The in vivo role of PtdIns(3,4,5)P3 binding to PDK1 PH domain defined by knockin mutation

We generated homozygous knockin ES cells expressing a form of 3-phosphoinositide-dependent protein kinase-1 (PDK1) with a mutation in its pleckstrin homology (PH) domain that abolishes phosphatidylinositol 3,4,5-tris-phosphate (PtdIns(3,4,5)P3) binding, without affecting catalytic activity. In the knockin cells, protein kinase B (PKB) was not activated by IGF1, whereas ribosomal S6 kinase (RSK) was activated normally, indicating that PtdIns(3,4,5)P3 binding to PDK1 is required for PKB but not RSK activation. Interestingly, amino acids and Rheb, but not IGF1, activated S6K in the knockin cells, supporting the idea that PtdIns(3,4,5)P3 stimulates S6K through PKB-mediated activation of Rheb. Employing PDK1 knockin cells in which either the PtdIns(3,4,5)P3 binding or substrate-docking 'PIF pocket' was disrupted, we established the roles that these domains play in regulating phosphorylation and stabilisation of protein kinase C isoforms. Moreover, mouse PDK1 knockin embryos in which either the PH domain or PIF pocket was disrupted died displaying differing phenotypes between E10.5 and E11.5. Although PDK1 plays roles in regulating cell size, cells derived from PH domain or PIF pocket knockin embryos were of normal size. These experiments establish the roles of the PDK1 regulatory domains and illustrate the power of knockin technology to probe the physiological function of protein-lipid and protein-protein interactions.     

High-resolution structure of the pleckstrin homology domain of protein kinase b/akt bound to phosphatidylinositol (3,4,5)-trisphosphate

The products of PI 3-kinase activation, PtdIns(3,4,5)P3 and its immediate breakdown product PtdIns(3,4)P2, trigger physiological processes, by interacting with proteins possessing pleckstrin homology (PH) domains. One of the best characterized PtdIns(3,4,5)P3/PtdIns(3,4)P2 effector proteins is protein kinase B (PKB), also known as Akt. PKB possesses a PH domain located at its N terminus, and this domain binds specifically to PtdIns(3,4,5)P3 and PtdIns(3,4)P2 with similar affinity. Following activation of PI 3-kinase, PKB is recruited to the plasma membrane by virtue of its interaction with PtdIns(3,4,5)P3/PtdIns(3,4)P2. PKB is then activated by the 3-phosphoinositide-dependent pro-tein kinase-1 (PDK1), which like PKB, possesses a PtdIns(3,4,5)P3/PtdIns(3,4)P2 binding PH domain. Here, we describe the high-resolution crystal structure of the isolated PH domain of PKB(alpha) in complex with the head group of PtdIns(3,4,5)P3. The head group has a significantly different orientation and location compared to other Ins(1,3,4,5)P4 binding PH domains. Mutagenesis of the basic residues that form ionic interactions with the D3 and D4 phosphate groups reduces or abolishes the ability of PKB to interact with PtdIns(3,4,5)P3 and PtdIns(3,4)P2. The D5 phosphate faces the solvent and forms no significant interactions with any residue on the PH domain, and this explains why PKB interacts with similar affinity with both PtdIns(3,4,5)P3 and PtdIns(3,4)P2.     

Targeting phospshatidylinositol 3-kinase signaling with novel phosphatidylinositol 3,4,5-triphosphate antagonists

The critical role of phopshatidylinositol-3-kinase (PtdIns3K) signaling in the regulation of a wide range of cellular functions, including cell survival and proliferation, autophagy, metabolism and cell migration, is well recognized. Activation of PtdIns3K leads to the generation of phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P₃). PtdIns(3,4,5)P₃ activates a complex signaling network controlling these diverse cellular functions through binding to Pleckstrin Homology (PH) domains of the effector proteins. We have recently described a new structural class of nonphosphoinositide small molecule inhibitors targeting binding of PtdIns(3,4,5) P₃ to PH domain targets. Using an in vitro PtdIns(3,4,5)P₃-PH domain binding assay, we identified two distinct PtdIns(3,4,5)P₃ antagonists, PIT-1 and PIT-2. Further cellular analysis revealed that both PITs inhibit PtdIns(3,4,5) P₃-dependent signaling mediated by Akt kinase, leading to the induction of apoptosis, metabolic stress and autophagy. An improved PIT-1 analog, DM-PIT-1, displays significant anticancer activity in the mouse syngeneic 4T1 breast cancer model in vivo. Discovery of PITs as well as other PtdIns(3,4,5)P₃ antagonists recently described by other laboratories suggest the possibility of targeting a key universal PtdIns(3,4,5)P₃/PH domain binding step in the PtdIns3K pathway using heterologous small molecule modulators.     

Targeting phospshatidylinositol 3-kinase signaling with novel phosphatidylinositol 3,4,5-triphosphate antagonists

The critical role of phopshatidylinositol-3-kinase (PtdIns3K) signaling in the regulation of a wide range of cellular functions, including cell survival and proliferation, autophagy, metabolism and cell migration, is well recognized. Activation of PtdIns3K leads to the generation of phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P 3). PtdIns(3,4,5)P 3 activates a complex signaling network controlling these diverse cellular functions through binding to Pleckstrin Homology (PH) domains of the effector proteins. We have recently described a new structural class of nonphosphoinositide small molecule inhibitors targeting binding of PtdIns(3,4,5) P 3 to PH domain targets. Using an in vitro PtdIns(3,4,5)P 3-PH domain binding assay, we identified two distinct PtdIns(3,4,5)P 3 antagonists, PIT-1 and PIT-2. Further cellular analysis revealed that both PITs inhibit PtdIns(3,4,5) P 3-dependent signaling mediated by Akt kinase, leading to the induction of apoptosis, metabolic stress and autophagy. An improved PIT-1 analog, DM-PIT-1, displays significant anticancer activity in the mouse syngeneic 4T1 breast cancer model in vivo. Discovery of PITs as well as other PtdIns(3,4,5)P 3 antagonists recently described by other laboratories suggest the possibility of targeting a key universal PtdIns(3,4,5)P 3/PH domain binding step in the PtdIns3K pathway using heterologous small molecule modulators.     

Elucidation of different inhibition mechanism of small chemicals on PtdInsP-binding domains using in silico docking experiments

Phosphatidylinositides, most negatively charged lipids in cellular membranes, regulate diverse effector proteins through the interaction with their lipid binding domains. We have previously reported inhibitory effect of small chemicals on the interaction between PtdIns(3,4,5)P₃ and Btk PH domain. Here, we report that the inhibitory effects of same sets of chemicals on Grp1 PH domain and epsin1 ENTH domain to elucidate diversity of inhibitory mechanisms upon different lipid binding domains. Among the chemicals, chemical 8 showed best inhibition in vitro assay for Grp1 PH domain and epsin1 ENTH domain, and then the interaction between small chemicals and lipid binding domains was further investigated by in silico docking experiments. As a result, it was concluded that the diverse inhibitory effects on different lipid binding domains were dependent on not only the number of interactions between small chemical and domain, but also additional interaction with positively charged surfaces as the secondary binding sites. This finding will help to develop lipid binding inhibitors as antagonists for lipid–protein interactions, and these inhibitors would be novel therapeutic drug candidates via regulating effector proteins involved in severe human diseases.     

Structural insights into the regulation of PDK1 by phosphoinositides and inositol phosphates

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.     

ARAP3 binding to phosphatidylinositol-(3,4,5)-trisphosphate depends on N-terminal tandem PH domains and adjacent sequences

Pleckstrin homology (PH) domains are modules characterised by a conserved three-dimensional protein fold. Several PH domains bind phosphoinositides with high affinity and specificity whilst most others do not. ARAP3 is a dual GTPase activating protein for Arf6 and RhoA which was identified in a screen for phosphatidylinositol-(3,4,5)-trisphophate (PtdIns(3,4,5)P₃) binding proteins. It is a regulator of cell shape and adhesion, and is itself regulated by PtdIns(3,4,5)P_3, which acts to recruit ARAP3 to the plasma membrane and to catalytically activate it. We show here that ARAP3 binds to PtdIns(3,4,5)P₃ in an unusual, PH domain-dependent manner. None of the five PH domains are sufficient to bind PtdIns(3,4,5)P₃ in isolation. Instead, the minimal PtdIns(3,4,5)P₃ binding fragment comprises ARAP3's N-terminal tandem PH domains, and an N-terminal linker region. For substantial binding, the N-terminal sterile alpha motif (SAM) domain is also required. Site-directed mutagenesis of either of the two N-terminal PH domains within the fragment greatly reduces binding to PtdIns(3,4,5)P₃, however, in the context of the full-length protein, point mutations in the second PH domain have a lesser effect on binding, whilst deletion of any one of the five PH domains abolishes PtdIns(3,4,5)P₃ binding. We propose a mechanism by which basic residues from the N-terminal tandem PH domains, and from elsewhere in the protein synergise to mediate strong, specific PtdIns(3,4,5)P₃ binding.     

Distinct polyphosphoinositide binding selectivities for pleckstrin homology domains of GRP1-like proteins based on diglycine versus triglycine motifs

GRP1 and the related proteins ARNO and cytohesin-1 are ARF exchange factors that contain a pleckstrin homology (PH) domain thought to target these proteins to cell membranes through binding polyphosphoinositides. Here we show the PH domains of all three proteins exhibit relatively high affinity for dioctanoyl phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P(3)), with K(D) values of 0.05, 1.6 and 1.0 micrometer for GRP1, ARNO, and cytohesin-1, respectively. However, the GRP1 PH domain was unique among these proteins in its striking selectivity for PtdIns(3,4, 5)P(3) versus phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P(2)), for which it exhibits about 650-fold lower apparent affinity. Addition of a glycine to the Gly(274)-Gly(275) motif in GRP1 greatly increased its binding affinity for PtdIns(4,5)P(2) with little effect on its binding to PtdIns(3,4,5)P(3), while deletion of a single glycine in the corresponding triglycine motif of the ARNO PH domain markedly reduced its binding affinity for PtdIns(4,5)P(2) but not for PtdIns(3,4,5)P(3). In intact cells, the hemagglutinin epitope-tagged PH domain of GRP1 was recruited to ruffles in the cell surface in response to insulin, as were full-length GRP1 and cytohesin-1, but the PH domain of cytohesin-1 was not. These data indicate that the unique diglycine motif in the GRP1 PH domain, as opposed to the triglycine in ARNO and cytohesin-1, directs its remarkable PtdIns(3,4,5)P(3) binding selectivity.     

Dissecting the role of the 3-phosphoinositide-dependent protein kinase-1 (PDK1) signalling pathways

The 3-phosphoinositide-dependent protein kinase-1 (PDK1) mediates the cellular effect of insulin and growth factors by activating a group of kinases including PKB/Akt, S6K, RSK, SGK and PKC isoforms. PDK1 possesses two regulatory domains namely a Pleckstrin Homology (PH) domain that binds to the phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] second messenger, and a substrate binding site termed the PIF-pocket. Employing a combination of biochemical, structural and mouse knock-in approaches we have been able to define the roles that the regulatory domains on PDK1 play. We have established that binding of PDK1 to PtdIns(3,4,5)P3 is essential for efficient activation of PKB isoforms as well as for maintaining normal cell size and insulin sensitivity. In contrast, the PIF-substrate binding pocket of PDK1 is not required for PKB activation, but is necessary for PDK1 to activate all of its other substrates.     

Interaction of PDK1 with Phosphoinositides Is Essential for Neuronal Differentiation but Dispensable for Neuronal Survival

3-Phosphoinositide-dependent protein kinase 1 (PDK1) operates in cells in response to phosphoinositide 3-kinase activation and phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P₃] production by activating a number of AGC kinases, including protein kinase B (PKB)/Akt. Both PDK1 and PKB contain pleckstrin homology (PH) domains that interact with the PtdIns(3,4,5)P₃ second messenger. Disrupting the interaction of the PDK1 PH domain with phosphoinositides by expressing the PDK1 K465E knock-in mutation resulted in mice with reduced PKB activation. We explored the physiological consequences of this biochemical lesion in the central nervous system. The PDK1 knock-in mice displayed a reduced brain size due to a reduction in neuronal cell size rather than cell number. Reduced BDNF-induced phosphorylation of PKB at Thr308, the PDK1 site, was observed in the mutant neurons, which was not rate limiting for the phosphorylation of those PKB substrates governing neuronal survival and apoptosis, such as FOXO1 or glycogen synthase kinase 3 (GSK3). Accordingly, the integrity of the PDK1 PH domain was not essential to support the survival of different embryonic neuronal populations analyzed. In contrast, PKB-mediated phosphorylation of PRAS40 and TSC2, allowing optimal mTORC1 activation and brain-specific kinase (BRSK) protein synthesis, was markedly reduced in the mutant mice, leading to impaired neuronal growth and differentiation.     

Regulation of P-Rex1 by phosphatidylinositol (3,4,5)-trisphosphate and Gbetagamma subunits

P-Rex1 is a guanine-nucleotide exchange factor (GEF) for the small GTPase Rac. We have investigated here the mechanisms of stimulation of P-Rex1 Rac-GEF activity by the lipid second messenger phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) and the Gbetagamma subunits of heterotrimeric G proteins. We show that a P-Rex1 mutant lacking the PH domain (DeltaPH) cannot be stimulated by PtdIns(3,4,5)P3, which implies that the PH domain confers PtdIns(3,4,5)P3 regulation of P-Rex1 Rac-GEF activity. Consistent with this, we found that PtdIns(3,4,5)P3 binds to the PH domain of P-Rex1 and that the DH/PH domain tandem is sufficient for PtdIns(3,4,5)P3-stimulated P-Rex1 activity. The Rac-GEF activities of the DeltaPH mutant and the DH/PH domain tandem can both be stimulated by Gbetagamma subunits, which infers that Gbetagamma subunits regulate P-Rex1 activity by binding to the catalytic DH domain. Deletion of the DEP, PDZ, or inositol polyphosphate 4-phosphatase homology domains has no major consequences on the abilities of either PtdIns(3,4,5)P3 or Gbetagamma subunits to stimulate P-Rex1 Rac-GEF activity. However, the presence of any of these domains impacts on the levels of basal and/or stimulated P-Rex1 Rac-GEF activity, suggesting that there are important functional interactions between the DH/PH domain tandem and the DEP, PDZ, and inositol polyphosphate 4-phosphatase homology domains of P-Rex1.     

Molecular mechanism of membrane targeting by the GRP1 PH domain

The general receptor for phosphoinositides isoform 1 (GRP1) is recruited to the plasma membrane in response to activation of phosphoinositide 3-kinases and accumulation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]. GRP1's pleckstrin homology (PH) domain recognizes PtdIns(3,4,5)P(3) with high specificity and affinity, however, the precise mechanism of its association with membranes remains unclear. Here, we detail the molecular basis of membrane anchoring by the GRP1 PH domain. Our data reveal a multivalent membrane docking involving PtdIns(3,4,5)P(3) binding, regulated by pH and facilitated by electrostatic interactions with other anionic lipids. The specific recognition of PtdIns(3,4,5)P(3) triggers insertion of the GRP1 PH domain into membranes. An acidic environment enhances PtdIns(3,4,5)P(3) binding and increases membrane penetration as demonstrated by NMR and monolayer surface tension and surface plasmon resonance experiments. The GRP1 PH domain displays a 28 nM affinity for POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/PtdIns(3,4,5)P(3) vesicles at pH 6.0, but binds 22-fold weaker at pH 8.0. The pH sensitivity is attributed in part to the His355 residue, protonation of which is required for the robust interaction with PtdIns(3,4,5)P(3) and significant membrane penetration, as illustrated by mutagenesis data. The binding affinity of the GRP1 PH domain for PtdIns(3,4,5)P(3)-containing vesicles is further amplified (by approximately 6-fold) by nonspecific electrostatic interactions with phosphatidylserine/phosphatidylinositol. Together, our results provide new insight into the multivalent mechanism of the membrane targeting and regulation of the GRP1 PH domain.     

Mechanistic basis of differential cellular responses of phosphatidylinositol 3,4-bisphosphate- and phosphatidylinositol 3,4,5-trisphosphate-binding pleckstrin homology domains

Phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) 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)P2 and PtdIns(3,4,5)P3. To understand how these PH domains differentially respond to PtdIns(3,4)P2 and PtdIns(3,4,5)P3 signals, we quantitatively determined the PtdIns(3,4)P2 and PtdIns(3,4,5)P3 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)P3 and PtdIns(3,4)P2 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)P3, 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)P2 and PtdIns(3,4,5)P3 signals.     

Engineering the phosphoinositide-binding profile of a class I pleckstrin homology domain

Pleckstrin homology (PH) domains are protein modules that bind with varying degrees of affinity and specificity membrane phosphoinositides. Previously we have shown that although the PH domains of the Ras GTPase-activating proteins GAP1m and GAP1IP4BP are 63% identical at the amino acid level they possess distinct phosphoinositide-binding profiles. The GAP1m PH domain binds phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), whereas the domain from GAP1IP4BP binds PtdIns(3,4,5)P3 and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) equally well. These phosphoinositide specificities are translated into distinct subcellular localizations. GAP1m is cytosolic and undergoes a rapid PtdIns(3,4,5)P3-dependent association with the plasma membrane following growth factor stimulation. In contrast, GAP1IP4BP is constitutively associated, in a PtdIns(4,5)P2-dependent manner, with the plasma membrane (Cozier, G. E., Lockyer, P. J., Reynolds, J. S., Kupzig, S., Bottomley, J. R., Millard, T., Banting, G., and Cullen, P. J. (2000) J. Biol. Chem. 275, 28261-28268). In the present study, we have used molecular modeling to identify residues in the GAP1IP4BP PH domain predicted to be required for high affinity binding to PtdIns(4,5)P2. This has allowed the isolation of a mutant, GAP1IP4BP-(K591T), which while retaining high affinity for PtdIns(3,4,5)P3 has a 6-fold reduction in its affinity for PtdIns(4,5)P2. Importantly, GAP1IP4BP-(K591T) is predominantly localized to the cytosol and undergoes a PtdIns(3,4,5)P3-dependent association with the plasma membrane following growth factor stimulation. We have therefore engineered the phosphoinositide-binding profile of the GAP1IP4BP PH domain, thereby emphasizing that subtle changes in PH domain structure can have a pronounced effect on phosphoinositide binding and the subcellular localization of GAP1IP4BP.     

Identification and analysis of PH domain-containing targets of phosphatidylinositol 3-kinase using a novel in vivo assay in yeast.

Phosphatidylinositol 3-kinase (PI3K) mediates a variety of cellular responses by generating PtdIns(3,4)P2 and PtdIns(3,4,5)P3. These 3-phosphoinositides then function directly as second messengers to activate downstream signaling molecules by binding pleckstrin homology (PH) domains in these signaling molecules. We have established a novel assay in the yeast Saccharomyces cerevisiae to identify proteins that bind PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in vivo which we have called TOPIS (Targets of PI3K Identification System). The assay uses a plasma membrane-targeted Ras to complement a temperature-sensitive CDC25 Ras exchange factor in yeast. Coexpression of PI3K and a fusion protein of activated Ras joined to a PH domain known to bind PtdIns(3,4)P2 (AKT) or PtdIns(3,4,5)P3 (BTK) rescues yeast growth at the non-permissive temperature of 37 degreesC. Using this assay, we have identified several amino acids in the beta1-beta2 region of PH domains that are critical for high affinity binding to PtdIns(3,4)P2 and/or PtdIns(3,4,5)P3, and we have proposed a structural model for how these PH domains might bind PI3K products with high affinity. From these data, we derived a consensus sequence which predicts high-affinity binding to PtdIns(3, 4)P2 and/or PtdIns(3,4,5)P3, and we have identified several new PH domain-containing proteins that bind PI3K products, including Gab1, Dos, myosinX, and Sbf1. Use of this assay to screen for novel cDNAs which rescue yeast at the non-permissive temperature should provide a powerful approach for uncovering additional targets of PI3K.     

Small chemicals with inhibitory effects on PtdIns(3,4,5)P3 binding of Btk PH domain

Phosphatidylinositol-3,4-5-triphosphates (PtdIns(3,4,5)P₃) formed by phosphoinositide-3-kinase (PI3K) had been known as a signaling molecule that plays important roles in diverse cellular processes such as cell signaling, metabolism, cell differentiation, and apoptosis. PtdIns(3,4,5)P₃ regulates diverse cellular processes by recruiting effector proteins to the specific cellular locations for correct functions. In this study, we reported the inhibitory effect of small chemicals on the interaction between PtdIns(3,4,5)P₃–Btk PH domain. Small chemicals were synthesized based on structural similarity of PtdInsP head-groups, and tested the inhibitory effects in vitro via surface plasmon resonance (SPR). As a result, the chemical 8 showed highest inhibitory effect with 17 μM of IC₅₀ value. To elucidate diverse inhibitory effects of different small chemicals we employed in silico docking experiment using molecular modeling and simulation. The result of docking experiments showed chemical 8 has more hydrogen bonding with the residues in PtdIns(3,4,5)P₃ binding site of Btk PH domain than others. Overall, our studies demonstrate the efficient approach to develop lipid binding inhibitors, and further we can use these chemicals to regulate effector proteins. In addition, our study would provide new insight that lipid binding domain may be the attractive therapeutic targets to treat severe human diseases.     

GAP1IP4BP contains a novel group I pleckstrin homology domain that directs constitutive plasma membrane association

The group I family of pleckstrin homology (PH) domains are characterized by their inherent ability to specifically bind phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and its corresponding inositol head-group inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). In vivo this interaction results in the regulated plasma membrane recruitment of cytosolic group I PH domain-containing proteins following agonist-stimulated PtdIns(3,4,5)P(3) production. Among group I PH domain-containing proteins, the Ras GTPase-activating protein GAP1(IP4BP) is unique in being constitutively associated with the plasma membrane. Here we show that, although the GAP1(IP4BP) PH domain interacts with PtdIns(3,4, 5)P(3), it also binds, with a comparable affinity, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) (K(d) values of 0.5 +/- 0.2 and 0.8 +/- 0.5 microm, respectively). Intriguingly, whereas this binding site overlaps with that for Ins(1,3,4,5)P(4), consistent with the constitutive plasma membrane association of GAP1(IP4BP) resulting from its PH domain-binding PtdIns(4,5)P(2), we show that in vivo depletion of PtdIns(4,5)P(2), but not PtdIns(3,4,5)P(3), results in dissociation of GAP1(IP4BP) from this membrane. Thus, the Ins(1,3,4,5)P(4)-binding PH domain from GAP1(IP4BP) defines a novel class of group I PH domains that constitutively targets the protein to the plasma membrane and may allow GAP1(IP4BP) to be regulated in vivo by Ins(1,3,4,5)P(4) rather than PtdIns(3,4,5)P(3).     

Peroxovanadate induces tyrosine phosphorylation of phosphoinositide-dependent protein kinase-1 potential involvement of src kinase.

Phosphoinositide-dependent protein kinase-1 (PDK1) is a recently identified kinase that phosphorylates and activates protein kinase B (PKB). Activation of PKB by insulin is linked to its translocation from the cytosol to the plasma membrane. However, no data are available yet concerning the localization of PDK1 in insulin-sensitive tissue. Using isolated adipocytes, we studied the effect of insulin and of an insulin-mimicking agent peroxovanadate on the subcellular localization of PDK1. In unstimulated adipocytes, overexpressed PDK1 was mostly cytosolic with a low amount associated to membranes. Peroxovanadate stimulation induced the redistribution of PDK1 to the membranes while insulin was without effect. This peroxovanadate effect was dependent on phosphatidylinositol 3,4,5 triphosphate [PtdIns(3,4,5)P3] production as inhibition of PtdIns 3-kinase by wortmannin or deletion of the PH domain of PDK1 prevented the peroxovanadate-induced translocation of PDK1. Further, peroxovanadate-treatment induced a tyrosine phosphorylation of PDK1 which was wortmannin insensitive and did not require the PH domain of PDK1. An inhibitor of Src kinase (PP2) decreased the peroxovanadate-induced PDK1 tyrosine phosphorylation and overexpression of v-Src stimulated this phosphorylation. Mutation of tyrosine 373 of PDK1 abolished the v-Src induced PDK1 tyrosine phosphorylation and partially reduced the effect of peroxovanadate. Our findings suggest that PDK1 could be a substrate for tyrosine kinases and identify Src kinase as one of the tyrosine kinases able to phosphorylate PDK1.     

Inositol pyrophosphates mediate chemotaxis in Dictyostelium via pleckstrin homology domain-PtdIns(3,4,5)P3 interactions

Inositol phosphates are well-known signaling molecules, whereas the inositol pyrophosphates, such as diphosphoinositol pentakisphosphate (InsP7/IP7) and bis-diphosphoinositol tetrakisphosphate (InsP8/IP8), are less well characterized. We demonstrate physiologic regulation of Dictyostelium chemotaxis by InsP7 mediated by its competition with PtdIns(3,4,5)P3 for binding pleckstrin homology (PH) domain-containing proteins. Chemoattractant stimulation triggers rapid and sustained elevations in InsP7/InsP8 levels. Depletion of InsP7 and InsP8 by deleting the gene for InsP6 kinase (InsP6K/IP6K), which converts inositol hexakisphosphate (InsP6/IP6) to InsP7, causes rapid aggregation of mutant cells and increased sensitivity to cAMP. Chemotaxis is mediated by membrane translocation of certain PH domain-containing proteins via specific binding to PtdIns(3,4,5)P3. InsP7 competes for PH domain binding with PtdIns(3,4,5)P3 both in vitro and in vivo. InsP7 depletion enhances PH domain membrane translocation and augments downstream chemotactic signaling activity.