Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.10.2 (focal adhesion kinase)
 44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Here we report that treatment with the anti-leukemic compound, LFM-A13 resulted in SYK kinase activation and caused distinct shape changes in platelets. Also provided is electron microscopic evidence that similar shape changes are observed in platelets from XID mice. We propose that LFM-A13 induces a conformational change in the PH domain of BTK and causes BTK to associate with PIP2 which effects actin bundling and shape change.
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PMID:A dual function anti-leukemic agent with anti-thrombotic activity. 1214 95

Growth factor binding events to receptor tyrosine kinases result in activation of phosphatidylinositol 3-kinase (PI3K), and activated PI3K generates the membrane-bound second messengers phosphatidylinositol 3,4-diphosphate [PI(3,4)P2] and PI(3,4,5)P3, which mediate membrane translocation of the phosphoinositide-dependent kinase-1 (PDK1) and protein kinase B (PKB, also known as Akt). In addition to the kinase domain, PDK1 and PKB contain a pleckstrin homology (PH) domain that binds to the second messenger, resulting in the phosphorylation and activation of PKB by PDK1. Recent evidence indicates that constitutive activation of PKB contributes to cancer progression by promoting proliferation and increased cell survival. The indicating of PDK1 and PKB as primary targets for discovery of anticancer drugs, together with the observations that both PDK1 and PKB contain small-molecule regulatory binding sites that may be in proximity to the kinase active site, make PDK1 and PKB ideal targets for the development of new strategies to structure-based drug design. While X-ray structures have been reported for the kinase domains of PDK1 and PKB, no suitable crystals have been obtained for either PDK1 or PKB with their PH domains intact. In this regard, a novel structure-based strategy is proposed, which utilizes segmental isotopic labeling of the PH domain in combination with site-directed spin labeling of the kinase active site. Then, long-range distance restraints between the 15N-labeled backbone amide groups of the PH domain and the unpaired electron of the active site spin label can be determined from magnetic resonance studies of the enhancement effect that the paramagnetic spin label has on the nuclear relaxation rates of the amide protons. The determination of the structure and position of the PH domain with respect to the known X-ray structure of the kinase active site could be useful in the rational design of potent and selective inhibitors of PDK1 and PKB by 'linking' the free energies of binding of substrate (ATP) analogs with analogs of the inositol polar head group of the phospholipid second messenger. The combined use of X-ray crystallography, segmental isotopic and spin labeling, and magnetic resonance studies can be further extended to the study of other dynamic multidomain proteins and targets for structure-based drug design.
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PMID:PDK1 and PKB/Akt: ideal targets for development of new strategies to structure-based drug design. 1282 87

Protein kinase B (PKB/Akt) is a key regulator of cell growth, proliferation and metabolism. It possesses an N-terminal pleckstrin homology (PH) domain that interacts with equal affinity with the second messengers PtdIns(3,4,5)P3 and PtdIns(3,4)P2, generated through insulin and growth factor-mediated activation of phosphoinositide 3-kinase (PI3K). The binding of PKB to PtdIns(3,4,5)P3/PtdIns(3,4)P2 recruits PKB from the cytosol to the plasma membrane and is also thought to induce a conformational change that converts PKB into a substrate that can be activated by the phosphoinositide-dependent kinase 1 (PDK1). In this study we describe two high-resolution crystal structures of the PH domain of PKBalpha in a noncomplexed form and compare this to a new atomic resolution (0.98 A, where 1 A=0.1 nm) structure of the PH domain of PKBalpha complexed to Ins(1,3,4,5)P4, the head group of PtdIns(3,4,5)P3. Remarkably, in contrast to all other PH domains crystallized so far, our data suggest that binding of Ins(1,3,4,5)P4 to the PH domain of PKB, induces a large conformational change. This is characterized by marked changes in certain residues making up the phosphoinositide-binding site, formation of a short a-helix in variable loop 2, and a movement of variable loop 3 away from the lipid-binding site. Solution studies with CD also provided evidence of conformational changes taking place upon binding of Ins(1,3,4,5)P4 to the PH domain of PKB. Our data provides the first structural insight into the mechanism by which the interaction of PKB with PtdIns(3,4,5)P3/PtdIns(3,4)P2 induces conformational changes that could enable PKB to be activated by PDK1.
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PMID:Binding of phosphatidylinositol 3,4,5-trisphosphate to the pleckstrin homology domain of protein kinase B induces a conformational change. 1296 41

Serine/threonine kinase Akt/PKB intracellular distribution undergoes rapid changes in response to agonists such as Platelet-derived growth factor (PDGF) or Insulin-like growth factor (IGF). The concept has recently emerged that Akt subcellular movements are facilitated by interaction with nonsubstrate ligands. Here we show that Akt is bound to the actin skeleton in in situ cytoskeletal matrix preparations from PDGF-treated Saos2 cells, suggesting an interaction between the two proteins. Indeed, by immunoprecipitation and subcellular fractioning, we demonstrate that endogenous Akt and actin physically interact. Using recombinant proteins in in vitro binding and overlay assays, we further demonstrate that Akt interacts with actin directly. Expression of Akt mutants strongly indicates that the N-terminal PH domain of Akt mediates this interaction. More important, we show that the partition between actin bound and unbound Akt is not constant, but is modulated by growth factor stimulation. In fact, PDGF treatment of serum-starved cells triggers an increase in the amount of Akt associated with the actin skeleton, concomitant with an increase in Akt phosphorylation. Conversely, expression of an Akt mutant in which both Ser473 and Thr308 have been mutated to alanine completely abrogates PDGF-induced binding. The small GTPases Rac1 and Cdc42 seem to facilitate actin binding, possibly increasing Akt phosphorylation.
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PMID:Targeting of the Akt/PKB kinase to the actin skeleton. 1468 94

The phosphatidylinositol 3-kinase (PI3K) pathway plays a critical role in B cell activation and differentiation. Recruitment of pleckstrin homology (PH) domain-containing signal transduction proteins to the plasma membrane through binding to 3-phosphoinositide second messengers represents a major effector mechanism for PI3Ks. We have found that the PH domains of Bam32 and tandem PH domain-containing protein 2 (TAPP2) specify a temporally distinct wave of membrane recruitment compared with that of Bruton's tyrosine kinase (Btk), with recruitment of these two adaptors representing a later stage of the response. In this study we provide direct evidence that PH domain-dependent recruitment of Btk to the membrane is blocked by coligation of the inhibitory receptor FcgammaRII in human B lymphoma cells. In contrast, recruitment specified by the Bam32 or TAPP2 PH domains is completely insensitive to FcgammaRII inhibition. This differential regulation can be accounted for by Src homology 2-containing inositol polyphosphate 5-phosphatase (SHIP) activity alone, as expression of membrane-targeted SHIP completely abrogated Btk recruitment, but had no inhibitory effect on Bam32 or TAPP2 recruitment. Strikingly, kinetic analysis revealed that membrane recruitment of Bam32 and TAPP2 is actually more rapid under "inhibitory" signaling conditions. Analysis of 3-phosphoinositide generation under activating and inhibitory signaling conditions indicated that recruitment of Bam32 and TAPP2 is inversely correlated with the SHIP substrate/product ratio (phosphatidylinositol 3,4,5-trisphosphate/phosphatidylinositol 3,4-bisphosphate). Overexpression of TAPP2 in B cells led to an increase in the sustained phase of the calcium response and increased NF-AT-dependent transcriptional activation after B cell Ag receptor ligation. Together, these results suggest that Bam32 and TAPP2 adaptors define a novel group of SHIP-activated targets of PI3K that regulate B cell Ag receptor signaling.
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PMID:Two distinct waves of membrane-proximal B cell antigen receptor signaling differentially regulated by Src homology 2-containing inositol polyphosphate 5-phosphatase. 1468 41

The programmed cell death occurs as part of normal mammalian development. The induction of developmental cell death is a highly regulated process and can be suppressed by a variety of extracellular stimuli. Recently, the ability of trophic factors to promote survival have been attributed, at least in part, to the phosphatidylinositide 3'-OH kinase (PI3K)/Protein Kinase B (PKB, also named Akt) cascade. Several targets of the PI3K/PKB signaling pathway have been identified that may underlie the ability of this regulatory cascade to promote cell survival. PKB possesses a N-terminal Pleckstrin Homology (PH) domain that binds specifically and with high affinity to PtIns(3,4,5)P(3) and PtIns(3,4)P(2), the PI3K second messengers. PKB is then recruited to the plasma membrane by virtue of its interaction with 3'-OH phosphatidylinositides and activated. Recent evidence indicates that PKB is active in various types of human cancer; constitutive PKB signaling activation is believed to promote proliferation and increased cell survival, thereby contributing to cancer progression. Thus, it has been shown that induction of PKB activity is augmented by the TCL1/MTCP1 oncoproteins through a physical association requiring the PKB PH domain. Here we present the three-dimensional solution structure of the PH domain of the human protein PKB (isoform beta). PKBbeta-PH is an electrostatically polarized molecule that adopts the same fold and topology as other PH-domains, consisting of a beta-sandwich of seven strands capped on one top by an alpha-helix. The opposite face presents three variable loops that appear poorly defined in the NMR structure. Measurements of (15)N spin relaxation times and heteronuclear (15)N[(1)H]NOEs showed that this poor definition is due to intrinsic flexibility, involving complex motions on different time scales. Chemical shift mapping studies correctly defined the binding site of Ins(1,3,4,5)P(4) (the head group of PtIns(3,4,5)P(3)), as was previously proposed from a crystallographic study. More interestingly, these studies allowed us to define a putative alternative low-affinity binding site for Ins(1,4,5)P(3). The binding of this sugar to PKBbeta-PH might also involve non-specific association that could explain the stabilization of the protein in solution in the presence of Ins(1,4,5)P(3).
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PMID:Solution structure and backbone dynamics of the pleckstrin homology domain of the human protein kinase B (PKB/Akt). Interaction with inositol phosphates. 1475 58

Grb7 is an adaptor molecule that mediates signal transduction from multiple cell surface receptors to various downstream signaling pathways. Grb7 and its related family member Grb10 and Grb14 share a conserved molecular architecture including an amino-terminal proline-rich region, a central segment termed the GM region (for Grb and Mig) which includes a PH domain and shares sequence homology with the Caenorhabditis elegans protein, Mig-10, and a carboxyl-terminal SH2 domain. Grb7/10/14 family proteins are phosphorylated on serine/threonine as well as tyrosine residues, although the functional significance of such phosphorylation is incompletely understood. Grb7/10/14 family proteins are mainly localized in the cytoplasm, but have been observed at the plasma membrane, focal contacts, or mitochondria under certain conditions. A large number of receptor tyrosine kinases and other signaling molecules can associate with Grb7/10/14 family proteins, mostly through the SH2 domains, although the functional consequences of such interactions have not been well characterized in most cases. Recent studies have suggested that various isoforms of Grb10 play important roles in mediating insulin/insulin-like growth factor regulation of cell proliferation and apoptosis, whereas Grb7 mediates signaling pathways from FAK and EphB1 receptor to regulate cell migration, which is also implicated in tumor progression. This review will discuss the current understanding of Grb7 mediated signal transduction pathways and their role in the regulation of various cellular functions.
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PMID:Grb7 in intracellular signaling and its role in cell regulation. 1476 59

Bruton's tyrosine kinase (Btk) is required for B cell development and signal transduction through cell-surface molecules such as BCR and IL-5 receptor. We have identified a Btk-associated molecule, BAM11 (hereafter referred to as BAM) that binds to the pleckstrin homology (PH) domain of Btk, and inhibits Btk activity both in vivo and in vitro. In this study, we demonstrate BAM's transcriptional co-activation activity and its functional interaction with Btk. By using transient transcription assays, we demonstrate that the enforced expression of BAM enhances transcriptional activity of the synthetic reporter gene. The C-terminus of BAM is essential for the transcriptional co-activation activity. The ectopic expression of Btk together with BAM enhances BAM's transcriptional co-activation activity. BAM's transcriptional co-activation activity is enhanced through interaction with Btk, and requires both its intact PH domain and functional kinase activity. We also show that enforced expression of TFII-I, another Btk-binding protein with transcriptional activity, together with BAM and Btk, further augments BAM- and Btk-dependent transcriptional co-activation. Furthermore, BAM can be co-immunoprecipitated with the INI1/SNF5 protein, a member of the SWI/SNF complex that remodels chromatin and activates transcription. We propose a model in which Btk regulates gene transcription in B cells by activating BAM and the SWI/SNF transcriptional complex via TFII-I activation.
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PMID:Bruton's tyrosine kinase (Btk) enhances transcriptional co-activation activity of BAM11, a Btk-associated molecule of a subunit of SWI/SNF complexes. 1509 81

ADP-ribosylation factor (Arf) family of small GTP-binding proteins plays a central role in membrane trafficking and cytoskeletal remodeling. ASAP1 (Arf-GAP containing SH3, ankyrin repeats, and PH domain) is a phospholipid-dependent Arf GTPase-activating protein (Arf-GAP) that binds to protein-tyrosine kinases Src and focal adhesion kinase. Using affinity chromatography and mass spectrometry (MS), we identified the adaptor protein CD2-associated protein (CD2AP) as a candidate binding partner of ASAP1. Both co-immunoprecipitation and GST pull-down experiments confirmed that CD2AP stably interacts with ASAP1 through its N-terminal SH3 domains. Using a mislocalization strategy, we show that sequestration of endogenous ASAP1 to mitochondria with a CD2AP SH3-mito fusion protein (the three N-terminal SH3 domains of CD2AP fused to Listeria monocytogenes ActA mitochondria-targeting sequence) inhibited REF52 cell spreading and migration in response to fibronectin stimulation. Using an alternative strategy we show that suppressing ASAP1 expression with small interfering RNA duplexes also significantly retarded cell spreading and inhibited cell migration. Furthermore, abrogation of ASAP1 function using either small interfering RNAs or mislocalization approaches caused an increase of GTP loading on Arf1 and loss of paxillin from adhesions. These results taken together with our previous observations that overexpression of ASAP1 inhibits cell spreading and alters paxillin localization to adhesions (Liu, Y., Loijens, J. C., Martin, K. H., Karginov, A. V., and Parsons, J. T. (2002) Mol. Biol. Cell. 13, 2147-2156) suggest that the recruitment of certain adhesion components such as paxillin requires dynamic GTP/GDP turnover of Arf1 GTPase.
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PMID:Mislocalization or reduced expression of Arf GTPase-activating protein ASAP1 inhibits cell spreading and migration by influencing Arf1 GTPase cycling. 1563 62

Akt/PKB is a serine/threonine protein kinase that functions as a critical regulator of cell survival and proliferation. Akt/PKB family comprises three highly homologous members known as PKBalpha/Akt1, PKBbeta/Akt2 and PKBgamma/Akt3 in mammalian cells. Similar to many other protein kinases, Akt/PKB contains a conserved domain structure including a specific PH domain, a central kinase domain and a carboxyl-terminal regulatory domain that mediates the interaction between signaling molecules. Akt/PKB plays important roles in the signaling pathways in response to growth factors and other extracellular stimuli to regulate several cellular functions including nutrient metabolism, cell growth, apoptosis and survival. This review surveys recent developments in understanding the molecular mechanisms of Akt/PKB activation and its roles in cell survival in normal and cancer cells.
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PMID:The activation of Akt/PKB signaling pathway and cell survival. 1578 65


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