scholarly journals Identification and characterization of pleckstrin-homology-domain-dependent and isoenzyme-specific Akt inhibitors

2005 ◽  
Vol 385 (2) ◽  
pp. 399-408 ◽  
Author(s):  
Stanley F. BARNETT ◽  
Deborah DEFEO-JONES ◽  
Sheng FU ◽  
Paula J. HANCOCK ◽  
Kathleen M. HASKELL ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Time Resolved ◽  
Akt Isoforms ◽  
Ic50 Values ◽  
Induced Apoptosis ◽  
A Site

We developed a high-throughput HTRF (homogeneous time-resolved fluorescence) assay for Akt kinase activity and screened approx. 270000 compounds for their ability to inhibit the three isoforms of Akt. Two Akt inhibitors were identified that exhibited isoenzyme specificity. The first compound (Akt-I-1) inhibited only Akt1 (IC50 4.6 μM) while the second compound (Akt-I-1,2) inhibited both Akt1 and Akt2 with IC50 values of 2.7 and 21 μM respectively. Neither compound inhibited Akt3 nor mutants lacking the PH (pleckstrin homology) domain at concentrations up to 250 μM. These compounds were reversible inhibitors, and exhibited a linear mixed-type inhibition against ATP and peptide substrate. In addition to inhibiting kinase activity of individual Akt isoforms, both inhibitors blocked the phosphorylation and activation of the corresponding Akt isoforms by PDK1 (phosphoinositide-dependent kinase 1). A model is proposed in which these inhibitors bind to a site formed only in the presence of the PH domain. Binding of the inhibitor is postulated to promote the formation of an inactive conformation. In support of this model, antibodies to the Akt PH domain or hinge region blocked the inhibition of Akt by Akt-I-1 and Akt-I-1,2. These inhibitors were found to be cell-active and to block phosphorylation of Akt at Thr308 and Ser473, reduce the levels of active Akt in cells, block the phosphorylation of known Akt substrates and promote TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand)-induced apoptosis in LNCap prostate cancer cells.

Structural Characterization of the Interaction between a Pleckstrin Homology Domain and Phosphatidylinositol 4,5-Bisphosphate

Biochemistry ◽  
1995 ◽  
Vol 34 (31) ◽  
pp. 9859-9864 ◽  
Author(s):  
John E. Harlan ◽  
Ho Sup Yoon ◽  
Philip J. Hajduk ◽  
Stephen W. Fesik
Keyword(s):  
Structural Characterization ◽  
Pleckstrin Homology Domain ◽  
Pleckstrin Homology

Stratifin, a keratinocyte specific 14–3-3 protein, harbors a pleckstrin homology (PH) domain and enhances protein kinase C activity

1995 ◽  
Vol 108 (11) ◽  
pp. 3569-3579
Author(s):  
E. Dellambra ◽  
M. Patrone ◽  
B. Sparatore ◽  
A. Negri ◽  
F. Ceciliani ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Terminal Differentiation ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Protein Database ◽  
Kinase C ◽  
Human Keratinocyte ◽  
Protein Kinase C Activity

The intrinsic signal(s) responsible for the onset of human keratinocyte terminal differentiation is not yet fully understood. Evidence has been recently accumulated linking the phospholipase-mediated activation of protein kinase C to the coordinate changes in gene expression occurring during keratinocyte terminal differentiation. Here we report the purification of a keratinocyte-derived protein enhancing protein kinase C enzymatic activity. The stimulator eluted as a peak with estimated molecular mass of approximately 70 kDa, while analysis by SDS-PAGE showed a 30 kDa protein migrating as a distinct doublet, suggesting the formation of a 30 kDa homodimer. The amino acid sequence analysis allowed the unambigous identification of the protein kinase C stimulator as a mixture of the highly homologous sigma (stratifin) and zeta isoforms of 14–3-3 proteins, which are homodimers of identical 30 kDa subunits. Mono Q anion exchange chromatography and immunoblot analysis further confirmed that stratifin enhances protein kinase C activity. Stratifin was originally sequenced from a human keratinocyte protein database, but its function was unknown. The pleckstrin homology domain has been recently related to protein translocation to the cell membrane as well as to functional interactions of intracellular proteins involved in signal transduction. We show here that stratifin (and 14–3-3 zeta) harbors a pleckstrin homology domain, and the consequent functional implications will be discussed.

scholarly journals The Yeast PH Domain Proteins Slm1 and Slm2 Are Targets of Sphingolipid Signaling during the Response to Heat Stress

2006 ◽  
Vol 27 (2) ◽  
pp. 633-650 ◽  
Author(s):  
Alexes Daquinag ◽  
Maria Fadri ◽  
Sung Yun Jung ◽  
Jun Qin ◽  
Jeannette Kunz
Keyword(s):  
Heat Stress ◽  
Signaling Pathways ◽  
Actin Polymerization ◽  
Mutational Analysis ◽  
Ph Domain ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein ◽  
A Site ◽  
Arginine Permease

ABSTRACT The PH domain-containing proteins Slm1 and Slm2 were previously identified as effectors of the phosphatidylinositol-4,5-bisphosphate (PI4,5P2) and TORC2 signaling pathways. Here, we demonstrate that Slm1 and Slm2 are also targets of sphingolipid signaling during the heat shock response. We show that upon depletion of cellular sphingolipid levels, Slm1 function becomes essential for survival under heat stress. We further demonstrate that Slm proteins are regulated by a phosphorylation/dephosphorylation cycle involving the sphingolipid-activated protein kinases Pkh1 and Pkh2 and the calcium/calmodulin-dependent protein phosphatase calcineurin. By using a combination of mass spectrometry and mutational analysis, we identified serine residue 659 in Slm1 as a site of phosphorylation. Characterization of Slm1 mutants that mimic dephosphorylated and phosphorylated states demonstrated that phosphorylation at serine 659 is vital for survival under heat stress and promotes the proper polarization of the actin cytoskeleton. Finally, we present evidence that Slm proteins are also required for the trafficking of the raft-associated arginine permease Can1 to the plasma membrane, a process that requires sphingolipid synthesis and actin polymerization. Together with previous work, our findings suggest that Slm proteins are subject to regulation by multiple signals, including PI4,5P2, TORC2, and sphingolipids, and may thus integrate inputs from different signaling pathways to temporally and spatially control actin polarization.

Molecular modelling and site-directed mutagenesis of the inositol 1,3,4,5-tetrakisphosphate-binding pleckstrin homology domain from the Ras GTPase-activating protein GAP1IP4BP

2000 ◽  
Vol 349 (1) ◽  
pp. 333-342 ◽  
Author(s):  
Gyles COZIER ◽  
Richard SESSIONS ◽  
Joanna R. BOTTOMLEY ◽  
Jon S. REYNOLDS ◽  
Peter J. CULLEN
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Inositol Phosphate ◽  
Site Directed Mutagenesis ◽  
Pleckstrin Homology Domain ◽  
Directed Mutagenesis ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Gtpase Activating Protein ◽  
Ras Gtpase

GAP1IP4BP is a Ras GTPase-activating protein (GAP) that in vitro is regulated by the cytosolic second messenger inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. We have studied Ins(1,3,4,5)P4 binding to GAP1IP4BP, and shown that the inositol phosphate specificity and binding affinity are similar to Ins(1,3,4,5)P4 binding to Bruton's tyrosine kinase (Btk), evidence which suggests a similar mechanism for Ins(1,3,4,5)P4 binding. The crystal structure of the Btk pleckstrin homology (PH) domain in complex with Ins(1,3,4,5)P4 has shown that the binding site is located in a partially buried pocket between the β1/β2- and β3/β4-loops. Many of the residues involved in the binding are conserved in GAP1IP4BP. Therefore we generated a model of the PH domain of GAP1IP4BP in complex with Ins(1,3,4,5)P4 based on the Btk-Ins(1,3,4,5)P4 complex crystal structure. This model had the typical PH domain fold, with the proposed binding site modelling well on the Btk structure. The model has been verified by site-directed mutagenesis of various residues in and around the proposed binding site. These mutations have markedly reduced affinity for Ins(1,3,4,5)P4, indicating a specific and tight fit for the substrate. The model can also be used to explain the specificity of inositol phosphate binding.

scholarly journals Plant Actin-binding Protein SCAB1 Is Dimeric Actin Cross-linker with Atypical Pleckstrin Homology Domain

2012 ◽  
Vol 287 (15) ◽  
pp. 11981-11990 ◽  
Author(s):  
Wei Zhang ◽  
Yang Zhao ◽  
Yan Guo ◽  
Keqiong Ye
Keyword(s):  
Functional Organization ◽  
Inositol Phosphates ◽  
Binding Protein ◽  
Actin Binding ◽  
Surface Patch ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Actin Binding Protein ◽  
Cross Linker

SCAB1 is a novel plant-specific actin-binding protein that binds, bundles, and stabilizes actin filaments and regulates stomatal movement. Here, we dissected the structure and function of SCAB1 by structural and biochemical approaches. We show that SCAB1 is composed of an actin-binding domain, two coiled-coil (CC) domains, and a fused immunoglobulin and pleckstrin homology (Ig-PH) domain. We determined crystal structures for the CC1 and Ig-PH domains at 1.9 and 1.7 Å resolution, respectively. The CC1 domain adopts an antiparallel helical hairpin that further dimerizes into a four-helix bundle. The CC2 domain also mediates dimerization. At least one of the coiled coils is required for actin binding, indicating that SCAB1 is a bivalent actin cross-linker. The key residues required for actin binding were identified. The PH domain lacks a canonical basic phosphoinositide-binding pocket but can bind weakly to inositol phosphates via a basic surface patch, implying the involvement of inositol signaling in SCAB1 regulation. Our results provide novel insights into the functional organization of SCAB1.

scholarly journals The Pleckstrin Homology Domain Proteins Slm1 and Slm2 Are Required for Actin Cytoskeleton Organization in Yeast and Bind Phosphatidylinositol-4,5-Bisphosphate and TORC2

2005 ◽  
Vol 16 (4) ◽  
pp. 1883-1900 ◽  
Author(s):  
Maria Fadri ◽  
Alexes Daquinag ◽  
Shimei Wang ◽  
Tao Xue ◽  
Jeannette Kunz
Keyword(s):  
Cell Growth ◽  
Actin Cytoskeleton ◽  
Membrane Dynamics ◽  
Effector Proteins ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Yeast Saccharomyces Cerevisiae ◽  
Actin Organization ◽  
Cytoskeleton Organization

Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] is a key second messenger that regulates actin and membrane dynamics, as well as other cellular processes. Many of the effects of PtdIns(4,5)P2are mediated by binding to effector proteins that contain a pleckstrin homology (PH) domain. Here, we identify two novel effectors of PtdIns(4,5)P2in the budding yeast Saccharomyces cerevisiae: the PH domain containing protein Slm1 and its homolog Slm2. Slm1 and Slm2 serve redundant roles essential for cell growth and actin cytoskeleton polarization. Slm1 and Slm2 bind PtdIns(4,5)P2through their PH domains. In addition, Slm1 and Slm2 physically interact with Avo2 and Bit61, two components of the TORC2 signaling complex, which mediates Tor2 signaling to the actin cytoskeleton. Together, these interactions coordinately regulate Slm1 targeting to the plasma membrane. Our results thus identify two novel effectors of PtdIns(4,5)P2regulating cell growth and actin organization and suggest that Slm1 and Slm2 integrate inputs from the PtdIns(4,5)P2and TORC2 to modulate polarized actin assembly and growth.

Expression and characterization of a pleckstrin homology domain in phospholipase C, PLC-η1

2007 ◽  
Vol 56 (2) ◽  
pp. 247-252 ◽  
Author(s):  
Toru Imai ◽  
Kouki Kasai ◽  
Junichi Kurita ◽  
Kiyoko Fukami ◽  
Mitsuru Tashiro ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Pleckstrin Homology Domain ◽  
Pleckstrin Homology

scholarly journals Expression of Gab1 Lacking the Pleckstrin Homology Domain Is Associated with Neoplastic Progression

2001 ◽  
Vol 21 (20) ◽  
pp. 6895-6905 ◽  
Author(s):  
Hideto Kameda ◽  
John I. Risinger ◽  
Bing-Bing Han ◽  
Seung Joon Baek ◽  
J. Carl Barrett ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Colony Formation ◽  
Soft Agar ◽  
Pleckstrin Homology Domain ◽  
Dependent Manner ◽  
Neoplastic Progression ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Amino Terminal ◽  
Endogenous Expression

ABSTRACT An in vitro transformation system of carcinogen-treated Syrian hamster embryo (SHE) cell cultures represents multistep genetic and nongenetic changes that develop during the neoplastic progression of normal cells to tumor cells in vivo. During this neoplastic progression, SHE cells demonstrate an altered response to epidermal growth factor (EGF). In the present report, we examined the role of the adapter protein Gab1 (Grb2-associated binder-1) in the neoplastic progression of SHE cells. We used two asbestos-transformed SHE cell clones in different neoplastic stages: a 10W+8 clone, which is immortal and retains the ability to suppress the tumorigenicity of tumor cells in cell-cell hybrid experiments, and a 10W−1 clone, which has lost this tumor suppressor ability. 10W+8 cells expressed full-length 100-kDa Gab1 and associated 5.2-kb mRNA. Upon repeated cell passaging, 10W−1 cells showed increasing expression of a novel 87-kDa form of Gab1 as well as 4.6-kb mRNA with diminishing expression of the original 100-kDa Gab1. cDNA encoding the 87-kDa Gab1 predicts a form of Gab1 lacking the amino-terminal 103 amino acids (Gab1Δ1-103), which corresponds to loss of most of the pleckstrin homology (PH) domain. Gab1Δ1-103 retains the ability to be phosphorylated in an EGF-dependent manner and to associate with the EGF receptor and SHP-2 upon EGF stimulation. The endogenous expression of Gab1Δ1-103 in 10W−1 cells appeared closely related to EGF-dependent colony formation in soft agar. Moreover, transfection and expression of Gab1Δ1-103, but not Gab1, in 10W+8 cells enhanced their EGF-dependent colony formation in soft agar. These results demonstrate that Gab1 is a target of carcinogen-induced transformation of SHE cells and that the expression of a Gab1 variant lacking most of the PH domain plays a specific role in the neoplastic progression of SHE cells.

scholarly journals Role for the Pleckstrin Homology Domain-Containing Protein CKIP-1 in Phosphatidylinositol 3-Kinase-Regulated Muscle Differentiation

2004 ◽  
Vol 24 (3) ◽  
pp. 1245-1255 ◽  
Author(s):  
Alexias Safi ◽  
Marie Vandromme ◽  
Sabine Caussanel ◽  
Laure Valdacci ◽  
Dominique Baas ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Active Form ◽  
Muscle Differentiation ◽  
C2c12 Cells ◽  
Pleckstrin Homology Domain ◽  
Dependent Manner ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

ABSTRACT In this work, we report the implication of the pleckstrin homology (PH) domain-containing protein CKIP-1 in phosphatidylinositol 3-kinase (PI3-K)-regulated muscle differentiation. CKIP-1 is upregulated during muscle differentiation in C2C12 cells. We show that CKIP-1 binds to phosphatidylinositol 3-phosphate through its PH domain and localizes to the plasma membrane in a PI3-K-dependent manner. Activation of PI3-K by insulin or expression of an active form of PI3-K p110 induces a rapid translocation of CKIP-1 to the plasma membrane. Conversely, expression of the 3-phosphoinositide phosphatase myotubularin or PI3-K inhibition by LY294002, wortmannin, or mutant p85 abolishes CKIP-1 binding to the membrane. Upon induction of differentiation in low-serum medium, CKIP-1 overexpression in C2C12 myoblasts first promotes proliferation and then stimulates the expression of myogenin and cell fusion in a manner reminiscent of the dual positive effect of insulin-like growth factors on muscle cells. Interference with the PI3-K pathway impedes the effect of CKIP-1 on C2C12 cell differentiation. Finally, silencing of CKIP-1 by RNA interference abolishes proliferation and delays myogenin expression. Altogether, these data strongly implicate CKIP-1 as a new component of PI3-K signaling in muscle differentiation.

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