Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Systematic identification of direct protein-protein interactions is often hampered by difficulties in expressing and purifying the corresponding full-length proteins. By taking advantage of the modular nature of many regulatory proteins, we attempted to simplify protein-protein interactions to the corresponding domain-ligand recognition and employed peptide arrays to identify such binding events. A group of 12 Src homology (SH) 3 domains from eight human proteins (Swiss-Prot ID: SRC, PLCG1, P85A, NCK1, GRB2, FYN, CRK) were used to screen a peptide target array composed of 1536 potential ligands, which led to the identification of 921 binary interactions between these proteins and 284 targets. To assess the efficiency of the peptide array target screening (PATS) method in identifying authentic protein-protein interactions, we examined a set of interactions mediated by the PLCgamma1 SH3 domain by coimmunoprecipitation and/or affinity pull-downs using full-length proteins and achieved a 75% success rate. Furthermore, we characterized a novel interaction between PLCgamma1 and hematopoietic progenitor kinase 1 (HPK1) identified by PATS and demonstrated that the PLCgamma1 SH3 domain negatively regulated HPK1 kinase activity. Compared to protein interactions listed in the online predicted human interaction protein database (OPHID), the majority of interactions identified by PATS are novel, suggesting that, when extended to the large number of peptide interaction domains encoded by the human genome, PATS should aid in the mapping of the human interactome.
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PMID:Systematic identification of SH3 domain-mediated human protein-protein interactions by peptide array target screening. 1747 47

Cell surface receptors and their associated signaling pathways on the plasma membrane are key targets in understanding cellular responses. However, the isolation and identification of receptor complexes has been elusive. The Fc receptor was captured from the surface of live cells using microbeads coated with the receptor's cognate ligand, gamma globulin (IgG), and analyzed by liquid chromatography and tandem mass spectrometry (LC-MS/MS) alongside several controls. Live-cell affinity receptor chromatography (LARC) resulted in a partially nonredundant list of 288 proteins that were specific to the Fc receptor complex. The proteins identified were in close agreement with previously determined factors in the Fc receptor complex as demonstrated by genetic and biochemical methods and revealed novel complex members. Confocal microscopy was used to confirm recruitment of SRC, SYK, PLC, PKC, PI3K, SHIP, TEC, CDC42, RAP, PAK, GAP, GEF, GRP, and CRK to the receptor complex upon activation by the same ligand microbeads. The expression of mutants and silencing RNA against specific isoforms were used to demonstrate a functional role for novel members of the Fc receptor complex, including RHOG (RAS homologue member G), p115 RhoGEF (protein of 115-kDa RAS homologue guanine exchange factor), and CRKL (CRK-like). The recruitment of AKT pleckstrin homology (PH) domain green fluorescent protein (GFP) was used to quantify the production of phosphorylated inositol at the activated receptor complex. We conclude that it is feasible to capture an activated receptor complex from the surface of live cells using ligand-coated microbeads for identification of members of a receptor complex or pathway by LC-MS/MS.
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PMID:Capture of an activated receptor complex from the surface of live cells by affinity receptor chromatography. 1860 92

ABL family tyrosine kinases are tightly regulated by autoinhibition and phosphorylation mechanisms. These kinases maintain an inactive conformation through intramolecular interactions involving SH3 and SH2 domains. RIN1, a downstream effector of RAS, binds to the ABL SH3 and SH2 domains and stimulates ABL tyrosine kinase activity. RIN1 binding to the ABL2 kinase resulted in a large decrease in Km and a small increase in Vmax toward an ABL consensus substrate peptide. The enzyme efficiency (k(cat)/Km) was increased more than 5-fold by RIN1. In addition, RIN1 strongly enhanced ABL-mediated phosphorylation of CRK, PSTPIP1, and DOK1, all established ABL substrates but with unique protein structures and distinct target sequences. Importantly RIN1-mediated stimulation of ABL kinase activity was independent of activation by SRC-mediated phosphorylation. RIN1 increased the kinase activity of both ABL1 and ABL2, and this occurred in the presence or absence of ABL regulatory domains outside the SH3-SH2-tyrosine kinase domain core. We further demonstrate that a catalytic site mutation associated with broad drug resistance, ABL1T315I, remains responsive to stimulation by RIN1. These findings are consistent with an allosteric kinase activation mechanism by which RIN1 binding promotes a more accessible ABL catalytic site through relief of autoinhibition. Direct disruption of RIN1 binding may therefore be a useful strategy to suppress the activity of normal and oncogenic ABL, including inhibitor-resistant mutants that confound current therapeutic strategies. Stimulation through derepression may be applicable to many other tyrosine kinases autoinhibited by coupled SH3 and SH2 domains.
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PMID:Enhancement of ABL kinase catalytic efficiency by a direct binding regulator is independent of other regulatory mechanisms. 1879 34

The adapter protein CRKL is required for the normal development of multiple tissues that rely on fibroblast growth factor 8 (FGF8). The precise role of CRKL in receptor signaling has been unclear, however. To address this issue, we first modeled the three-dimensional structure of CRKL by molecular dynamics. By taking advantage of structural simulations, we performed in silico analysis of the interactions of the autophosphorylation sites of FGR receptor 1 (FGFR1) with the SH2 domain of CRKL or a highly related protein, CRK. As predicted by simulations, we confirm the specific physical interaction of phosphorylated Y463 (pY463) in FGFR1 with the CRKL SH2 domain at an affinity approximately 30-fold stronger than that of CRK. We also provide evidence that interactions outside of the core YXXP motif have a significant impact on physical association, which is consistent with predictions from molecular-dynamics simulations. Furthermore, we identify CRKL as an essential component of an FGF8-induced feed-forward loop permissive for efficient activation of the mitogen-activated protein kinase Erk1/2, as well as FGF8-induced anchorage-independent cell growth, using Crkl-deficient cells or a pY463 synthetic peptide. Although many cells generally require cell-matrix adhesion, our results demonstrate that CRKL permits cells to bypass the strict need for adhesion in response to FGF8 through direct interaction with receptor.
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PMID:Structural and functional basis of a role for CRKL in a fibroblast growth factor 8-induced feed-forward loop. 1930 7

To demonstrate the utility of phage display in generating highly specific antibodies, affinity selections were conducted on 20 related Src Homology 2 (SH2) domains (ABL1, ABL2, BTK, BCAR3, CRK, FYN, GRB2, GRAP2, LYN, LCK, NCK1, PTPN11 C, PIK3R1 C, PLCgamma1 C, RASA1 C, SHC1, SH2D1A, SYK N, VAV1 and the tandem domains of ZAP70). The domains were expressed in Escherichia coli, purified and used in affinity selection experiments. In total, 1292/3800 of the resultant antibodies were shown to bind the target antigen. Of the 695 further evaluated in specificity ELISAs against all 20 SH2 domains, 379 antibodies were identified with unique specificity (i.e. monospecific). Sequence analysis revealed that there were at least 150 different clones with 1-19 different antibodies/antigen. This includes antibodies that distinguish between ABL1 and ABL2, despite their 89% sequence identity. Specificity was confirmed for many on protein arrays fabricated with 432 different proteins. Thus, even though the SH2 domains share a common three-dimensional structure and 20-89% identity at the primary structure level, we were able to isolate antibodies with exquisite specificity within this family of structurally related domains.
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PMID:Generating a panel of highly specific antibodies to 20 human SH2 domains by phage display. 2016 16

CRKL (CRK-like) is an adapter protein predominantly phosphorylated in cells that express the tyrosine kinase p210(BCR-ABL), the fusion product of a (9;22) chromosomal translocation causative for chronic myeloid leukemia. It has been unclear, however, whether CRKL plays a functional role in p210(BCR-ABL) transformation. Here, we show that CRKL is required for p210(BCR-ABL) to support interleukin-3-independent growth of myeloid progenitor cells and long-term outgrowth of B-lymphoid cells from fetal liver-derived hematopoietic progenitor cells. Furthermore, a synthetic phosphotyrosyl peptide that binds to the CRKL SH2 domain with high affinity blocks association of endogenous CRKL with the p210(BCR-ABL) complex and reduces c-MYC levels in K562 human leukemic cells as well as in mouse hematopoietic cells transformed by p210(BCR-ABL) or the imatinib-resistant mutant T315I. These results indicate that the function of CRKL as an adapter protein is essential for p210(BCR-ABL)-induced transformation.
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PMID:A specific need for CRKL in p210BCR-ABL-induced transformation of mouse hematopoietic progenitors. 2080 13

Mapping protein interactions by immunoprecipitation is limited by the availability of antibodies recognizing available native epitopes within protein complexes with sufficient affinity. Here we demonstrate a scalable approach for generation of such antibodies using phage display and affinity maturation. We combined antibody variable heavy (V(H)) genes from target-specific clones (recognizing Src homology 2 (SH2) domains of LYN, VAV1, NCK1, ZAP70, PTPN11, CRK, LCK, and SHC1) with a repertoire of 10(8) to 10(9) new variable light (V(L)) genes. Improved binders were isolated by stringent selections from these new "chain-shuffled" libraries. We also developed a predictive 96-well immunocapture screen and found that only 12% of antibodies had sufficient affinity/epitope availability to capture endogenous target from lysates. Using antibodies of different affinities to the same epitope, we show that affinity improvement was a key determinant for success and identified a clear affinity threshold value (60 nM for SHC1) that must be breached for success in immunoprecipitation. By combining affinity capture using matured antibodies to SHC1 with mass spectrometry, we identified seven known binding partners and two known SHC1 phosphorylation sites in epidermal growth factor (EGF)-stimulated human breast cancer epithelial cells. These results demonstrate that antibodies capable of immunoprecipitation can be generated by chain shuffling, providing a scalable approach to mapping protein-protein interaction networks.
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PMID:Mapping protein interactions by combining antibody affinity maturation and mass spectrometry. 2170 3

The signaling adapter protein CRK is an indispensable molecule involved in regulating the malignant potential of human cancers. CRK-like (CRKL) is a hematopoietic cell-dominant homologue of CRK that is reported to be phosphorylated by BCR-ABL tyrosine kinase in chronic myelogenous leukemia patients, but its biological function in non-hematopoietic tumors remains unclear. In this study, we explored the tumorigenic role of CRKL in head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo. Immunoprecipitation analysis of HNSCC cell line, HSC-3 cells, showed that the dominant binding partner for C3G was CRKL, not CRK. To clarify the molecular function of CRKL, we established lentiviral shRNA-mediated CRKL-knockdown HNSCC cell lines. In CRKL-knockdown HSC-3 and HSC-4 cells, cell growth and motility were diminished compared to control cells. Cell adhesion assays showed that cell attachment onto both fibronectin- and collagen-coated dishes was significantly suppressed in CRKL-knockdown HSC-3 cells, while no significant change was observed for poly-l-lysine-coated dishes. Immunofluorescence staining revealed that focal adhesion was reduced in CRKL-knockdown HSC-3 cells. With a pulldown assay, CRKL-knockdown HSC-3 cells showed decreased amounts of active Rap1 compared to control cells. Moreover, in an in vivo assay, tumor formation of CRKL-knockdown HSC-3 cells in nude mice was significantly abrogated. Our results indicate that CRKL regulates HNSCC-cell growth, motility, and integrin-dependent cell adhesion, suggesting that CRKL plays a principal role in HNSCC tumorigenicity.
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PMID:CRKL plays a pivotal role in tumorigenesis of head and neck squamous cell carcinoma through the regulation of cell adhesion. 2224 89

To identify novel signaling pathways necessary for rhabdomyosarcoma (RMS) survival, we performed a loss-of-function screen using an inducible small hairpin RNA (shRNA) library in an alveolar and an embryonal RMS cell line. This screen identified CRKL expression as necessary for growth of alveolar RMS and embryonal RMS both in vitro and in vivo. We also found that CRKL was uniformly highly expressed in both RMS cell lines and tumor tissue. As CRKL is a member of the CRK adapter protein family that contains an SH2 and two SH3 domains and is involved in signal transduction from multiple tyrosine kinase receptors, we evaluated CRKL interaction with multiple tyrosine kinase receptor signaling pathways in RMS cells. While we saw no interaction of CRKL with IGFIR, MET or PI3KAKT/mTOR pathways, we determined that CRKL signaling was associated with SRC family kinase (SFK) signaling, specifically with YES kinase. Inhibition of SFK signaling with dasatinib or another SFK inhibitor, sarcatinib, suppressed RMS cell growth in vitro and in vivo. These data identify CRKL as a novel critical component of RMS growth. This study also demonstrates the use of functional screening to identify a potentially novel therapeutic target and treatment approach for these highly aggressive pediatric cancers.
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PMID:Loss-of-function screen in rhabdomyosarcoma identifies CRKL-YES as a critical signal for tumor growth. 2331 29

Autism is a pervasive neurodevelopmental disorder diagnosed in early childhood. The genetic factors might play an important role in its pathogenesis. Previous studies revealed that Reelin (RELN) polymorphisms were associated with autism. However, the roles of genes in Reelin signaling pathway for autism are largely unknown. As several knockout mice models in which the Reelin pathway genes (i.e. DAB1, VLDLR/APOER2, FYN/SRC and CRK/CRKL) are deficient have the similar phenotype as the reeler mice (Reelin(-/-)), we hypothesized that the Reelin signaling pathway genes might play roles in the etiology of autism. Therefore, we conducted a family-based association study. Sixty-two tagged single nucleotide polymorphisms (SNPs) covering 15 genes in Reelin pathway were genotyped in 239 trios, and 14 significant SNPs were further investigated in the additional 188 trios. In the total 427 trios, we found significant genetic association between autism and four SNPs in DAB1 (rs12035887 G: p=0.0006; rs3738556 G: p=0.0044; rs1202773 A: p=0.0048; rs12740765 T: p=0.0196). After the Bonferroni correction, SNP rs12035887 remained significant. Furthermore, the haplotype constructed with rs1202773 and rs12023109 in DAB1 showed significant excess transmission in both individual and global haplotype analyses (p=0.0052 and 0.0279, respectively). Our findings suggested that variations in DAB1 involved in the Reelin signaling pathway might contribute to genetic susceptibility to autism with Chinese Han decent, supporting the defect in the Reelin signaling pathway as a predisposition factor for autism.
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PMID:Association study between genes in Reelin signaling pathway and autism identifies DAB1 as a susceptibility gene in a Chinese Han population. 2333 77


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