scholarly journals Binding of phosphatidylinositol 3,4,5-trisphosphate to the pleckstrin homology domain of protein kinase B induces a conformational change

2003 ◽  
Vol 375 (3) ◽  
pp. 531-538 ◽  
Author(s):  
Christine C. MILBURN ◽  
Maria DEAK ◽  
Sharon M. KELLY ◽  
Nick C. PRICE ◽  
Dario R. ALESSI ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Binding Site ◽  
Conformational Change ◽  
Conformational Changes ◽  
Protein Kinase B ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Variable Loop ◽  
Solution Studies

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 PKBα in a noncomplexed form and compare this to a new atomic resolution (0.98 Å, where 1 Å=0.1 nm) structure of the PH domain of PKBα 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 α-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.

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.

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 Involvement of the Pleckstrin Homology Domain in the Insulin-stimulated Activation of Protein Kinase B

1998 ◽  
Vol 273 (45) ◽  
pp. 29600-29606 ◽  
Author(s):  
Carol L. Sable ◽  
Nathalie Filippa ◽  
Chantal Filloux ◽  
Brian A. Hemmings ◽  
Emmanuel Van Obberghen
Keyword(s):  
Protein Kinase ◽  
Protein Kinase B ◽  
Pleckstrin Homology Domain ◽  
Pleckstrin Homology

scholarly journals In vitro and In vivo Activity of Novel Small-Molecule Inhibitors Targeting the Pleckstrin Homology Domain of Protein Kinase B/AKT

2009 ◽  
Vol 69 (12) ◽  
pp. 5073-5081 ◽  
Author(s):  
Sylvestor A. Moses ◽  
M. Ahad Ali ◽  
Song Zuohe ◽  
Lei Du-Cuny ◽  
Li Li Zhou ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Protein Kinase B ◽  
Pleckstrin Homology Domain ◽  
Pleckstrin Homology

scholarly journals Myo1c Binds Phosphoinositides through a Putative Pleckstrin Homology Domain

2006 ◽  
Vol 17 (11) ◽  
pp. 4856-4865 ◽  
Author(s):  
David E. Hokanson ◽  
Joseph M. Laakso ◽  
Tianming Lin ◽  
David Sept ◽  
E. Michael Ostap
Keyword(s):  
Binding Site ◽  
Membrane Trafficking ◽  
Cellular Localization ◽  
Membrane Binding ◽  
Pleckstrin Homology Domain ◽  
Dependent Manner ◽  
Ph Domain ◽  
Pleckstrin Homology

Myo1c is a member of the myosin superfamily that binds phosphatidylinositol-4,5-bisphosphate (PIP2), links the actin cytoskeleton to cellular membranes and plays roles in mechano-signal transduction and membrane trafficking. We located and characterized two distinct membrane binding sites within the regulatory and tail domains of this myosin. By sequence, secondary structure, and ab initio computational analyses, we identified a phosphoinositide binding site in the tail to be a putative pleckstrin homology (PH) domain. Point mutations of residues known to be essential for polyphosphoinositide binding in previously characterized PH domains inhibit myo1c binding to PIP2 in vitro, disrupt in vivo membrane binding, and disrupt cellular localization. The extended sequence of this binding site is conserved within other myosin-I isoforms, suggesting they contain this putative PH domain. We also characterized a previously identified membrane binding site within the IQ motifs in the regulatory domain. This region is not phosphoinositide specific, but it binds anionic phospholipids in a calcium-dependent manner. However, this site is not essential for in vivo membrane binding.

scholarly journals Ceramide Disables 3-Phosphoinositide Binding to the Pleckstrin Homology Domain of Protein Kinase B (PKB)/Akt by a PKCζ-Dependent Mechanism

2003 ◽  
Vol 23 (21) ◽  
pp. 7794-7808 ◽  
Author(s):  
Darren J. Powell ◽  
Eric Hajduch ◽  
Gursant Kular ◽  
Harinder S. Hundal
Keyword(s):  
Cell Death ◽  
Protein Kinase ◽  
Cell Fate ◽  
Protein Kinase B ◽  
Cellular Responses ◽  
Inhibitory Effect ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Hormonal Activation ◽  
Dependent Mechanism

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 PKCζ, which physically interacts with PKB in unstimulated cells. Insulin reduces the PKB-PKCζ 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 PKCζ, leading to phosphorylation of the PKB-PH domain on Thr34. This phosphorylation inhibited phosphatidylinositol 3,4,5-trisphosphate (PIP3) 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 PIP3 even in the presence of a ceramide-activated PKCζ and, as such, expression of PKB T34A mutant in L6 cells was resistant to inhibition by ceramide treatment. Inhibitors of PKCζ and a kinase-dead PKCζ 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 PKCζ-dependent process may be one mechanism by which ceramide promotes cell death and induces insulin resistance.

Sign in / Sign up

Export Citation Format

Share Document