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)

The thrombopoietin receptor (TpoR) regulates hematopoietic stem cell renewal, megakaryocyte differentiation, and platelet formation. TpoR signals by activating Janus kinases JAK2 and Tyk2. Here we show that, in addition to signaling downstream from the activated TpoR, JAK2 and Tyk2 strongly promote cell surface localization and enhance total protein levels of the TpoR. This effect is caused by stabilization of the mature endoglycosidase H-resistant form of the receptor. Confocal microscopy indicates that TpoR colocalizes partially with recycling transferrin in Ba/F3 cells. The interaction with JAK2 or Tyk2 appears to protect the receptor from proteasome degradation. Sequences encompassing Box1 and Box2 regions of the receptor cytosolic domain and an intact JAK2 or Tyk2 FERM domain are required for these effects. We discuss the relevance of our results to the reported defects of TpoR processing in myeloproliferative diseases and to the mechanisms of Tpo signaling and clearance via the TpoR.
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PMID:Janus kinases affect thrombopoietin receptor cell surface localization and stability. 1589 90

FIP200 is a novel protein inhibitor for focal adhesion kinase (FAK), which binds to FAK directly and inhibits its kinase activity and associated cellular functions, such as cell adhesion, spreading, and motility in fibroblasts. Here we show that FIP200 inhibits G1-S phase progression, proliferation, and clonogenic survival in human breast cancer cells. Consistent with the G1 arrest induced by FIP200, we found that FIP200 increased p21 and decreased cyclin D1 protein levels in breast cancer cells. In addition, FIP200 significantly induced p21 promoter activity in MCF-7 cells and this response was abolished upon deletion of p53 binding sites within p21 promoter. Furthermore, we found that FIP200 could interact with exogenous and endogenous p53 protein and significantly increase its half-life compared with the control cells. We also found that the NH2-terminal 154 residues of FIP200 were sufficient to mediate p53 interaction and G1 arrest in cells. The increase in p53 half-life correlated with the increased phosphorylation at Ser15 and decreased proteasomal degradation via ubiquitin and Hdm2-independent mechanism. Stabilization of p53 by FIP200 could be partially reversed by NQO1 inhibitor, dicoumarol. In contrast to p53, FIP200 decreased cyclin D1 protein half-life by promoting proteasome-dependent degradation of cyclin D1. In summary, our results suggest that FIP200 increases p21 protein levels via stabilization of its upstream regulator p53 and decreases cyclin D1 protein by promoting its degradation. Both effects are critical for FIP200-induced G1 arrest and may contribute to the putative antitumor activities of FIP200 in breast cancer.
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PMID:Mechanism of cell cycle regulation by FIP200 in human breast cancer cells. 1606 48

The abundance of surface GH receptor (GHR) is an important determinant of cellular GH sensitivity and is regulated at both transcriptional and posttranscriptional levels. In previous studies of GHR-expressing Janus kinase 2 (JAK2)-deficient human fibrosarcoma cells (gamma2A-GHR), we demonstrated that stable transfection with JAK2 resulted in increased steady-state levels of mature GHR (endoH-resistant; relative molecular mass, 115-140 kDa) relative to precursor GHR (endoH-sensitive; relative molecular mass, 100 kDa). We now examine further the effects of JAK2 on GHR trafficking by comparing gamma2A-GHR to gamma2A-GHR cells stably reconstituted with JAK2 (C14 cells). In the presence of JAK2, GHR surface expression was increased, as assessed by surface biotinylation, 125I-labeled human GH cell surface binding, and immunofluorescence microscopy assays. Although the absence of JAK2 precluded GH-stimulated signaling, GH-induced GHR disulfide linkage (a proxy for the GH-induced conformational changes in the GHR dimer) proceeded independent of JAK2 expression, indicating that the earliest steps in GH-induced GHR triggering are not prevented by the absence of JAK2. RNA interference-mediated knockdown of JAK2 in C14 cells resulted in a decreased mature to precursor ratio, supporting a primary role for JAK2 either in enhancing GHR biogenesis or dampening mature GHR degradation. To address these potential mechanisms, metabolic pulse-chase labeling experiments and experiments in which the fate of previously synthesized GHR was followed by anti-GHR immunoblotting after cycloheximide treatment (cycloheximide chase experiments) were performed. These indicated that the presence of JAK2 conferred modest enhancement (1.3- to 1.5-fold) in GHR maturation but substantially prolonged the t1/2 of the mature GHR, suggesting a predominant effect on mature GHR stability. Cycloheximide chase experiments with metalloprotease, proteasome, and lysosome inhibitors indicated that the enhanced stability of mature GHR conferred by JAK2 is not related to effects on constitutive receptor metalloproteolysis but rather is a result of reduced constitutive endosomal/lysosomal degradation of the mature GHR. These results are discussed in the context of emerging information on how JAK-family members modulate surface expression of other cytokine receptors.
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PMID:Janus kinase 2 enhances the stability of the mature growth hormone receptor. 1608 39

Androgen receptor plays a critical role in the development of primary as well as advanced hormone-refractory prostate cancer. Therefore, ablation of androgen receptor from prostate cancer cells is an interesting concept for developing a new therapy not only for androgen-dependent prostate cancer but also for metastatic hormone-refractory prostate cancer, for which there is no effective treatment available. We report here that LAQ824, a cinnamyl hydroxamatic acid histone deacetylase inhibitor currently in human clinical trials, effectively depleted androgen receptor in prostate cancer cells at nanomolar concentrations. LAQ824 seemed capable of depleting both the mutant and wild-type androgen receptors in either androgen-dependent and androgen-independent prostate cancer cells. Although LAQ824 may exert its effect through multiple mechanisms, several lines of evidence suggest that inactivation of the heat shock protein-90 (Hsp90) molecular chaperone is involved in LAQ824-induced androgen receptor depletion. Besides androgen receptor, LAQ824 reduced the level of Hsp90 client proteins HER-2 (ErbB2), Akt/PKB, and Raf-1 in LNCaP cells. Another Hsp90 inhibitor, 17-allyamino-17-demethoxygeldanamycin (17-AAG), also induced androgen receptor diminution. LAQ824 induced Hsp90 acetylation in LNCaP cells, which resulted in inhibition of its ATP-binding activity, dissociation of Hsp90-androgen receptor complex, and proteasome-mediated degradation of androgen receptor. Consequently, LAQ824 blocked androgen-induced prostate-specific antigen production in LNCaP cells. LAQ824 effectively inhibited cell proliferation and induced apoptosis of these prostate cancer cells. These results reveal that LAQ824 is a potent agent for depletion of androgen receptor and a potential new drug for prostate cancer.
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PMID:Chemical ablation of androgen receptor in prostate cancer cells by the histone deacetylase inhibitor LAQ824. 1617 22

A deficit in proteasome function in Parkinson's disease has been speculated. We characterized the ubiquitin-proteasome system in three regions of brain from transgenic and nontransgenic littermates. Mice expressing a doubly mutated form of human alpha-synuclein had significant impairments whereas mice expressing the wild-type gene had lesser changes compared to nontransgenic littermates. Significant abnormalities in line hm2 alpha-SYN-39 included declines in 20S-mediated proteolytic activity, the level of the 19S proteasome subunits Rpt1 and Rpn2, and the level of soluble total high MW ubiquitin cross-reacting proteins. Line hw alpha-SYN-5 had significant, but restricted proteasome abnormalities. The severity of impairment was proportional to the substantia nigra dopaminergic neuronal loss previously identified. There were significant correlations between the level of Rpn2 with the level of Rpt1, the activity of the 20S proteasome, and the level of soluble high MW ubiquitin cross-reacting proteins. These abnormalities in symptomatic line hm2 alpha-SYN-39 mice are consistent with abnormalities identified in tissue from patients with Parkinson's disease.
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PMID:Proteasome dysfunction in aged human alpha-synuclein transgenic mice. 1671 78

Serum- and glucocorticoid-induced protein kinase-1 (SGK-1) plays a critical role in regulation of the epithelial sodium channel, ENaC. SGK-1 also shares significant catalytic domain homology with protein kinase B (PKB/AKT-1) and is a downstream effector of antiapoptotic phosphoinositide 3-kinase signaling. Steady-state levels of an active SGK-1 are tightly regulated by rapid transcriptional activation and post-translational modification including phosphorylation. We show here that endogenous SGK-1 protein is polyubiquitinated and rapidly degraded by the 26S proteasome. In contrast to other rapidly degraded kinases, neither the catalytic activity of SGK-1 nor activation site phosphorylation was required for its ubiquitin modification and degradation. Instead, SGK-1 degradation required a lysine-less six-amino-acid (amino acids 19-24) hydrophobic motif (GMVAIL) within the N-terminal domain. Deletion of amino acids 19-24 significantly increased the half-life of SGK1 and prevented its ubiquitin modification. Interestingly, this minimal region was also required for the association of SGK-1 with the endoplasmic reticulum. Ubiquitin modification and degradation of SGK-1 were increasingly inhibited by the progressive mutation of six N-terminal lysine residues surrounding the GMVAIL motif. Mutation of all six lysines to arginine did not disrupt the subcellular localization of SGK-1 despite a significant decrease in ubiquitination, implying that this modification per se was not required for targeting to the endoplasmic reticulum. These results suggest that constitutive ubiquitin-mediated degradation of SGK-1 is an important mechanism regulating its biological activity.
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PMID:A novel N-terminal hydrophobic motif mediates constitutive degradation of serum- and glucocorticoid-induced kinase-1 by the ubiquitin-proteasome pathway. 1681 52

Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALK+ ALCL) is characterized by constitutive activation of the Janus kinase (JAK)3/signal transducers and activators of transcription 3 (STAT3) signaling pathway. SHP1, a tyrosine phosphatase that negatively regulates JAK/STAT, is frequently absent in ALK+ ALCL owing to gene methylation. To test the hypothesis that loss of SHP1 contributes to JAK3/STAT3 activation in ALK+ ALCL cells, we induced SHP1 expression using 5-aza-2'-deoxycytidine (5-AZA), an inhibitor of DNA methyltransferase, in ALK+ ALCL cell lines, and correlated with changes in the JAK3/STAT3 pathway. 5-AZA gradually restored SHP1 expression in Karpas 299 and SU-DHL-1 cells over 5 days. The initially low level of SHP1 expression did not result in significant changes to the expression or tyrosine phosphorylation of JAK3 and STAT3. However, higher levels of SHP1 seen subsequently correlated with substantial decreases in JAK3 and pJAK3, followed by pSTAT3 (but not STAT3). Importantly, the decrease in JAK3 was abrogated by MG132, a proteasome inhibitor. 5-AZA induced no significant increase in apoptosis but it sensitized ALCL cells to doxorubicin-induced apoptosis. Our findings support the concept that loss of SHP1 contributes to the constitutive activation of JAK3/STAT3 in ALK+ ALCL cells. SHP1 appears to downregulate JAK3 by two mechanisms: tyrosine dephosphorylation and increased degradation via the proteasome pathway.
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PMID:Restoration of shp1 expression by 5-AZA-2'-deoxycytidine is associated with downregulation of JAK3/STAT3 signaling in ALK-positive anaplastic large cell lymphoma. 1687 Dec 83

For immunotherapy of residual disease in patients with Philadelphia-positive leukemias, the BCR-ABL fusion regions are attractive disease-specific T-cell targets. We analyzed these regions for the prevalence of cytotoxic T lymphocyte (CTL) epitopes by an advanced reverse immunology procedure. Seventeen novel BCR-ABL fusion peptides were identified to bind efficiently to the human lymphocyte antigen (HLA)-A68, HLA-B51, HLA-B61 or HLA-Cw4 HLA class I molecules. Comprehensive enzymatic digestion analysis showed that 10 out of the 28 HLA class I binding fusion peptides were efficiently excised after their C-terminus by the proteasome, which is an essential requirement for efficient cell surface expression. Therefore, these peptides are prime vaccine candidates. The other peptides either completely lacked C-terminal liberation or were only inefficiently excised by the proteasome, rendering them inappropriate or less suitable for inclusion in a vaccine. CTL raised against the properly processed HLA-B61 epitope AEALQRPVA from the BCR-ABL e1a2 fusion region, expressed in acute lymphoblastic leukemia (ALL), specifically recognized ALL tumor cells, proving cell surface presentation of this epitope, its applicability for immunotherapy and underlining the accuracy of our epitope identification strategy. Our study provides a reliable basis for the selection of optimal peptides to be included in immunotherapeutic BCR-ABL vaccines against leukemia.
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PMID:BCR-ABL fusion regions as a source of multiple leukemia-specific CD8+ T-cell epitopes. 1693 47

The interaction of the P-beta-Cat(19-44) peptide, a 26 amino acid peptide (K(19)AAVSHWQQQSYLDpSGIHpSGATTTAP(44)) that mimics the phosphorylated beta-Catenin antigen, has been studied with its monoclonal antibody BC-22, by transferred nuclear Overhauser effect NMR spectroscopy (TRNOESY) and saturation transfer difference NMR (STD NMR) spectroscopy. This antibody is specific to diphosphorylated beta-Catenin and does not react with the non-phosphorylated protein. Phosphorylation of beta-Catenin at sites Ser33 and Ser37 on the DSGXXS motif is required for the interaction of beta-Catenin with the ubiquitin ligase SCF(beta-TrCP). beta-TrCP is involved in the ubiquitination and proteasome targeting of the oncogenic protein beta-Catenin, the accumulation of which has been implicated in various human cancers. The three-dimensional structure of the P-beta-Cat(19-44) in the bound conformation was determined by TRNOESY NMR experiments; the peptide adopts a compact structure in the presence of mAb with formation of turns around Trp25 and Gln26, with a tight bend created by the DpS(33)GIHpS(37) motif; the peptide residues (D32-pS37) forming this bend are recognized by the antibody as demonstrated by STD NMR experiments. STD NMR studies provide evidence for the existence of a conformational epitope containing tandem repeats of phosphoserine motifs. The peptide's epitope is predominantly located in the large bend and in the N-terminal segment, implicating bidentate association. These findings are in excellent agreement with a recently published NMR structure required for the interaction of beta-Catenin with the SCF(beta-TrCP) protein.
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PMID:STD and TRNOESY NMR studies for the epitope mapping of the phosphorylation motif of the oncogenic protein beta-catenin recognized by a selective monoclonal antibody. 1699 60

IL-5, IL-3, and GM-CSF are related hematopoietic cytokines, which regulate the function of myeloid cells and are mediators of the allergic inflammatory response. These cytokines signal through heteromeric receptors containing a specific alpha chain and a shared signaling chain, betac. Previous studies demonstrated that the ubiquitin (Ub) proteasome degradation pathway was involved in signal termination of the betac-sharing receptors. In this study, the upstream molecular events leading to proteasome degradation of the IL-5 receptor (IL-5R) were examined. By using biochemical and flow cytometric methods, we show that JAK kinase activity is required for betac ubiquitination and proteasome degradation but only partially required for IL-5R internalization. Furthermore, we demonstrate the direct ubiquitination of the betac cytoplasmic domain and identify lysine residues 566 and 603 as sites of betac ubiquitination. Lastly, we show that ubiquitination of the betac cytoplasmic domain begins at the plasma membrane, increases after receptor internalization, and is degraded by the proteasome after IL-5R internalization. We propose an updated working model of IL-5R down-regulation, whereby IL-5 ligation of its receptor activates JAK2/1 kinases, resulting in betac tyrosine phosphorylation, ubiquitination, and IL-5R internalization. Once inside the cell, proteasomes degrade the betac cytoplasmic domain, and the truncated receptor complex is terminally degraded in the lysosomes. These data establish a critical role for JAK kinases and the Ub/proteasome degradation pathway in IL-5R down-regulation.
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PMID:JAK kinases control IL-5 receptor ubiquitination, degradation, and internalization. 1722 23


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