Phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2 ) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) are lipid second messengers that regulate various cellular processes by recruiting a wide range of downstream effector proteins to membranes. Several pleckstrin homology (PH) domains have been reported to interact with PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 . To understand how these PH domains differentially respond to PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 signals, we quantitatively determined the PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 binding properties of several PH domains, including Akt, ARNO, Btk, DAPP1, Grp1, and C-terminal TAPP1 PH domains by surface plasmon resonance and monolayer penetration analyses. The measurements revealed that these PH domains have significant different phosphoinositide specificities and affinities. Btk-PH and TAPP1-PH showed genuine PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 specificities, respectively, whereas other PH domains exhibited less pronounced specificities. Also, the PH domains showed different degrees of membrane penetration, which greatly affected the kinetics of their membrane dissociation. Mutational studies showed that the presence of two proximal hydrophobic residues on the membrane-binding surface of the PH domain is important for membrane penetration and sustained membrane residence. When NIH 3T3 cells were stimulated with platelet-derived growth factor to generate PtdIns(3,4,5)P 3 , reversible translocation of Btk-PH, Grp1-PH, ARNO-PH, DAPP1-PH, and its L177A mutant to the plasma membrane was consistent with their in vitro membrane binding properties. Collectively, these studies provide new insight into how various PH domains would differentially respond to cellular PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 signals. Phosphoinositides (PIs)2 are mono-and polyphosphorylated derivatives of phosphatidylinositol (PtdIns) (1-3). AlthoughPIs are minor components of membrane lipids, they regulate a wide range of biological processes, including cell proliferation, cell survival, differentiation, signal transduction, cytoskeleton organization, and membrane trafficking (1-3). PIs regulate these cellular processes primarily by serving as site-specific membrane signals that mediate the membrane recruitment and regulation of effector proteins. The PI-mediated cellular processes allow exceptional spatiotemporal specificity because the spatial and temporal distribution of various PIs is dynamically and tightly regulated by a series of PI-modifying enzymes, such as phospholipases, lipid kinases, and lipid phosphatases, located in different cell membranes (1-3). For instance, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) are mainly found in the plasma membrane, whereas phosphatidylinositol 3-phosphate (PtdIns3P), phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 5-phosphate (PtdIns5P), and phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P 2 ) are primarily present in endosomes, Golgi, nucl...

Cu(II) complexes of three tridentate ligands, L(1), L(2) and L(3), [L(1), N-((1H-imidazole-2-yl)methyl)-2-(pyridine-2-yl)ethanamine; L(2), N-((1-methyl-1H-imidazole-2-yl)methyl)-2-(pyridine-2-yl)ethanamine; L(3), 2-(pyridine-2-yl)-N-((pyridine-2-yl)methyl)ethanamine] respectively, were synthesized and characterized. The single crystal X-ray structure of complex 1 reveals the pseudo octahedral coordination geometry around the copper center. Absorption and fluorescence experimental evidence show good DNA binding propensity (in the order of 10(5) M(-1)) of the complexes. Thermal denaturation and circular dichroism (CD) analyses reveal minor structural changes of calf thymus (CT) DNA in presence of complexes and groove and/or surface binding of the complexes to CT-DNA. Kinetic DNA cleavage assay shows pseudo-first-order kinetic reaction between the complex and supercoiled (SC) DNA. In addition, mechanistic SC DNA cleavage results show higher DNA cleavage activity in presence of reducing agent, due to the presence of hydroxyl radicals. In vitro cytotoxicity assay of the complexes demonstrate that the complexes have low toxicity for different cancer cell lines and IC(50) values were between 37 and 156 μM.

Many cytosolic proteins are recruited to the plasma membrane (PM) during cell signaling and other cellular processes. Recent reports have indicated that phosphatidylserine (PS), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) that are present in the PM play important roles for their specific PM recruitment. To systematically analyze how these lipids mediate PM targeting of cellular proteins, we performed biophysical, computational, and cell studies of the Ca 2؉ -dependent C2 domain of protein kinase C␣ (PKC␣) that is known to bind PS and phosphoinositides. In vitro membrane binding measurements by surface plasmon resonance analysis show that PKC␣-C2 nonspecifically binds phosphoinositides, including PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 , but that PS and Ca 2؉ binding is prerequisite for productive phosphoinositide binding. PtdIns(4,5)P 2 or PtdIns(3,4,5)P 3 augments the Ca 2؉ -and PSdependent membrane binding of PKC␣-C2 by slowing its membrane dissociation. Molecular dynamics simulations also support that Ca 2؉ -dependent PS binding is essential for membrane interactions of PKC␣-C2. PtdIns(4,5)P 2 alone cannot drive the membrane attachment of the domain but further stabilizes the Ca 2؉ -and PS-dependent membrane binding. When the fluorescence protein-tagged PKC␣-C2 was expressed in NIH-3T3 cells, mutations of phosphoinositide-binding residues or depletion of PtdIns(4,5)P 2 and/or PtdIns(3,4,5)P 3 from PM did not significantly affect the PM association of the domain but accelerated its dissociation from PM. Also, local synthesis of PtdIns(4,5)P 2 or PtdIns(3,4,5)P 3 at the PM slowed membrane dissociation of PKC␣-C2. Collectively, these studies show that PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 augment the Ca 2؉ -and PS-dependent membrane binding of PKC␣-C2 by elongating the membrane residence of the domain but cannot drive the PM recruitment of PKC␣-C2. These studies also suggest that effective PM recruitment of many cellular proteins may require synergistic actions of PS and phosphoinositides.One of the hallmarks of cell signaling proteins is their reversible recruitment to the plasma membrane (PM) 5 in response to receptor activation (1-4). Although some of these proteins are known to translocate to the PM through protein-protein interactions, a large number of proteins are recruited to the PM by directly interacting with lipids present in the PM (4). The inner leaflet of the PM of mammalian cells is known to be rich in anionic phospholipids, PS (about 20 mol %) (5) and inositol phospholipids, including PtdIns(4,5)P 2 (about 1 mol %) (6 -8). These anionic lipids have been shown to play key roles in PM recruitment of a wide variety of cytosolic proteins. A series of in vitro membrane binding and cellular translocation studies of various proteins, including protein kinase C (PKC) (9) and sphingosine kinase (10), as well as their isolated lipid binding domains (11), have indicated that PS-selective proteins are targeted to the PM through direct ...

pH regulates the transmembrane transport of Cl− ions by bis(iminourea) derivatives.

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