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1.
J Biol Chem ; 283(41): 27653-27667, 2008 Oct 10.
Article in English | MEDLINE | ID: mdl-18669636

ABSTRACT

Protein kinase B (PKB)/Akt has been strongly implicated in the insulin-dependent stimulation of GLUT4 translocation and glucose transport in skeletal muscle and fat cells. Recently an allosteric inhibitor of PKB (Akti) that selectively targets PKBalpha and -beta was reported, but as yet its precise mechanism of action or ability to suppress key insulin-regulated events such as glucose and amino acid uptake and glycogen synthesis in muscle cells has not been reported. We show here that Akti ablates the insulin-dependent regulation of these processes in L6 myotubes at submicromolar concentrations and that inhibition correlates tightly with loss of PKB activation/phosphorylation. Similar findings were obtained using 3T3-L1 adipocytes. Akti did not inhibit IRS1 tyrosine phosphorylation, phosphatidylinositol 3-kinase signaling, or activation of Erks, ribosomal S6 kinase, or atypical protein kinases C but significantly impaired regulation of downstream PKB targets glycogen synthase kinase-3 and AS160. Akti-mediated inhibition of PKB requires an intact kinase pleckstrin homology domain but does not involve suppression of 3-phosphoinositide binding to this domain. Importantly, we have discovered that Akti inhibition is critically dependent upon a solvent-exposed tryptophan residue (Trp-80) that is present within the pleckstrin homology domain of all three PKB isoforms and whose mutation to an alanine (PKB(W80A)) yields an Akti-resistant kinase. Cellular expression of PKB(W80A) antagonized the Akti-mediated inhibition of glucose and amino acid uptake. Our findings support a critical role for PKB in the hormonal regulation of glucose and system A amino acid uptake and indicate that use of Akti and expression of the drug-resistant kinase will be valuable tools in delineating cellular PKB functions.


Subject(s)
Adipocytes/metabolism , Amino Acid Transport System A/metabolism , Amino Acids/metabolism , Drug Resistance , Glucose Transporter Type 4/metabolism , Glucose/metabolism , Hypoglycemic Agents/pharmacology , Insulin/pharmacology , Muscle, Skeletal/metabolism , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , 3T3-L1 Cells , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adipocytes/cytology , Amino Acid Transport System A/genetics , Amino Acids/genetics , Animals , Biological Transport/genetics , Drug Resistance/genetics , Enzyme Activation/genetics , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , GTPase-Activating Proteins/genetics , GTPase-Activating Proteins/metabolism , Glucose Transporter Type 4/genetics , Glycogen/genetics , Glycogen/metabolism , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3/metabolism , Insulin Receptor Substrate Proteins , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , Isoenzymes/metabolism , Mice , Muscle, Skeletal/cytology , Mutation, Missense , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Rats , Ribosomal Protein S6 Kinases/genetics , Ribosomal Protein S6 Kinases/metabolism
2.
Biochem J ; 399(3): 427-34, 2006 Nov 01.
Article in English | MEDLINE | ID: mdl-16879102

ABSTRACT

TAB1 [TAK1 (transforming growth factor-beta-activated kinase 1)-binding protein 1] is one of the regulatory subunits of TAK1, a protein kinase that lies at the head of three pro-inflammatory kinase cascades. In the current study we report the crystal structure of the N-terminal domain of TAB1. Surprisingly, TAB1 possesses a fold closely related to that of the PPM (Mg2+- or Mn2+-dependent protein phosphatase) family as demonstrated by the close structural similarity with protein phosphatase 2C alpha. However, we were unable to detect any phosphatase activity for TAB1 using a phosphopeptide or p-nitrophenyl phosphate as substrate. Although the overall protein phosphatase 2C alpha fold is conserved in TAB1, detailed structural analyses and mutagenesis studies show that several key residues required for dual metal-binding and catalysis are not present in TAB1, although binding of a single metal is supported by soaking experiments with manganese and isothermal titration calorimetry. Thus, it appears that TAB1 is a 'pseudophosphatase', possibly binding to and regulating accessibility of phosphorylated residues on substrates downstream of TAK1 or on the TAK1 complex itself.


Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Sequence , Calorimetry , Catalysis , Crystallography, X-Ray , Manganese/metabolism , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Nitrophenols/metabolism , Organophosphorus Compounds/metabolism , Phosphoprotein Phosphatases/chemistry , Phosphoprotein Phosphatases/genetics , Phosphoproteins/metabolism , Protein Binding , Protein Conformation , Protein Folding , Protein Phosphatase 2C , Protein Structure, Tertiary , Protein Subunits , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Structure-Activity Relationship , Substrate Specificity
3.
J Biol Chem ; 280(19): 18797-802, 2005 May 13.
Article in English | MEDLINE | ID: mdl-15741170

ABSTRACT

3-Phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates the T-loop of several AGC (cAMP-dependent, cGMP-dependent, protein kinase C) family protein kinases, resulting in their activation. Previous structural studies have revealed that the alpha C-helix, located in the small lobe of the kinase domain of PDK1, is a key regulatory element, as it links a substrate interacting site termed the hydrophobic motif (HM) pocket with the phosphorylated Ser-241 in the T-loop. In this study we have demonstrated by mutational analysis that interactions between the phosphorylated Ser-241 and the alpha C-helix are not required for PDK1 activity or substrate binding through the HM-pocket but are necessary for PDK1 to be activated or stabilized by a peptide that binds to this site. The structure of an inactive T-loop mutant of PDK1, in which Ser-241 is changed to Ala, was also determined. This structure, together with surface plasmon resonance binding studies, demonstrates that the PDK1(S241A)-inactive mutant possesses an intact HM-pocket as well as an ordered alpha C-helix. These findings reveal that the integrity of the alpha C-helix and HM-pocket in PDK1 is not regulated by T-loop phosphorylation.


Subject(s)
Protein Serine-Threonine Kinases/chemistry , 3-Phosphoinositide-Dependent Protein Kinases , Adenosine Triphosphate/chemistry , Amino Acid Motifs , Binding Sites , Cell Line , Cyclic AMP/metabolism , DNA Mutational Analysis , Humans , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Mutation , Peptides/chemistry , Phosphorylation , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Serine/chemistry , Substrate Specificity , Surface Plasmon Resonance , Temperature
4.
EMBO J ; 23(20): 3918-28, 2004 Oct 13.
Article in English | MEDLINE | ID: mdl-15457207

ABSTRACT

3-phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates and activates many kinases belonging to the AGC subfamily. PDK1 possesses a C-terminal pleckstrin homology (PH) domain that interacts with PtdIns(3,4,5)P3/PtdIns(3,4)P2 and with lower affinity to PtdIns(4,5)P2. We describe the crystal structure of the PDK1 PH domain, in the absence and presence of PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4. The structures reveal a 'budded' PH domain fold, possessing an N-terminal extension forming an integral part of the overall fold, and display an unusually spacious ligand-binding site. Mutagenesis and lipid-binding studies were used to define the contribution of residues involved in phosphoinositide binding. Using a novel quantitative binding assay, we found that Ins(1,3,4,5,6)P5 and InsP6, which are present at micromolar levels in the cytosol, interact with full-length PDK1 with nanomolar affinities. Utilising the isolated PDK1 PH domain, which has reduced affinity for Ins(1,3,4,5,6)P5/InsP6, we perform localisation studies that suggest that these inositol phosphates serve to anchor a portion of cellular PDK1 in the cytosol, where it could activate its substrates such as p70 S6-kinase and p90 ribosomal S6 kinase that do not interact with phosphoinositides.


Subject(s)
Inositol Phosphates/metabolism , Phosphatidylinositols/metabolism , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , 3-Phosphoinositide-Dependent Protein Kinases , Amino Acid Sequence , Binding Sites , Binding, Competitive , Cell Line , Crystallography, X-Ray , Cytosol/chemistry , Cytosol/metabolism , Fluorescence Resonance Energy Transfer , Glutathione Transferase/metabolism , Humans , Hydrophobic and Hydrophilic Interactions , Ligands , Lipid Metabolism , Models, Molecular , Mutagenesis, Site-Directed , Mutation , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Spectrum Analysis, Raman , Water/chemistry
5.
Biochem J ; 382(Pt 3): 857-65, 2004 Sep 15.
Article in English | MEDLINE | ID: mdl-15242348

ABSTRACT

Binding of the Rac1-specific guanine-nucleotide-exchange factor, Tiam1, to the plasma membrane requires the N-terminal pleckstrin homology domain. In the present study, we show that membrane-association is mediated by binding of PtdIns(4,5)P(2) to the pleckstrin homology domain. Moreover, in 1321N1 astrocytoma cells, translocation of Tiam1 to the cytosol, following receptor-mediated stimulation of PtdIns(4,5)P(2) breakdown, correlates with decreased Rac1-GTP levels, indicating that membrane-association is required for GDP/GTP exchange on Rac1. In addition, we show that platelet-derived growth factor activates Rac1 in vivo by increasing PtdIns(3,4,5)P(3) concentrations, rather than the closely related lipid, PtdIns(3,4)P(2). Finally, the data demonstrate that PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) bind to the same pleckstrin homology domain in Tiam1 and that soluble inositol phosphates appear to compete with lipids for this binding. Together, these novel observations provide strong evidence that distinct phosphoinositides regulate different functions of this enzyme, indicating that local concentrations of signalling lipids and the levels of cytosolic inositol phosphates will play crucial roles in determining its activity in vivo.


Subject(s)
Guanine Nucleotide Exchange Factors/metabolism , Phosphatidylinositol Phosphates/metabolism , Proteins/metabolism , rac1 GTP-Binding Protein/metabolism , Androstadienes/pharmacology , Cell Line, Tumor , Cell Membrane/metabolism , Cytosol/metabolism , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Humans , Peptide Fragments/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphatidylinositol Phosphates/physiology , Phosphoinositide-3 Kinase Inhibitors , Platelet-Derived Growth Factor/pharmacology , Protein Binding , Protein Structure, Tertiary , Protein Transport , Receptors, Cytoplasmic and Nuclear/metabolism , Recombinant Fusion Proteins/metabolism , T-Lymphoma Invasion and Metastasis-inducing Protein 1 , Thrombin/metabolism , Wortmannin
6.
Nat Cell Biol ; 6(5): 393-404, 2004 May.
Article in English | MEDLINE | ID: mdl-15107860

ABSTRACT

The molecular mechanisms underlying the formation of carriers trafficking from the Golgi complex to the cell surface are still ill-defined; nevertheless, the involvement of a lipid-based machinery is well established. This includes phosphatidylinositol 4-phosphate (PtdIns(4)P), the precursor for phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). In yeast, PtdIns(4)P exerts a direct role, however, its mechanism of action and its targets in mammalian cells remain uncharacterized. We have identified two effectors of PtdIns(4)P, the four-phosphate-adaptor protein 1 and 2 (FAPP1 and FAPP2). Both proteins localize to the trans-Golgi network (TGN) on nascent carriers, and interact with PtdIns(4)P and the small GTPase ADP-ribosylation factor (ARF) through their plekstrin homology (PH) domain. Displacement or knockdown of FAPPs inhibits cargo transfer to the plasma membrane. Moreover, overexpression of FAPP-PH impairs carrier fission. Therefore, FAPPs are essential components of a PtdIns(4)P- and ARF-regulated machinery that controls generation of constitutive post-Golgi carriers.


Subject(s)
ADP-Ribosylation Factors/metabolism , Carrier Proteins/metabolism , Cell Membrane/metabolism , Fungal Proteins/metabolism , Golgi Apparatus/metabolism , Phosphatidylinositol Phosphates/metabolism , trans-Golgi Network/metabolism , Adaptor Proteins, Signal Transducing , Animals , Biological Transport/physiology , COS Cells , Carrier Proteins/chemistry , Carrier Proteins/genetics , Fungal Proteins/genetics , Golgi Apparatus/ultrastructure , Humans , Molecular Sequence Data , Protein Structure, Tertiary , RNA, Small Interfering/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Subcellular Fractions/chemistry , Subcellular Fractions/metabolism
7.
Structure ; 12(2): 215-26, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14962382

ABSTRACT

LY333531, BIM-1, BIM-2, BIM-3, and BIM-8 are bisindolyl maleimide-based, nanomolar protein kinase C inhibitors. LY333531, a PKCbeta-specific inhibitor, is in clinical trials against diabetes and cardiac ventricular hypertrophy complications. Specificity analysis with a panel of 29 protein kinases reveals that these bisindolyl maleimide inhibitors also inhibit PDK1, a key kinase from the insulin signaling pathway, albeit in the lower microM range. To understand the molecular basis of inhibition, the PDK1 kinase domain was cocrystallized with these bisindolyl maleimide inhibitors. The inhibitor complexes represent the first structural description of this class of compounds, revealing their unusual nonplanar conformation within the ATP binding site and also explaining the higher inhibitory potential of LY33331 compared to the BIM compounds toward PDK1. A combination of site-directed mutagenesis and essential dynamics analysis gives further insight into PDK1 and also PKC inhibition by these compounds, and may aid inhibitor design.


Subject(s)
Indoles/pharmacology , Maleimides/pharmacology , Models, Molecular , Protein Serine-Threonine Kinases/metabolism , 3-Phosphoinositide-Dependent Protein Kinases , Binding Sites , Crystallography, X-Ray , Enzyme Inhibitors/pharmacology , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Structure, Tertiary , Signal Transduction
8.
Mol Cell Biol ; 23(21): 7794-808, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14560023

ABSTRACT

Ceramide is generated in response to numerous stress-inducing stimuli and has been implicated in the regulation of diverse cellular responses, including cell death, differentiation, and insulin sensitivity. Recent evidence indicates that ceramide may regulate these responses by inhibiting the stimulus-mediated activation of protein kinase B (PKB), a key determinant of cell fate and insulin action. Here we show that inhibition of this kinase involves atypical PKCzeta, which physically interacts with PKB in unstimulated cells. Insulin reduces the PKB-PKCzeta interaction and stimulates PKB. However, dissociation of the kinase complex and the attendant hormonal activation of PKB were prevented by ceramide. Under these circumstances, ceramide activated PKCzeta, leading to phosphorylation of the PKB-PH domain on Thr(34). This phosphorylation inhibited phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) binding to PKB, thereby preventing activation of the kinase by insulin. In contrast, a PKB-PH domain with a T34A mutation retained the ability to bind PIP(3) even in the presence of a ceramide-activated PKCzeta and, as such, expression of PKB T34A mutant in L6 cells was resistant to inhibition by ceramide treatment. Inhibitors of PKCzeta and a kinase-dead PKCzeta both antagonized the inhibitory effect of ceramide on PKB. Since PKB confers a prosurvival signal and regulates numerous pathways in response to insulin, suppressing its activation by a PKCzeta-dependent process may be one mechanism by which ceramide promotes cell death and induces insulin resistance.


Subject(s)
Ceramides/metabolism , Phosphatidylinositol Phosphates/metabolism , Protein Kinase C/metabolism , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/metabolism , Amino Acid Sequence , Animals , Cell Line , Enzyme Activation , Enzyme Inhibitors/metabolism , Insulin/metabolism , Macromolecular Substances , Models, Molecular , Molecular Sequence Data , Peptides/genetics , Peptides/metabolism , Phosphatidylinositol Phosphates/chemistry , Phosphorylation , Protein Binding , Protein Isoforms/metabolism , Protein Kinase C/genetics , Protein Structure, Tertiary , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-akt , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Signal Transduction
9.
Biochem J ; 375(Pt 2): 255-62, 2003 Oct 15.
Article in English | MEDLINE | ID: mdl-12892559

ABSTRACT

PDK1 (3-phosphoinositide-dependent protein kinase-1) is a member of the AGC (cAMP-dependent, cGMP-dependent, protein kinase C) family of protein kinases, and has a key role in insulin and growth-factor signalling through phosphorylation and subsequent activation of a number of other AGC kinase family members, such as protein kinase B. The staurosporine derivative UCN-01 (7-hydroxystaurosporine) has been reported to be a potent inhibitor for PDK1, and is currently undergoing clinical trials for the treatment of cancer. Here, we report the crystal structures of staurosporine and UCN-01 in complex with the kinase domain of PDK1. We show that, although staurosporine and UCN-01 interact with the PDK1 active site in an overall similar manner, the UCN-01 7-hydroxy group, which is not present in staurosporine, generates direct and water-mediated hydrogen bonds with active-site residues. Inhibition data from UCN-01 tested against a panel of 29 different kinases show a different pattern of inhibition compared with staurosporine. We discuss how these differences in inhibition could be attributed to specific interactions with the additional 7-hydroxy group, as well as the size of the 7-hydroxy-group-binding pocket. This information could lead to opportunities for structure-based optimization of PDK1 inhibitors.


Subject(s)
Protein Serine-Threonine Kinases/chemistry , Staurosporine/analogs & derivatives , Staurosporine/chemistry , 3-Phosphoinositide-Dependent Protein Kinases , Animals , Binding Sites , Catalytic Domain , Cell Line , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Models, Molecular , Molecular Conformation , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Protein Structure, Tertiary , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spodoptera , Staurosporine/pharmacology , Structure-Activity Relationship
11.
Biochem J ; 363(Pt 3): 657-66, 2002 May 01.
Article in English | MEDLINE | ID: mdl-11964166

ABSTRACT

Ptd(4,5)P(2) is thought to promote and organize a wide range of cellular functions, including vesicular membrane traffic and cytoskeletal dynamics, by recruiting functional protein complexes to restricted locations in cellular membranes. However, little is known about the distribution of PtdIns(4,5)P(2) in the cell at high resolution. We have used the pleckstrin homology (PH) domain of phospholipase delta(1) (PLCdelta(1)), narrowly specific for PtdIns(4,5)P(2), to map the distribution of the lipid in astrocytoma and A431 cells. We applied the glutathione S-transferase-tagged PLCdelta(1) PH domain (PLCdelta(1)PH-GST) in an on-section labelling approach which avoids transfection procedures. Here we demonstrate PtdIns(4,5)P(2) labelling in the plasma membrane, and also in intracellular membranes, including Golgi (mainly stack), endosomes and endoplasmic reticulum, as well as in electron-dense structures within the nucleus. At the plasma membrane, labelling was more concentrated over lamellipodia, but not in caveolae, which contained less than 10% of the total cell-surface labelling. A dramatic decrease in signal over labelled compartments was observed on preincubation with the cognate headgroup [Ins(1,4,5)P(3)], and plasma-membrane labelling was substantially decreased after stimulation with thrombin-receptor-activating peptide (SFLLRN in the one-letter amino acid code), a treatment which markedly diminishes PtdIns(4,5)P(2) levels. Thus we have developed a highly selective method for mapping the PtdIns(4,5)P(2) distribution within cells at high resolution, and our data provide direct evidence for this lipid at key functional locations.


Subject(s)
Intracellular Membranes/metabolism , Isoenzymes/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Type C Phospholipases/metabolism , Blood Proteins/metabolism , Cell Line , Cell Membrane/metabolism , Humans , Lipid Metabolism , Microscopy, Immunoelectron , Peptide Fragments/metabolism , Phosphatidylinositol 4,5-Diphosphate/chemistry , Phospholipase C delta , Phosphoproteins/metabolism , Sequence Homology, Amino Acid , Tumor Cells, Cultured
12.
Sci STKE ; 2002(129): pl6, 2002 Apr 23.
Article in English | MEDLINE | ID: mdl-11972359

ABSTRACT

The Protein Lipid Overlay (PLO) assay enables the identification of the lipid ligands with which lipid binding proteins interact. This assay also provides qualitative information on the relative affinity with which a protein binds to a lipid. In the PLO assay, serial dilutions of different lipids are spotted onto a nitrocellulose membrane to which they attach. These membranes are then incubated with a lipid binding protein possessing an epitope tag. The membranes are washed and the protein, still bound to the membrane by virtue of its interaction with lipid(s), is detected by immunoblotting with an antibody recognizing the epitope tag. This procedure requires only a few micrograms of protein and is quicker and cheaper to perform than other methods that have been developed to assess protein-lipid interactions. The reagents required for the PLO assay are readily available from commercial sources and the assay can be performed in any laboratory, even by those with no prior expertise in this area.


Subject(s)
Carrier Proteins/metabolism , Lipid Metabolism , Lipids/isolation & purification , Biological Transport/physiology , Humans , Kidney/cytology , Kidney/embryology , Ligands , Phospholipids/isolation & purification , Phospholipids/metabolism
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