Detection of Inositol Phosphates by Split PH Domains

Abstract Pleckstrin-2, a widely expressed paralog of pleckstrin, is composed of two Pleckstrin Homology (PH) domains and Disheveled-Egl 10-Pleckstrin (DEP) domain. Although the activity of pleckstrin is regulated by its’ phosphorylation state, pleckstrin-2 is not a phospho-protein suggesting that it possesses a different mechanism of regulation. Previous reports have shown that many PH domains mediate protein binding to inositol phosphates and phospholipids, thus regulating protein function. Therefore, we speculated that localized production of specific polyphosphatidylinositols might bind, and activate, pleckstrin-2. Using a lipid-binding assay, we found that pleckstrin-2 bound with greatest affinity to the products of phosphatidylinositol 3-kinase and phosphatidylinositol 5-kinase. The individual PH domains of pleckstrin-2 bound the same products but with lower affinity, implying that both PH domains cooperate for maximal lipid affinity of the full-length protein. GFP-tagged pleckstrin-2 had a cytoplasmic distribution in non-adherent Jurkat cells, but through a pathway dependent on the phospholipid-binding pocket of its PH domains, rapidly moved to the cell membrane following adhesion to immobilized fibronectin. Once bound to the cell membrane, pleckstrin-2 enhanced Jurkat cell spreading 2-fold and increased membrane ruffling. The membrane association of pleckstrin-2, and its resultant cell spreading, were dependent on D3-phosphoinositides since these effects were disrupted by pharmacologic inhibition of PI3K with either wortmannin or LY294002. To investigate the role of this protein within platelets, we generated mice containing a null mutation within the pleckstrin-2 gene. Pleckstrin-2 null mice were born at the expected frequency, and had no overt spontaneous hemorrhagic events. Mice lacking pleckstrin-2 had normal platelet counts and morphologic appearance of their megakaryocytes. Following stimulation of the PAR-4 (thrombin) receptor, pleckstrin-2 knockout platelets displayed normal assembly of filamentous actin. However, pleckstrin-2 null platelets had impaired aggregation following stimulation by collagen or submaximal doses of the PAR-4 activating peptide. Since pleckstrin-2 deficient platelets aggregated normally in response to ADP, these results suggested that these platelets might have an impaired ability to secrete dense granules. Accordingly, we found that pleckstrin-2 null platelets had a defect in their ability to secrete ATP in response to stimulation by 5μM collagen or 200μM of the PAR-4 activating peptide. However, pleckstrin-2 knockout platelets did incorporate 14C-serotonin as efficiently as wild type platelets. This latter observation suggested that the secretion defect in pleckstrin-2 null platelets was not attributable to a deficiency of dense granules, but instead is due to a defect in exocytosis of granules. Together, these data suggest that the PH domains of pleckstrin-2 cooperatively bind PI3K generated phospholipids on the cell membrane, and help mediate platelet secretion.

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Computational Analysis of the Binding Specificities of PH Domains

BioMed Research International ◽

10.1155/2015/792904 ◽

2015 ◽

Vol 2015 ◽

pp. 1-11 ◽

Cited By ~ 9

Author(s):

Zhi Jiang ◽

Zhongjie Liang ◽

Bairong Shen ◽

Guang Hu

Keyword(s):

Inositol Phosphates ◽

Structural Aspect ◽

Genomic Research ◽

Ph Domain ◽

Domain Structures ◽

Ph Domains ◽

Cellular Signal ◽

Structure Relationship ◽

Insight Into ◽

Structure Properties

Pleckstrin homology (PH) domains share low sequence identities but extremely conserved structures. They have been found in many proteins for cellular signal-dependent membrane targeting by binding inositol phosphates to perform different physiological functions. In order to understand the sequence-structure relationship and binding specificities of PH domains, quantum mechanical (QM) calculations and sequence-based combined with structure-based binding analysis were employed in our research. In the structural aspect, the binding specificities were shown to correlate with the hydropathy characteristics of PH domains and electrostatic properties of the bound inositol phosphates. By comparing these structure properties with sequence-based profiles of physicochemical properties, PH domains can be classified into four functional subgroups according to their binding specificities and affinities to inositol phosphates. The method not only provides a simple and practical paradigm to predict binding specificities for functional genomic research but also gives new insight into the understanding of the basis of diseases with respect to PH domain structures.

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PI3K Regulates Pleckstrin-2 in T-Cell Cytoskeletal Re-Organization.

Blood ◽

10.1182/blood.v106.11.3305.3305 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3305-3305

Author(s):

Tami L. Bach ◽

Wesley T. Kerr ◽

Charles S. Abrams

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Membrane Trafficking ◽

Inositol Phosphates ◽

Point Mutations ◽

Cell Spreading ◽

Lipid Binding ◽

Membrane Association ◽

Ph Domains

Abstract Pleckstrin-2, a paralog of pleckstrin-1, is composed of two Pleckstrin Homology (PH) domains and a Disheveled-Egl 10-Pleckstrin (DEP) domain. Several studies have shown that PH domains mediate binding of their host proteins to inositol phosphates and phospholipids, and thus regulate protein function. PH domains are found in many molecules involved in cellular signaling, cytoskeletal organization, membrane trafficking, and phospholipid modification. Proteins containing the DEP domain also regulate a broad range of cellular functions and evidence is emerging that several signaling proteins may rely on their DEP domains for membrane association. We speculated that the function of pleckstrin-2 is dependent upon its ability to bind to specific polyphosphatidylinositols. A lipid-binding assay revealed that pleckstrin-2 binds with greatest affinity to the products of phosphatidylinositol 3-kinase (PI3K) and phosphatidylinositol 5-kinase. The individual PH domains of pleckstrin-2 bind to the same products but with lower affinity, implying that both PH domains cooperate for maximal lipid affinity of the full-length protein. To examine the effect of pleckstrin-2 in human T-cells, Jurkat T-cells were transfected with GFP-tagged plasmids that direct the expression of pleckstrin-2 variants. Using confocal video microscopy we demonstrated that upon activation of the T-cell antigen receptor or the integrin α4β1, pleckstrin-2 rapidly moves from the cytoplasm to the cellular membrane and enhances membrane ruffling. Quantitation of cell footprint size revealed a two-fold increase in cell spreading. Furthermore, the membrane association of pleckstrin-2 and its resultant cell spreading were dependent on D3-phosphoinositides since these effects were disrupted by pharmacologic inhibition of PI3K with either wortmannin or LY294002. Consistent with this observation, a pleckstrin-2 variant containing point mutations in both of its PH domains failed to associate with the cell membrane and had no effect on spreading under the same conditions, suggesting that pleckstrin-2 membrane association occurs through a pathway dependent on the phospholipid-binding pocket of its PH domains. Although still membrane-bound, a pleckstin-2 variant containing point mutations in the second β-turn and second α-helical coil of the DEP domain demonstrated a decreased ability of pleckstrin-2 to induce membrane ruffles and lamellipodia, without decreasing filopodia formation. The cell footprint size of the DEP domain mutants was also decreased compared to that of wild type pleckstrin-2. These results suggest that the pleckstrin-2 DEP domain may function to promote actin-rich membrane extensions and ruffling. The localization of receptors, signaling intermediates, and cytoskeletal components at the T-cell/APC interface is thought to be a major determinant of efficient T-cell activation. Our data indicate that in T-lymphocytes, pleckstrin-2 uses modular motifs to bind to membrane-associated phosphatidylinositols, such as those generated by PI3K, to organize the actin cytoskeleton and to promote lymphocyte spreading.

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Phosphotyrosine protein of molecular mass 30 kDa binds specifically to the positively charged region of the pleckstrin N-terminal pleckstrin homology domain

Biochemical Journal ◽

10.1042/bj3420423 ◽

1999 ◽

Vol 342 (2) ◽

pp. 423-430

Author(s):

Limin LIU ◽

Mary MAKOWSKE

Keyword(s):

Binding Site ◽

Inositol Phosphates ◽

Site Directed Mutagenesis ◽

Pleckstrin Homology Domain ◽

Ph Domain ◽

Pleckstrin Homology ◽

Glutathione S Transferase ◽

Protein Protein Interaction ◽

Ph Domains ◽

Positively Charged

It has been proposed that phosphoinositides and inositol phosphates serve as general ligands for members of the structurally related pleckstrin homology (PH) domain family. The N-terminal PH domain of pleckstrin (N-PH), in contrast with other PH domains, does not bind to any of these ligands with the high affinity expected for a physiological interaction. To examine whether N-PH might instead mediate protein-protein interaction, a fusion protein with glutathione S-transferase (GST) expressing N-PH (GST-N-PH) was used to screen [35S]methionine metabolically labelled HL-60 and Bac1.2F5 cell lysates for potential binding partners. A 30 kDa binding protein was identified in both cell lines. Binding to N-PH demonstrated specificity, because binding was approx. 10-fold higher than when an equimolar amount of pleckstrin C-terminal PH domain (GST-C-PH) was used as probe. The 30 kDa protein could also be metabolically labelled with [32P]Pi and proved to be a tyrosine-phosphorylated protein. Binding to N-PH could be specifically inhibited with phosphotyrosine but not with phosphothreonine; the inhibition was concentration-dependent. Site-directed mutagenesis indicated that a positively charged region previously identified as the phosphoinositide-binding site in N-PH and other PH domains, rather than a putative phosphotyrosine-binding region previously identified in structurally similar phosphotyrosine-binding (PTB) domains, served as the binding site. These results suggest that the positively charged region of N-PH has the potential to interact with a protein ligand that contains phosphotyrosine.

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The regulation of membrane to cytosol partitioning of signalling proteins by phosphoinositides and their soluble headgroups

Biochemical Society Transactions ◽

10.1042/bst0331303 ◽

2005 ◽

Vol 33 (6) ◽

pp. 1303-1307 ◽

Cited By ~ 10

Author(s):

C.P. Downes ◽

A. Gray ◽

A. Fairservice ◽

S.T. Safrany ◽

I.H. Batty ◽

...

Keyword(s):

Ligand Binding ◽

Inositol Phosphates ◽

Nucleotide Exchange ◽

Ph Domain ◽

Inositol Polyphosphates ◽

Signalling Molecules ◽

Nucleotide Exchange Factor ◽

Inositol Phospholipids ◽

Ph Domains ◽

Pi Ligands

Inositol phospholipids [PIs (phosphoinositides)] represent a group of membrane-tethered signalling molecules which differ with respect to the number and distribution of monoester phosphate groups around the inositol ring. They function by binding to proteins which possess one of several domains that bind a particular PI species, often with high affinity and specificity. PH (pleckstrin homology) domains for example possess ligand-binding pockets that are often lined with positively charged residues and which bind PIs with varying degrees of specificity. Several PH domains bind not only PIs, but also their cognate headgroups, many of which occur naturally in cells as relatively abundant cytosolic inositol phosphates. The subcellular distributions of proteins possessing such PH domains are therefore determined by the relative levels of competing membrane-bound and soluble ligands. A classic example of the latter is the PH domain of phospholipase Cδ1, which binds both phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate. We have shown that the N-terminal PH domain of the Rho family guanine nucleotide-exchange factor, Tiam 1, binds PI ligands promiscuously allowing multiple modes of regulation. We also recently analysed the ligand-binding specificity of the PH domain of PI-dependent kinase 1 and found that it could bind abundant inositol polyphosphates such as inositol hexakisphosphate. This could explain the dual distribution of this key signalling component, which needs to access substrates at both the plasma membrane and in the cytosol.

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Eicosapentaenoic Acid Interferes with U46619-Stimulated Formation of Inositol Phosphates in Washed Rabbit Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647129 ◽

1989 ◽

Vol 62 (04) ◽

pp. 1116-1120 ◽

Cited By ~ 9

Author(s):

N Chetty ◽

J D Vickers ◽

R L Kinlough-Rathbone ◽

M A Packham ◽

J F Mustard

Keyword(s):

Signal Transduction ◽

Fatty Acid Composition ◽

Eicosapentaenoic Acid ◽

Inositol Phosphates ◽

Inositol Phosphate ◽

Dense Granule ◽

Detectable Change ◽

Membrane Phospholipids ◽

Rabbit Platelets ◽

Stimulation Of

SummaryEicosapentaenoic acid (EPA) inhibits platelet responsiveness to aggregating agents. To investigate the reactions that are affected by EPA, we examined the effect of preincubating aspirintreated rabbit platelets with EPA on stimulation of inositol phosphate formation in response to the TXA2 analogue U46619. Stimulation of platelets with U46619 (0.5 μM) caused aggregation and slight release of dense granule contents; aggregation and release were inhibited by preincubation of the platelets with EPA (50 μM) for 1 h followed by washing to remove unincorporated EPA. Incubation with EPA (50 μM) for 1 h did not cause a detectable increase in the amount of EPA in the platelet phospholipids. When platelets were prelabelled with [3H]inositol stimulation with U46619 of control platelets that had not been incubated with EPA significantly increased the labelling of mos1tol phosphates. The increases in inositol phosphate labelling due to U46619 at 10 and 60 s were partially inhibited by premcubat10n of the platelets with 50 μM EPA. Since the activity of cyclo-oxygenase was blocked with aspirin, inhibition of inositol phosphate labelling in response to U46619 indicates either that there may be inhibition of signal transduction without a detectable change in the amount of EPA in platelet phospholipids, that changes in signal transduction require only minute changes in the fatty acid composition of membrane phospholipids, or that after a 1 h incubation with EPA, activation of phospholipase C is affected by a mechanism that is not directly related to incorporation of EPA.

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