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)

Chromosomal translocations leading to overexpression of p14(TCL1) and its homologue p13(MTCP1) are hallmarks of several human T-cell malignancies (1). p14(TCL1)/p13(MTCP1) co-activate protein kinase B (PKB, also named Akt) by binding to its pleckstrin homology (PH) domain, suggesting that p14(TCL1)/p13(MTCP1) induce T-cell leukemia by promoting anti-apoptotic signals via PKB (2, 3). Here we combined fluorescence anisotropy, NMR, and small angle x-ray-scattering measurements to determine the affinities, molecular interfaces, and low resolution structure of the complex formed between PKBbeta-PH and p14(TCL1)/p13(MTCP1). We show that p14(TCL1)/p13(MTCP1) target PKB-PH at a site that has not yet been observed in PH-protein interactions. Located opposite the phospholipid binding pocket and distal from known protein-protein interaction sites on PH domains, the binding of dimeric TCL1 proteins to this site would allow the crosslinking of two PKB molecules at the cellular membrane in a preactivated conformation without disrupting certain PH-ligand interactions. Thus this interaction could serve to strengthen membrane association, promote trans-phosphorylation, hinder deactivation of PKB, and involve PKB in a multi-protein complex, explaining the array of known effects of TCL1. The binding sites on both proteins present attractive drug targets against leukemia caused by TCL1 proteins.
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PMID:Structural basis for the co-activation of protein kinase B by T-cell leukemia-1 (TCL1) family proto-oncoproteins. 1516 87

14-3-3 proteins are dimeric phophoserine-binding molecules that participate in important cellular processes such as cell proliferation, cell-cycle control and the stress response. In this work, we report that several isoforms of 14-3-3s are expressed in neonatal rat cardiomyocytes. To understand their function, we utilized a general 14-3-3 peptide inhibitor, R18, to disrupt 14-3-3 functions in cardiomyocytes. Cardiomyocytes infected with adenovirus-expressing YFP-R18 (AdR18) exhibited markedly increased protein synthesis and atrial natriuretic peptide production and potentiated the responses to norepinephrine stimulation. This response was blocked by the pretreatment with LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor. Consistent with a role of PI3K in the R18 effect, R18 induced phosphorylation of a protein cloned from the vakt oncogene of retrovirus AKT8 (Akt - also called protein kinase B, PKB) at Ser473 and glycogen synthase 3beta (GSK3beta) at Ser9, but not extracellular signal-regulated kinase 1/2 (ERK1/2). AdR18-induced PKB and GSK3beta phosphorylation was completely blocked by LY294002. In addition, a member of the nuclear factor of activated T cells (NFAT) family, NFAT3, was converted into faster mobility forms and translocated into the nucleus upon the treatment of AdR18. These results suggest that 14-3-3s inhibits cardiomyocytes hypertrophy through regulation of the PI3K/PKB/GSK3beta and NFAT pathway.
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PMID:14-3-3 proteins regulate glycogen synthase 3beta phosphorylation and inhibit cardiomyocyte hypertrophy. 1581 80

The present work reports a new mode of action of the naturally occurring flavanols catechin and epicatechin and their dimers B2 and B5, in the breast cancer T47D cell line, namely, their interaction with membrane androgen receptors. We show that monomeric and dimeric flavanols are complete (B2) or partial displacers of radiolabeled testosterone bound on T47D membranes, with affinities ranging from 1.7 (B5) to 82.2 nM (B2). In addition, they trigger the phosphorylation of the same signaling molecules (FAK, PI3K) as testosterone-BSA, minutes after binding to membrane receptors, leading to actin cytoskeleton polymerization and redistribution, with formation of filopodia and lamellipodia. The PI3K inhibitor wortmannin reverts the effect of polyphenols and testosterone-BSA, providing additional evidence about activation of a similar signaling cascade. Incubation of T47D cells for more than 2 h with polyphenols or testosterone-BSA induces apoptosis, which follows the same time-dependent pattern. We conclude that flavanols (monomers or dimers) are agonists of membrane androgen receptors and could be used as testosterone-protein conjugates for the management of tumors, in which, application of testosterone-BSA induces regression, providing additional data about the mechanism of their antiproliferative action.
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PMID:Monomeric and oligomeric flavanols are agonists of membrane androgen receptors. 1603 99

Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.
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PMID:Model for growth hormone receptor activation based on subunit rotation within a receptor dimer. 1611 38

An angle Omega is defined to serve as a metric for global side-chain orientations, which reflects the orientation of the side chain relative to the radial vector from the center of the protein to an amino acid. The side-chain orientations of buried residues exhibit characteristically different orientations than do exposed residues, in both monomeric and dimeric structures. Overall, buried side chains point mostly inward, whereas surface side chains tend to point outward from the surface. This difference in behavior also correlates well with the residue hydrophobicity; so a global side-chain orientation can be viewed as a direct structural manifestation of hydrophobicity. When various solvent-accessible layers are considered, the behavior is relatively continuous between centrally located and exposed residues. In the case of interfacial residues between subunits, there are statistically significant differences between exposed residues and interface residues for ALA, ARG, ASN, ASP, GLU, HIS, LYS, THR, VAL, MET, PRO, and overall the interface residues have an increased tendency to point inward. Presumably, these substantial differences in orientations of side chains may be a manifestation of hydrophobic forces.
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PMID:How do side chains orient globally in protein structures? 1615 44

In the absence of erythropoietin (Epo) cell surface Epo receptors (EpoR) are dimeric; dimerization is mediated mainly by the transmembrane domain. Binding of Epo changes the orientation of the two receptor subunits. This conformational change is transmitted through the juxtamembrane and transmembrane domains, leading to activation of JAK2 kinase and induction of proliferation and survival signals. To define the active EpoR conformation(s) we screened libraries of EpoRs with random mutations in the transmembrane domain and identified several point mutations that activate the EpoR in the absence of ligand, including changes of either of the first two transmembrane domain residues (Leu(226) and Ile(227)) to cysteine. Following this discovery, we performed cysteine-scanning mutagenesis in the EpoR juxtamembrane and transmembrane domains. Many mutants formed disulfide-linked receptor dimers, but only EpoR dimers linked by cysteines at positions 223, 226, or 227 activated EpoR signal transduction pathways and supported proliferation of Ba/F3 cells in the absence of cytokines. These data suggest that activation of dimeric EpoR by Epo binding is achieved by reorienting the EpoR transmembrane and the connected cytosolic domains and that certain disulfide-bonded dimers represent the activated dimeric conformation of the EpoR, constitutively activating downstream signaling. Based on our data and the previously determined structure of Epo bound to a dimer of the EpoR extracellular domain, we present a model of the active and inactive conformations of the Epo receptor.
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PMID:Active conformation of the erythropoietin receptor: random and cysteine-scanning mutagenesis of the extracellular juxtamembrane and transmembrane domains. 1641 57

Endothelial nitric oxide synthase (eNOS) is regulated by phosphorylation of Ser(1177) and Thr(495), which affects NO bioavailability. Cigarette smoke disturbs the eNOS-cGMP-NO pathway and causes decreased NO production. Here the authors investigated the acute effects of cigarette smoke on eNOS phosphorylation, focusing on protein kinases (PKs). Endothelial cell culture was concentration- and time-dependently treated first with cigarette smoke buffer (CSB), then with reduced glutathione (GSH) or various PK inhibitors (H-89, LY-294002, Ro-318425, and ruboxistaurin). eNOS, phospho-Ser(1177)-eNOS, phospho-Thr(495)-eNOS, Akt(PKB), and phospho-Akt protein levels were determined by Western blot. CSB increased the phosphorylation of eNOS at Ser(1177) and more at Thr(495) in a concentration- and time-dependent manner (p < .01, p < .05 versus control, respectively) and resulted in the dissociation of the active dimeric form of eNOS (p < .05). GSH decreased the phosphorylation of eNOS at both sites (p < .05 versus CSB without GSH) and prevented the decrease of dimer eNOS level. CSB treatment also decreased the level of phospho-Ser(473)-Akt (p < .05 versus control). Inhibition of PKA by H-89 did not affect CSB-induced phosphorylation, whereas the PKB inhibitor LY-294002 enhanced it at Ser(1117). The PKC blockers Ro-318425 and ruboxistaurin augmented the CSB-induced phosphorylation at Ser(1177) but decreased phosphorylation at Thr(495) (p < .05 versus CSB). Cigarette smoke causes a disruption of the enzymatically active eNOS dimers and shifts the eNOS phosphorylation to an inhibitory state. Both effects might lead to reduced NO bioavailability. The shift of the eNOS phosphorylation pattern to an inhibitory state seems to be independent of the PKA and phosphoinositol 3-kinase (PI3-K)/Akt pathways, whereas PKC appears to play a key role.
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PMID:Cigarette smoke-induced alterations in endothelial nitric oxide synthase phosphorylation: role of protein kinase C. 1792 42

GH binds dimerized GH receptors (GHRs) to form a trimolecular complex and induces downstream signaling events. The mechanism by which GH binding converts the inactive predimerized GHR to its active signaling conformation is uncertain. GH has no axis of symmetry. Its interaction with GHR is mediated by two asymmetric binding sites on GH, each with distinct affinity. Site 1 is of high affinity and is thought to mediate the first binding step. Mutation of binding site 2 (as in the human GH mutant, G120R) disrupts the second binding but leaves site 1 binding intact. G120R is a GH antagonist; it binds only one GHR and thus fails to signal, and it prevents productive GHR binding by normal GH. We previously demonstrated that prolactin receptor signaling was achieved by a dimeric version of a prolactin antagonist. We now employ assays of cellular signaling and receptor conformational changes to examine whether GH molecules harboring two site 1 regions can trigger GHR activation. We used recombinantly produced GH-GH and G120R-G120R dimers in which monomers in tandem are connected by a short linker peptide. Rabbit GHR-expressing human fibrosarcoma cells (C14) were treated with GH, G120R, GH-GH, or G120R-G120R. As expected, GH and GH-GH, but not G120R, induced GHR disulfide linkage, as assessed by anti-GHR blotting of cell extracts resolved by SDS-PAGE under nonreducing conditions. Disulfide linkage of GHRs reflects attainment of the active signaling conformation. Likewise, GH and GH-GH, but not G120R, caused Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5) activation. Notably, G120R-G120R, despite its lack of an intact site 2 in either dimer partner, also promoted GHR disulfide linkage and JAK2 and STAT5 activation, albeit less potently than either GH or GH-GH. Time-course responses of the three agonists were similar in terms of JAK2 and STAT5 activation. Pretreatment of cells with our conformation-sensitive inhibitory monoclonal antibody, anti-GHR ext-mAb, prevented ligand-induced receptor activation for all three agonists. GHR was also rendered less immunoprecipitable by anti-GHR ext-mAb after treatment with these agonists. These results are important in that they indicate that a ligand with two intact binding sites 1 causes GHR to adopt similar conformational changes as does GH and thus triggers activation of JAK2 and downstream signaling. Furthermore, we infer that there is substantial flexibility in the GHR extracellular domain, such that it productively accommodates GH dimers that are much larger than GH.
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PMID:Activation of growth hormone receptors by growth hormone and growth hormone antagonist dimers: insights into receptor triggering. 1809 90

BCR-ABL negative myeloproliferative neoplasms (MPNs; polycythemia vera, essential thrombocythemia, primary myelofibrosis) are malignant diseases arising from a multipotent hematopoietic progenitor, frequently altered by JAK2 V617F or other JAK/STAT activating mutations. The thrombopoietin receptor (TpoR, MPL) is one of the major dimeric cytokine receptors that use JAK2 in the myeloid lineage, and was found to be down-modulated in certain MPN patients. We searched for negative regulators of MPL expression. Here we report that miR-28 targets the 3' untranslated (3'UTR) region of MPL, inhibiting its translation, as well as other proteins potentially involved in megakaryocyte differentiation, such as E2F6. Expression of miR-28 in CD34-derived megakaryocytes inhibited terminal differentiation. miR-28 was found to be overexpressed in platelets of a fraction of MPN patients, while it was expressed at constant low levels in platelets from healthy subjects. Constitutive activation of STAT5 leading to autonomous growth of hematopoietic cell lines was associated with increased miR-28 expression. We discuss how down-modulating MPL and other targets of miR-28, and of related miR-708 and miR-151, could contribute to MPN pathogenicity.
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PMID:miR-28 is a thrombopoietin receptor targeting microRNA detected in a fraction of myeloproliferative neoplasm patient platelets. 2044 18

The JAK2 V617F mutation present in over 95% of Polycythemia Vera patients and in 50% of Essential Thrombocythemia and Primary Myelofibrosis patients renders the kinase constitutively active. In the absence of a three-dimensional structure for the full-length protein, the mechanism of activation of JAK2 V617F has remained elusive. In this study, we used functional mutagenesis to investigate the involvement of the JH2 alphaC helix in the constitutive activation of JAK2 V617F. We show that residue F595, located in the middle of the alphaC helix of JH2, is indispensable for the constitutive activity of JAK2 V617F. Mutation of F595 to Ala, Lys, Val or Ile significantly decreases the constitutive activity of JAK2 V617F, but F595W and F595Y are able to restore it, implying an aromaticity requirement at position 595. Substitution of F595 to Ala was also able to decrease the constitutive activity of two other JAK2 mutants, T875N and R683G, as well as JAK2 K539L, albeit to a lower extent. In contrast, the F595 mutants are activated by erythropoietin-bound EpoR. We also explored the relationship between the dimeric conformation of EpoR and several JAK2 mutants. Since residue F595 is crucial to the constitutive activation of JAK2 V617F but not to initiation of JAK2 activation by cytokines, we suggest that small molecules that target the region around this residue might specifically block oncogenic JAK2 and spare JAK2 wild-type.
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PMID:JAK2 V617F constitutive activation requires JH2 residue F595: a pseudokinase domain target for specific inhibitors. 2058 91


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