IP4 Binding Research Articles

Positive regulation of Itk PH domain function by soluble IP4 is required for thymocyte positive selection but dispensable for negative selection

Membrane recruitment of Itk and PLCγ1 require interactions between their Pleckstrin homology (PH) domains and PIP3, the phospholipid product of PI‐3 kinase. Dual anchoring via SH2 and PH domains are required for optimal Itk‐mediated activation of PLCγ1 and the subsequent generation of IP3 and DAG. We have identified a novel mechanism in which conversion of IP3 into IP4 by ItpkB creates a physiologically important soluble ligand for the Itk PH domain. IP4 specifically promotes PH domain‐dependent recruitment of Itk but not PLCγ1 to PIP3 and is required for optimal Itk activation. PH‐domain mediated oligomerization of Itk leads us to propose a model in which IP4 binding to one Itk PH domain can allosterically increase the affinity of associated Itk PH domains for membrane PIP3. IP4‐mediated positive feedback of Itk activation is required for positive selection during T cell development. Dose dependent reduction of single positive thymocytes suggests that ItpkB limits the efficiency of T cell maturation. Interestingly, our data indicates that thymocyte negative selection is normal in the absence of ItpkB and suggests that ItpkB participates in distinguishing TCR‐dependent death or survival signals.

The requirement of a Ku‐IP6 (inositol hexakisphosphate) complex for efficient repair of DNA double strand breaks by non‐homologous end joining in mammals

Non-Homologous-End-Joining (NHEJ) is the major pathway in mammalian cell that repairs DNA Double-Strand-Breaks (DSBs) and plays a critical role in maintaining genomic stability. Inositol hexakisphosphate (IP6) was previously identified to stimulate NHEJ and bind to the Ku70/Ku80 heterodimer (Ku). To study the role of Ku-IP6 interaction in NHEJ, selected lysine residues of the putative Ku-IP6 binding site were mutated to alanine. Ku wild type and IP6-binding mutants were expressed using the baculovirus expression system and purified to > 90% pure. The IP6-binding mutants have 2 – 10 fold decrease in IP6 binding compare to wild type (Kd ≈ 100 nM); they also fail to complement NHEJ in vitro. Interestingly, all the Ku-IP6 binding mutants bind to dsDNA ends and activate DNA-PKcs kinase activity comparable to wild type. In summary, we have identified the IP6 binding site in Ku and this Ku-IP6 interaction is essential for efficient NHEJ in vitro but unrelated to its role in binding dsDNA ends or activating DNA-PKcs kinase activity. This research was funded by the National Institutes of Health GM070639.

Interactions between HIV-1 Gag Molecules in Solution: An Inositol Phosphate-mediated Switch

Retrovirus particle assembly is mediated by the Gag protein. Gag is a multi-domain protein containing discrete domains connected by flexible linkers. When recombinant HIV-1 Gag protein (lacking myristate at its N terminus and the p6 domain at its C terminus) is mixed with nucleic acid, it assembles into virus-like particles (VLPs) in a fully defined system in vitro. However, this assembly is defective in that the radius of curvature of the VLPs is far smaller than that of authentic immature virions. This defect can be corrected to varying degrees by addition of inositol phosphates to the assembly reaction. We have now explored the binding of inositol hexakisphosphate (IP6) to Gag and its effects upon the interactions between Gag protein molecules in solution. Our data indicate that basic regions at both ends of the protein contribute to IP6 binding. Gag is in monomer-dimer equilibrium in solution, and mutation of the previously described dimer interface within its capsid domain drastically reduces Gag dimerization. In contrast, when IP6 is added, Gag is in monomer-trimer rather than monomer-dimer equilibrium. The Gag protein with a mutation at the dimer interface also remains almost exclusively monomeric in IP6; thus the “dimer interface” is essential for the trimeric interaction in IP6. We discuss possible explanations for these results, including a change in conformation within the capsid domain induced by the binding of IP6 to other domains within the protein. The participation of both ends of Gag in IP6 interaction suggests that Gag is folded over in solution, with its ends near each other in three-dimensional space; direct support for this conclusion is provided in a companion manuscript. As Gag is an extended rod in immature virions, this apparent proximity of the ends in solution implies that it undergoes a major conformational change during particle assembly.

Specific interaction of IP6 with human Ku70/80, the DNA-binding subunit of DNA-PK.

In eukaryotic cells, DNA double-strand breaks can be repaired by non-homologous end-joining, a process dependent upon Ku70/80, XRCC4 and DNA ligase IV. In mammals, this process also requires DNA-PK(cs), the catalytic subunit of the DNA-dependent protein kinase DNA-PK. Previously, inositol hexakisphosphate (IP6) was shown to be bound by DNA-PK and to stimulate DNA-PK-dependent end-joining in vitro. Here, we localize IP6 binding to the Ku70/80 subunits of DNA- PK, and show that DNA-PK(cs) alone exhibits no detectable affinity for IP6. Moreover, proteolysis mapping of Ku70/80 in the presence and absence of IP6 indicates that binding alters the conformation of the Ku70/80 heterodimer. The yeast homologue of Ku70/80, yKu70/80, fails to bind IP6, indicating that the function of IP6 in non-homologous end-joining, like that of DNA-PK(cs), is unique to the mammalian end-joining process.

Mutagenesis analysis of IP4 binding sites in the PH domain of Bruton's tyrosine kinase

Mutagenesis analysis of IP4 binding sites in the PH domain of Bruton's tyrosine kinase

Identification of the Ras GTPase-activating protein GAP1m as a phosphatidylinositol-3,4,5-trisphosphate-binding protein in vivo

GAP1m is a member of the GAP1 family of Ras GTPase-activating proteins (GAPs) [1]. In vitro, it has been shown to bind inositol 1,3,4,5-tetrakisphosphate (IP4), the water-soluble inositol head group of the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3) [2,3]. This has led to the suggestion that GAP1m might function as a PIP3 receptor in vivo[4]. Here, using rat pheochromocytoma PC12 cells transiently transfected with a plasmid expressing a chimera of green fluorescent protein fused to GAP1m (GFP–GAP1m), we show that epidermal growth factor (EGF) induces a rapid (less than 60 seconds) recruitment of GFP–GAP1m from the cytosol to the plasma membrane. This recruitment required a functional GAP1m pleckstrin homology (PH) domain, because a specific point mutation (R629C) in the PH domain that inhibits IP4 binding in vitro[5] totally blocked EGF-induced GAP1m translocation. Furthermore, the membrane translocation was dependent on PI 3-kinase, and the time course of translocation paralleled the rate by which EGF stimulates the generation of plasma membrane PIP3[6]. Significantly, the PIP3-induced recruitment of GAP1m did not appear to result in any detectable enhancement in its basal Ras GAP activity. From these results, we conclude that GAP1m binds PIP3in vivo, and it is recruited to the plasma membrane, but does not appear to be activated, following agonist stimulation of PI 3-kinase.

Inositol 1,3,4,5-Tetrakisphosphate Binding Activities of Neuronal and Non-neuronal Synaptotagmins: IDENTIFICATION OF CONSERVED AMINO ACID SUBSTITUTIONS THAT ABOLISH INOSITOL 1,3,4,5-TETRAKISPHOSPHATE BINDING TO SYNAPTOTAGMINS III, V, AND X

Synaptotagmins I and II are essential for Ca2+-regulated exocytosis of synaptic vesicles from neurons, probably serving as Ca2+ sensors. This Ca2+-sensing function is thought to be disrupted by binding of an inositol 1,3,4,5-tetrakisphosphate (IP4) to the C2B domain of synaptotagmin I or II (Fukuda, M., Moreira, J. E., Lewis, F. M. T., Sugimori, M., Niinobe, M., Mikoshiba, K., and Llinás, R. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10708-10712). Recently, several synaptotagmin isoforms, expressed outside the nervous system, have been identified in rats and proposed to be involved in constitutive vesicle traffic. To test whether the inositol high polyphosphates also regulate constitutive vesicle traffic by binding to the non-neuronal synaptotagmins, we examined the IP4 binding properties of the recombinant C2 domains of both neuronal (III, V, X, and XI) and non-neuronal (VI-VIII and IX) synaptotagmins. The C2B domains of synaptotagmins VII-IX and XI had strong IP4 binding activity, but the C2B domain of synaptotagmin VI showed very weak IP4 binding activity. In contrast, there was no significant IP4 binding activity of the C2B domains of synaptotagmins III, V, and X or any of the C2A domains. A phylogenetic tree of the C2 domains of 11 isoforms revealed that synaptotagmins III, V, VI, and X (IP4-insensitive or very weak IP4-binding isoforms) belong to the same branch. Based on the sequence comparison between the IP4-sensitive and -insensitive isoforms, we performed site-directed mutagenesis of synaptotagmin III and identified several amino acid substitutions that abolish IP4 binding activity. Our data suggest that the inositol high polyphosphates might also regulate constitutive vesicle traffic via binding to the IP4-sensitive non-neuronal synaptotagmins.

The relation between the no-generative neurons and the central circuitry for rhythmic feeding in the pond snail, Lymnaea stagnalis

Synaptotagmin I (or II), a possible Ca2+-sensor of synaptic vesicles, has two functionally distinct C2 domains: the C2A domain binds Ca2+and the C2B domain binds inositol high polyphosphates (IP4, IP5, and IP6). Ca2+-regulated exocytosis of secretory vesicles is proposed to be activated by Ca2+ binding to the C2A domain and inhibited by inositol polyphosphate binding to the C2B domain. Synaptotagmins now constitute a large family and are thought to be involved in both regulated and constitutive vesicular trafficking. They are classified from their distribution as neuronal (synaptotagmin I–V, X, and XI) and the ubiquitous type (synaptotagmin VI–IX). Among them, synaptotagmins III, V, VI and X are deficient in IP4 binding activity due to the amino acid substitutions in the C-terminal region of the C2B domain, suggesting that these isoforms can work for vesicular trafficking even in the presence of inositol high polyphosphates. Synaptotagmin I is also known to be present in neuronal growth cone vesicles. Antibody against the C2A domain (anti-C2A) that inhibits Ca2+-regulated exocytosis also blocked neurite outgrowth of the chick dorsal root ganglion (DRG) neuron, suggesting that Ca2+-dependent synaptotagmin activation is also crucial for neurite outgrowth.

Activation of protein kinase C potentiates transmitter release from goldfish retinal bipolar cells

Synaptotagmin I (or II), a possible Ca2+-sensor of synaptic vesicles, has two functionally distinct C2 domains: the C2A domain binds Ca2+and the C2B domain binds inositol high polyphosphates (IP4, IP5, and IP6). Ca2+-regulated exocytosis of secretory vesicles is proposed to be activated by Ca2+ binding to the C2A domain and inhibited by inositol polyphosphate binding to the C2B domain. Synaptotagmins now constitute a large family and are thought to be involved in both regulated and constitutive vesicular trafficking. They are classified from their distribution as neuronal (synaptotagmin I–V, X, and XI) and the ubiquitous type (synaptotagmin VI–IX). Among them, synaptotagmins III, V, VI and X are deficient in IP4 binding activity due to the amino acid substitutions in the C-terminal region of the C2B domain, suggesting that these isoforms can work for vesicular trafficking even in the presence of inositol high polyphosphates. Synaptotagmin I is also known to be present in neuronal growth cone vesicles. Antibody against the C2A domain (anti-C2A) that inhibits Ca2+-regulated exocytosis also blocked neurite outgrowth of the chick dorsal root ganglion (DRG) neuron, suggesting that Ca2+-dependent synaptotagmin activation is also crucial for neurite outgrowth.

Selective Photoaffinity Labeling of the Inositol Polyphosphate Binding C2B Domains of Synaptotagmins

Synaptotagmin (Syt) II, a synaptic vesicle protein containing two copies of highly conserved protein kinase C homology regions known as the C2A and C2B domains, acts as a Ca2+ sensor and provides both phospholipid and inositol polyphosphate (IPn) recognition domains important in endo- and exocytosis. Four photoaffinity analogues of IP3, IP4, and IP6 containing a P-1- or P-2-linked 4-benzoyldihydrocinnamidyl (BZDC) photophore were used to label glutathione S-transferase (GST) fusion constructs of the Syt II-C2A and C2B domains. The P-2-linked [3H]BZDC-IP6 showed efficient, IP6-displaceable labeling of the GST-Syt II-C2B. The rank order of photocovalent modification paralleled the order of competitive displacement: IP6 (P-2-linked) > IP4 > IP3. The P-1-linked [3H]BZDC-IP6 failed to label the C2B domains. The GST-Syt III-C2B domain, which lacks IP6 binding affinity, also failed to undergo labeling by P-2-linked [3H]BZDC-IP6. When mixtures of the 32-amino acid basic peptide corresponding to the essential IPn binding region of the Syt II-C2B domain and GST-Syt II-C2B were labeled by a stoichiometric amount of P-2-linked [3H]BZDC-IP6, the two polypeptides showed equivalent affinity for the photolabel. Although the CD spectrum of this 32-mer at two pH values showed a random coil, the photoaffinity analogue of IP6 appeared to induce a binding-compatible structure in the short peptide.

Structure-Function Relationships of the Mouse Gap1m: DETERMINATION OF THE INOSITOL 1,3,4,5-TETRAKISPHOSPHATE-BINDING DOMAIN

Gap1(IP4BP), one of a member of Ras GTPase-activating proteins, has been identified as a specific inositol 1,3,4,5-tetrakisphosphate (IP4)-binding protein (Cullen, P. J., Hsuan, J. J., Truong, O., Letcher, A. J., Jackson, T. R., Dawson, A. P., and Irvine, R. F. (1995) Nature 386, 527-530). In this paper we describe Gap1(m), which is closely related to Gap1(IP4BP), to also be an IP4-binding protein and show that the pleckstrin homology domain (PH) is the central IP4-binding domain by expressing fragments of the mouse Gap1(m) in Escherichia coli as fusion proteins and examining their activities. However, in addition to the PH domain, an adjacent GAP-related domain and carboxyl terminus are required for high affinity specific IP4 binding. The PH domain is highly conserved in the Gap1 family and also has striking homology to the amino-terminal region of Bruton's tyrosine kinase. Substitution of Cys for Arg at position 628 in the PH domain corresponding to the mutation of Bruton's tyrosine kinase observed in X-linked immunodeficiency mice results in a dramatic reduction of IP4 binding activity as well as phospholipid binding capacity of Gap1(m). This mutant also showed the GAP activity against Ha-Ras to be similar to that of the wild type Gap1(m). Our results suggest that the PH domain of Gap1(m) functions as a modulatory domain of GAP activity by binding IP4 and phospholipids.

Functional Diversity of C2 Domains of Synaptotagmin Family: MUTATIONAL ANALYSIS OF INOSITOL HIGH POLYPHOSPHATE BINDING DOMAIN

Synaptotagmins I and II are inositol high polyphosphate series (inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate) binding proteins, which are thought to be essential for Ca(2+)-regulated exocytosis of neurosecretory vesicles. In this study, we analyzed the inositol high polyphosphate series binding site in the C2B domain by site-directed mutagenesis and compared the IP4 binding properties of the C2B domains of multiple synaptotagmins (II-IV). The IP4 binding domain of synaptotagmin II is characterized by a cluster of highly conserved, positively charged amino acids (321 GKRLKKKKTTVKKK 324). Among these, three lysine residues, at positions 327, 328, and 332 in the middle of the C2B domain, which is not conserved in the C2A domain, were found to be essential for IP4 binding in synaptotagmin II. When these lysine residues were altered to glutamine, the IP4 binding ability was completely abolished. The primary structures of the IP4 binding sites are highly conserved among synaptotagmins I through IV. However, synaptotagmin III did not show significant binding ability, which may be due to steric hindrance by the C-terminal flanking region. These functional diversities of C2B domains suggest that not all synaptotagmins function as inositol high polyphosphate sensors at the synaptic vesicle.

Inositol-1,3,4,5-tetrakisphosphate binding to C2B domain of IP4BP/synaptotagmin II.

IP4BP/Synaptotagmin II is an inositol-1,3,4,5-tetrakisphosphate (IP4) or inositol polyphosphate-binding protein, which is accumulated at nerve terminals. Here we report a novel function of the C2B domain, which was originally thought to be responsible for Ca(2+)-dependent binding to phospholipid membranes. A study of deletion mutants showed that about 30 amino acids of the central region of the C2B domain of mouse IP4BP/synaptotagmin II (315 IHLMQNGKRLKKKKTTVKKKTLNPYFNESFSF 346) are essential for inositol polyphosphate binding. This binding domain includes a sequence corresponding to the squid Pep20 peptide, which is also known to be essential for neurotransmitter release (Bommert, K., Charlton, M. P., DeBello, W. M., Chin, G. J., Betz, H., and Augustine, G. J. (1993) Nature 363, 163-165), suggesting that inositol polyphosphate has some effect on neurotransmitter release. Rabphilin 3A, another neuronal protein containing C2 domains, cannot bind IP4, indicating that the IP4 binding property is specific to the C2B domain of synaptotagmin. Phospholipid and IP4 binding experiments clearly indicated that the C2A and C2B domains have different functions. The C2A domain binds phospholipid in a Ca(2+)-dependent manner, but the C2B domain binds inositol polyphosphate and phospholipid irrespective of the presence of Ca2+. Our data suggest that the C2B domain of synaptotogamin is the inositol polyphosphate sensor at the synaptic vesicle and may be involved in synaptic function.

Golgi coatomer binds, and forms K(+)-selective channels gated by, inositol polyphosphates.

Coatomer is a distinct type of coat protein complex involved in the formation of specific Golgi intercisternal transport vesicles. Direct binding studies using purified coatomer isolated from bovine liver cytosol show that coatomer specifically binds both inositol 1,3,4,5-tetrakisphosphate ((1,3,4,5)IP4) and inositol hexakisphosphate (IP6) with subnanomolar affinities (0.1 and 0.2 nM, respectively). Diphosphoinositol pentakisphosphate (PP-IP5) is an efficient competitor for both (1,3,4,5)IP4 and IP6 binding to coatomer. Inositol 1,3,4,5,6-pentakisphosphate ((1,3,4,5,6)IP5) is a poor inhibitor of IP6 binding, whereas little or no competition is detected with inositol 1,4,5-trisphosphate ((1,4,5)I-P3). Coatomer displays ion channel activity when reconstituted into planar bilayers which is preferentially permeable to K+. Permeability ratios of the channel are PK+/PCl- approximately 8.0 and PK+/PNa+ approximately 7.1, indicating a cation-selective channel with selectivity of K+ over Na+. In symmetrical 500 mM KCl, the smallest observable unitary channel conductance is 8.3 picosiemens. The coatomer channel activity is normally active with long open times (0.1 to several seconds) and is selectively blocked by 10 microM (1,3,4,5)IP4, 1 microM IP6, and 0.27 microM PP-IP5; even lower concentrations are sufficient to induce channel flicker. The channel activity is not affected by (1,4,5)IP3, or (1,3,4,5,6)IP5. Thus, the channel activity of coatomer is modulated by the inositol polyphosphates which exhibit tight binding to the complex.

Isolation and characterization of a novel inositol hexakisphosphate binding protein from mammalian cell cytosol.

Inositol hexakisphosphate (IP6) is present in most mammalian cells, although its intracellular function is as yet undefined. We find that the total protein fraction from bovine brain cytosol contains a significant level of specific binding for IP6 precipitable with 40% saturated ammonium sulfate. A protein complex has been isolated from this fraction that specifically binds IP6 and is purified about 500-fold over the cytosol. The IP6 binding protein (IP6BP) chromatographs as a single peak of binding activity on a gel exclusion column, with a Stokes radius equivalent to 266 +/- 14 kDa. The IP6BP is a heterooligomeric complex composed of a number of subunits with molecular weights varying from 23,000 to 60,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Scatchard analyses of IP6 binding of both the crude ammonium sulfate fraction and the purified complex show the presence of a similar high-affinity binding site (Kd approximately 6.0 nM). Bmax for the purified fraction is 1.8 nmol of IP6/mg of protein or 0.48 mol of IP6 bound/mol of complex. Other inositol polyphosphates, such as inositol 1,3,4,5,6-pentakisphosphate, inositol 1,3,4,5-tetrakisphosphate, and inositol 1,4,5-trisphosphate, are poor competitors for IP6 binding to the purified complex. The purification scheme, when applied to a rat liver cytosol fraction, yields a similar IP6BP. This complex has an apparent size of 512,000 using gel exclusion chromatography and contains an additional protein band with M(r) = 97,000 by SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential Localization and pH Dependency of Phosphoinositide 1,4,5‐IP3, 1,3,4,5‐IP4 and IP6 Receptors in Rat and Human Brains

It is well established that the inositol lipids mediate signal transduction in several cellular populations. Many neurotransmitters, hormones and growth factors act at plasma membrane receptors to induce the hydrolysis of phosphatidylinositols and hence the generation of various inositol phosphates (IP). The best known member of this family is 1,4,5-IP3, which is associated with the release of Ca2+ from intracellular pools. It has also been proposed that two others inositides, 1,3,4,5-IP4 and IP6, may be involved in Ca2+ homeostasis. In order to study the possible relevance of these various inositides in neuronal tissues, we have localized the respective receptors in rat and human brain under both acidic and basic pH conditions. In the hippocampal formation, [3H]1,3,4,5-IP4 binding sites are concentrated in the hilus and the molecular layer while a clearly different pattern of distribution is seen for [3H]1,4,5-IP3, its highest concentration of labelling being concentrated in the oriens and radiatum laminae. This contrasting profile of distribution is also observed in other brain areas such as the caudate-putamen, the septo-hippocampal area, and the molecular and granular layers of the cerebellum. Moreover, while highest amounts of specific [3H]1,4,5-IP3 binding are obtained at pH 8.5, the opposite is found for [3H]1,3,4,5-IP4, with high binding levels seen under acidic conditions. [3H]IP6 binding sites are broadly distributed with specific labelling concentrated in areas enriched with neuronal perikarya such as the granular cell layer of the dentate gyrus, the pyramidal cell layers of the hippocampus and the granular cell layer of the cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for stereospecific inositol 1,3,4,5-[3H]tetrakisphosphate binding sites on rat liver nuclei. Delineating inositol 1,3,4,5-tetrakisphosphate interaction in nuclear calcium signaling process.

3H-Labeled inositol 1,3,4,5-tetrakisphosphate (IP4) binding sites are observed on nuclei isolated from rat liver and devoid of any microsomal, mitochondrial, or plasma membrane constituents. A pH of about 6.5 is found optimum for maximum [3H]IP4 specific binding that is sensitive to changes in pH. The [3H]IP4 binding on the nuclei can be distinguished into a high affinity site and a low affinity site. The two binding sites are characterized by distinct KD and Bmax (1.6 nM versus 57.0 nM KD; 0.25 pmol/mg protein and 3.7 pmol/mg protein Bmax). IP4 is capable of 45Ca2+ uptake even in the absence of ATP. The calcium uptake by nuclei is highly sensitive to IP4 since it is achieved even at 1 nM IP4 concentration. Furthermore, data are documented demonstrating that a rapid and transient 45Ca2+ release by inositol 1,4,5-trisphosphate (IP3) from the intact nuclei can be reversed by IP4. The presence of IP3 potentiates the action of IP4 in nuclear calcium reuptake as attested by the rate of calcium uptake by IP4 in the absence of IP3 (0.16 nmol/s/mg of protein) and in the presence of IP3 (4.0 nmol/s/mg of protein). A novel mechanism of nuclear calcium signaling is proposed where IP4 brings calcium into the nuclei mediated by its specific putative binding sites.