in an ITAM-dependent manner. Thus, our data provide evidence for the existence of two opposite signaling pathways upon PIR aggregation. PIR-A induces the stimulatory signal by using ITAM in the associated gamma chain, whereas PIR-B mediates the inhibitory signal through its ITIMs. 相似文献
17.
A novel human cDNA encoding a cytosolic 62-kDa protein (p62) that binds to the Src homology 2 (SH2) domain of p56lck in a phosphotyrosine-independent manner has been cloned. The cDNA is composed of 2074 nucleotides with an open reading frame encoding 440 amino acids. Northern analysis suggests that p62 is expressed ubiquitously in all tissues examined. p62 is not homologous to any known protein in the data base. However, it contains a cysteine-rich region resembling a zinc finger motif, a potential G-protein-binding region, a PEST motif, and several potential phosphorylation sites. Using T7-epitope tagged p62 expression in HeLa cells, the expressed protein was shown to bind to the lck SH2 domain. Deletion of the N-terminal 50 amino acids abolished binding, but mutagenesis of the single tyrosine residue in this region had no effect on binding. Thus, the cloned cDNA indeed encodes the p62 protein, which is a phosphotyrosine-independent ligand for the lck SH2 domain. Its binding mechanism is unique with respect to binding modes of other known ligands for SH2 domains. 相似文献
18.
G Siegal B Davis SM Kristensen A Sankar J Linacre RC Stein G Panayotou MD Waterfield PC Driscoll 《Canadian Metallurgical Quarterly》1998,276(2):461-478
Heterodimeric class IA phosphoinositide 3-kinase (PI 3-kinase) plays a crucial role in a variety of cellular signalling events downstream of a number of cell-surface receptor tyrosine kinases. Activation of the enzyme is effected in part by the binding of two Src homology-2 domains (SH2) of the 85 kDa regulatory subunit to specific phosphotyrosine-containing peptide motifs within activated cytoplasmic receptor domains. The solution structure of the uncomplexed C-terminal SH2 (C-SH2) domain of the p85 alpha subunit of PI 3-kinase has been determined by means of multinuclear, double and triple-resonance NMR experiments and restrained molecular-dynamics simulated-annealing calculations. The solution structure clearly indicates that the uncomplexed C-SH2 domain conforms to the consensus polypeptide fold exhibited by other SH2 domains, with an additional short helical element at the N terminus. In particular, the C-SH2 structure is very similar to both the p85 alpha N-terminal SH2 domain (N-SH2) and the Src SH2 domain with a root mean square difference (rmsd) for 44 C alpha atoms of 1.09 and 0.89 A, respectively. The canonical BC, EF and BG loops are less well-defined by the experimental restraints and show greater variability in the ensemble of C-SH2 conformers. The lower level of definition in these regions may reflect the presence of conformational disorder, an interpretation supported by the absence or broadening of backbone and side-chain NMR resonances for some of these residues. NMR experiments were performed, where C-SH2 was titrated with phosphotyrosine-containing peptides corresponding to p85 alpha recognition sites in the cytoplasmic domain of the platelet-derived growth-factor receptor. The ligand-induced chemical-shift perturbations indicate the amino-acid residues in C-SH2 involved in peptide recognition follow the pattern predicted from homologous complexes. A series of C-SH2 mutants was generated and tested for phosphotyrosine peptide binding by surface plasmon resonance. Mutation of the invariant Arg36 (beta B5) to Met completely abolishes phosphopeptide binding. Mutation of each of Ser38, Ser39 or Lys40 in the BC loop to Ala reduces the affinity of C-SH2 for a cognate phosphopeptide, as does mutation of His93 (BG5) to Asn. These effects are consistent with the involvement of the BC loop and BG loops regions in ligation of phosphopeptide ligands. Mutation of Cys57 (beta D5) in C-SH2 to Ile, the corresponding residue type in the p85 alpha N-SH2 domain, results in a change in peptide binding selectivity of C-SH2 towards that demonstrated by p85 alpha N-SH2. This pattern of p85 alpha phosphopeptide binding specificity is interpreted in terms of a model of the p85 alpha/PDGF-receptor interaction. 相似文献
19.
P Franken S Arold A Padilla M Bodeus F Hoh MP Strub M Boyer M Jullien R Benarous C Dumas 《Canadian Metallurgical Quarterly》1997,6(12):2681-2683
Human immunodeficiency virus Nef protein accelerates virulent progression of AIDS by its interaction with specific cellular proteins involved in cellular activation and signal transduction. Here we report the purification and crystallization of the conserved core of HIV-1LAI Nef protein in the unliganded form and in complex with the wild-type SH3 domain of the P59fyn protein-tyrosine kinase. One-dimensional NMR experiments show that full-length protein and truncated fragment corresponding to the product of HIV-1 protease cleavage have a well-folded compact tertiary structure. The ligand-free HIV-1 Nefcore protein forms cubic crystals belonging to space group P23 with unit cell dimensions of a = b = c = 86.4 A. The Nef-Fyn SH3 cocrystals belong to the space group P6(1)22 or its enantiomorph, P6(5)22, with unit cell dimensions of a = b = 108.2 A and c = 223.7 A. Both crystal forms diffract to a resolution limit of 3.0 A resolution using synchrotron radiation, and are thus suitable for X-ray structure determination. 相似文献
20.
T Trüb JD Frantz M Miyazaki H Band SE Shoelson 《Canadian Metallurgical Quarterly》1997,272(2):894-902
We have characterized an SH3-SH2-SH3 linker protein that is prominently expressed in lymphoid tissues. This protein has 58% sequence identity to Grb2. An identical protein called Grap has been found in hematopoietic cells. In Jurkat cells, T cell receptor activation leads to the association of Grap with phosphoproteins p36/38 and, to a lesser degree, Shc. This interaction is mediated by the Grap SH2 domain, which has similar binding specificity to the Grb2 SH2 domain. Grap also associates via its SH3 domains with Sos, the Ras guanine nucleotide exchange factor; with dynamin, a GTPase involved in membrane protein trafficking; and with Sam68, a nuclear RNA-binding protein that serves as a substrate of Src kinases during mitosis. T cell activation effects an increase in Grap association with p36/38, Shc, Sos, and dynamin. Sam68 binding is constitutive. Phospholipase C-gamma1 and Fyn are also found in activated Grap signaling complexes, although these interactions may not be direct. We conclude that Grap is a prominent component of lymphocyte receptor signaling. Based on the known functions of bound effector molecules, Grap-mediated responses to antigen challenge may include endocytosis of the T cell receptor, cellular proliferation, and regulated entry into the cell cycle. 相似文献