Phosphoinositides (PtdInsPs) play critical roles in cytoplasmic signal transduction pathways. However, their functions in the nucleus are unclear, as specific nuclear receptors for PtdInsPs have not been identified. Here, we show that ING2, a candidate tumor suppressor protein, is a nuclear PtdInsP receptor. ING2 contains a plant homeodomain (PHD) finger, a motif common to many chromatin-regulatory proteins. We find that the PHD fingers of ING2 and other diverse nuclear proteins bind in vitro to PtdInsPs, including the rare PtdInsP species, phosphatidylinositol 5-phosphate (PtdIns(5)P). Further, we demonstrate that the ING2 PHD finger interacts with PtdIns(5)P in vivo and provide evidence that this interaction regulates the ability of ING2 to activate p53 and p53-dependent apoptotic pathways. Together, our data identify the PHD finger as a phosphoinositide binding module and a nuclear PtdInsP receptor, and suggest that PHD-phosphoinositide interactions directly regulate nuclear responses to DNA damage.

Oxysterol binding proteins (OSBPs) comprise a large conserved family of proteins in eukaryotes. Their ubiquity notwithstanding, the functional activities of these proteins remain unknown. Kes1p, one of seven members of the yeast OSBP family, negatively regulates Golgi complex secretory functions that are dependent on the action of the major yeast phosphatidylinositol/phosphatidylcholine Sec14p. We now demonstrate that Kes1p is a peripheral membrane protein of the yeast Golgi complex, that localization to the Golgi complex is required for Kes1p function in vivo, and that targeting of Kes1p to the Golgi complex requires binding to a phosphoinositide pool generated via the action of the Pik1p, but not the Stt4p, PtdIns 4-kinase. Localization of Kes1p to yeast Golgi region also requires function of a conserved motif found in all members of the OSBP family. Finally, we present evidence to suggest that Kes1p may regulate adenosine diphosphate-ribosylation factor (ARF) function in yeast, and that it may be through altered regulation of ARF that Kes1p interfaces with Sec14p in controlling Golgi region secretory function.

. Although the CD spectrum of this 32-mer at two pH values showed a random coil, the photoaffinity analogue of IP 6 appeared to induce a binding-compatible structure in the short peptide.The synaptotagmins (Syts) 1 are synaptic vesicle proteins that play essential roles in nucleating the clathrin coat during endocytosis and in acting as Ca 2ϩ sensors (1-3) and phosphoinositide sensors (4) during exocytosis. They are a critical part of a complex machinery of intracellular protein transport (5, 6) and the synaptic vesicle cycle (7). In addition to a short Nterminal intravesicular region and single transmembrane domain, Syts have two copies of highly conserved repeats, known as the C2A and C2B domains, which are homologous to the C2 regulatory region of protein kinase C (8). In particular, the C2B domain appears to play several roles. First, the C2B domain of mouse Syt II shows specific binding to high polyphosphate inositols (IP n s) (9), a feature that is not shared by the highly homologous C2A domain nor with the C2 domains of other proteins such as rabphilin. Moreover, the C2B domain is necessary but not sufficient for IP n binding. Although Syt II and IV show high affinity binding of IP 4 and IP 6 , the Syt III-C2B domain shows negligible binding, despite a high sequence identity with the Syt II-C2B domain, including the 32-residue region examined by mutational analysis (10). Inhibition of C2B function in the squid giant axon disrupts synaptic vesicle release and recycling (11). Similarly, Caenorhabditis elegans mutants lacking Syt or simply the C2B domain cannot recycle synaptic vesicles (12), and the presence of Syt I in cerebrospinal fluid of Alzheimer's patients (13) may provide clues to the disruption of synaptic function in humans. The C2B domain acts as a high affinity receptor for clathrin assembly protein AP-2 (14), and Ca 2ϩ appears to mediate Syt dimerization via the C2B domain (15).Synaptotagmin II isolated from mouse cerebellum shows high affinity binding to Ins(1,3,4,5)P 4 with a K D of 30 M (16) and 117 nM for the GST-Syt II-C2B construct (9). Curiously, the rank order of IP n s binding for native Syt II was Ins-(1,3,4,5,6)-P 5 Ͼ (1,3,4,5)-P 4 Ͼ Ins(1,2,3,4,5,6)-P 6 Ͼ Ins(1,4,5)-P 3 , whereas for the GST-Syt II-C2B domain the order was IP 6 Ͼ IP 5 Ͼ IP 4 Ͼ IP 3 . Deletion mutants allowed mapping of the IP n binding site to the central region of the mouse Syt II-C2B domain, specifically residues 315-346 (IHLMQNGKRLKKKK-TTVKKKTLNPYFNESFSF) (9). In order to obtain direct evidence for the binding site of IP n s in the C2B domain, to examine the InsP n selectivity of the domains, to explore the Ca 2ϩ dependence of binding, to determine the role of the central 315-346 peptide, and to evaluate the reason for the failure of Syt III-C2B domain to bind IP n s, we undertook a series of photoaffinity labeling experiments using four tritium-labeled, benzophenone-containing derivatives of IP 3 , IP 4 , and IP 6 (17). EXPERIMENTAL PROCEDURES Chemicals-P-1-Tethered

Synaptotagmin II (Syt II) is a key protein in the calcium-dependent exocytosis of synaptic vesicles. It contains two domains homologous to the C2 regulatory region of protein kinase C. The C2A domain acts as a calcium sensor, while the C2B domain has high affinity for inositol polyphosphates (InsP(n)()s) and phosphoinositide polyphosphates (PtdInsP(n)()s). We describe the use of a surface plasmon resonance biosensor in determining the binding kinetics of the C2B domain with InsP(n)() and PtdInsP(n) ligands. Biosensor surfaces were prepared with covalently attached Ins(1,4,5)P(3), Ins(1,3,4,5)P(4), and InsP(6) ligands. The interactions of bacterially expressed His(6)-tagged C2B and (C2A+C2B) domains of Syt II were examined in the presence and absence of competing InsP(n)s and PtdInsP(n)s. Both His(6)-C2B and His(6)-(C2A+C2B) exhibited the highest affinity for the Ins(1,3,4,5)P(4)-modified surface with a K(D) value of 6 nM. The His(6)-(C2A+C2B) had a 10-fold lower association rate constant for the InsP(6)-linked surface (k(a) = 4.6 x 10(3) M(-1) s(-1)) than for the Ins(1,3,4,5)P(4)-modified surface (k(a) = 6.8 x 10(4) M(-1) s(-1)). Two water-soluble phosphoinositides, dioctanoyl-PtdIns(3,4,5)P(3) and dioctanoyl-PtdIns(4,5)P(2), were superior to the soluble InsP(n)s in displacing binding to the Ins(1,3,4,5)P(4)-modified surface. The binding of His(6)-C2B and His(6)-(C2A+C2B) to InsP(n) surfaces did not show significant calcium dependence. These data support a model in which the binding of the C2B domain of Syt II to PtdInsP(n)s is important for the docking and/or fusion of the secretory vesicles to the synaptic plasma membrane.