Understanding the relative affinity and specificity of the pleckstrin homology domain of protein kinase B for inositol phosphates

2012 ◽  
Vol 14 (2) ◽  
pp. 929-936 ◽  
Author(s):  
Sarah A. J. Rosen ◽  
Piers R. J. Gaffney ◽  
Bernard Spiess ◽  
Ian R. Gould
Keyword(s):  
Protein Kinase ◽  
Inositol Phosphates ◽  
Protein Kinase B ◽  
Pleckstrin Homology Domain ◽  
Pleckstrin Homology ◽  
Relative Affinity

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 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.

scholarly journals Dual Phosphorylation of Btk by Akt/Protein Kinase B Provides Docking for 14-3-3ζ, Regulates Shuttling, and Attenuates both Tonic and Induced Signaling in B Cells

2013 ◽  
Vol 33 (16) ◽  
pp. 3214-3226 ◽  
Author(s):  
Dara K. Mohammad ◽  
Beston F. Nore ◽  
Alamdar Hussain ◽  
Manuela O. Gustafsson ◽  
Abdalla J. Mohamed ◽  
...  
Keyword(s):  
Protein Kinase ◽  
B Cell ◽  
Tyrosine Phosphorylation ◽  
Nuclear Translocation ◽  
Kinase Inhibitor ◽  
Protein Kinase B ◽  
Negative Regulator ◽  
Pleckstrin Homology Domain ◽  
Loss Of Function ◽  
Interaction Sites

Bruton's tyrosine kinase (Btk) is crucial for B-lymphocyte activation and development. Mutations in theBtkgene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Using tandem mass spectrometry, 14-3-3ζ was identified as a new binding partner and negative regulator of Btk in both B-cell lines and primary B lymphocytes. The activated serine/threonine kinase Akt/protein kinase B (PKB) phosphorylated Btk on two sites prior to 14-3-3ζ binding. The interaction sites were mapped to phosphoserine pS51 in the pleckstrin homology domain and phosphothreonine pT495 in the kinase domain. The double-alanine, S51A/T495A, replacement mutant failed to bind 14-3-3ζ, while phosphomimetic aspartate substitutions, S51D/T495D, caused enhanced interaction. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 abrogated S51/T495 phosphorylation and binding. A newly characterized 14-3-3 inhibitor, BV02, reduced binding, as did the Btk inhibitor PCI-32765 (ibrutinib). Interestingly, in the presence of BV02, phosphorylation of Btk, phospholipase Cγ2, and NF-κB increased strongly, suggesting that 14-3-3 also regulates B-cell receptor (BCR)-mediated tonic signaling. Furthermore, downregulation of 14-3-3ζ elevated nuclear translocation of Btk. The loss-of-function mutant S51A/T495A showed reduced tyrosine phosphorylation and ubiquitination. Conversely, the gain-of-function mutant S51D/T495D exhibited intense tyrosine phosphorylation, associated with Btk ubiquitination and degradation, likely contributing to the termination of BCR signaling. Collectively, this suggests that Btk could become an important new candidate for the general study of 14-3-3-mediated regulation.

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