IP recognition by IPK 1 1 Roles of Phosphate Recognition in Inositol

Inositol phosphate kinases (IPKs) sequentially phosphorylate inositol phosphates (IPs) to yield a group of small signaling molecules involved in diverse cellular processes. Inositol 1,3,4,5,6pentakisphosphate 2-kinase (IPK1) phosphorylates IP5 to IP6; however, the mechanism of IP recognition employed by IPK1 is currently unresolved. We previously demonstrated that IPK1 possesses an unstable N-lobe in the absence of IP, which led us to propose that the phosphate profile of the IP was linked to stabilization of IPK1. Here, we describe a systematic study to determine the roles of the 1, 3, 5 and 6 phosphate groups of IP5 in IP binding and IPK1 activation. The 5and 6-phosphate groups were the most important for IP binding to IPK1, and the 1and 3-phosphate groups were more important for IPK1 activation than others. Moreover, we demonstrate that there are three critical residues (R130, K170, and K411) necessary for IPK1 activity. R130 is the only substratebinding N-lobe residue that can render IPK1 inactive; its 1-phosphate is critical for full IPK1 activity and for stabilization of the active conformation of IPK1. Taken together, our results support the model for recognition of the IP substrate by IPK1 in which (i) the 4-, 5-, and 6-phosphates are initially recognized by the Clobe and, subsequently, (ii) the interaction between the 1-phosphate and R130 stabilizes the N-lobe and activates IPK1. This model of IP recognition, believed to be unique among IPKs, could be exploited for selective inhibition of IPK1 in future studies that investigate the role of higher IPs. Inositol phosphates (IPs) are a group of small molecules that play critical roles in cellular signaling (1). IP signaling regulates DNA editing and repair (2), vesicle transport (3), and ion channel regulation (4) and has been implicated in diseases such as cancer and diabetes (5). IPs are produced by sequential phosphorylation of inositol 1,4,5-trisphosphate (IP3) by a family of enzymes known as IP kinases (IPKs) (1). Similarity between IPs, which sometimes differ by only one phosphate group on the inositol ring, demands that IPKs use mechanisms to recognize and phosphorylate specific positions of their IP substrates while excluding highly similar http://www.jbc.org/cgi/doi/10.1074/jbc.M113.487777 The latest version is at JBC Papers in Press. Published on July 24, 2013 as Manuscript M113.487777 Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Jauary 2, 2018 hp://w w w .jb.org/ D ow nladed from IP recognition by IPK1 2 molecules. Crystal structures from each of the IPK subfamilies have revealed that the structural determinants for IP discrimination vary between IPKs. Inositol 1,4,5-trisphosphate 3-kinase (IP3K) employs shape complementarity to recognize precisely positioned phosphate and hydroxyl groups of IP3 (6). In contrast, inositol 1,3,4trisphosphate 5/6-kinase/ inositol 3,4,5,6tetrakisphosphate 1-kinase (ITPK1) discriminates among IPs using phosphate affinity and stereochemical features to establish contacts with phosphates that are sufficient for substrate recognition (7). Crystal structures of inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) in its IP substrateand product-bound forms reveal extensive contacts with all phosphate groups of the bound IPs (8). These structures reveal how inositol 1,3,4,5,6-pentakisphosphate (IP5) is phosphorylated on its 2’ axial hydroxyl, yielding inositol 1,2,3,4,5,6-hexakisphosphate (IP6), but they do not suggest a mechanism through which IPK1 selectively recognizes IP5 as its substrate while excluding other highly phosphorylated IPs with free axial 2’ hydroxyl groups. We recently determined the crystal structure of wild-type IPK1 in an IP-free state, which exhibited disorder within its N-lobe of the kinase, centered at R130 (9). This IP-free structure suggests that binding of IP substrate plays a role in stabilization of the Nand C-lobes of the kinase, which is an important step in the activation of protein kinases (10-12). Our current objective was to define the contributions of the individual phosphate groups of the IP to binding and to recognition of the bound IP as a substrate. The results demonstrate that each phosphate group of the IP plays a different role in binding and activation of IPK1 and that there are three critical contacts formed between IPK1 and the IP that mediate IPK1 activation. EXPERIMENTAL PROCEDURES Generation of alanine mutants – Residues with interactions to IP5, either directly or through solvent molecules, were identified using previous crystal structures (9). Mutation of these residues to alanine was performed by site-directed mutagenesis using the quick-change method (Stratagene). A pET28a vector containing wildtype Arabidopsis thaliana IPK1 and a hexahistidine tag was used as a template (a kind gift from C. Brearely). All mutations were verified by DNA sequencing. Protein expression and purification – Wild-type IPK1 and alanine mutants were expressed in BL21 AI cells (Invitrogen) that were grown in Terrific Broth to an OD600 = 1.5 and induced with 0.5 mM IPTG and 0.1% L-arabinose at 18 oC for 20 h. Cells were lysed in 10 mM Tris-HCl (pH 8.0), 250 mM NaCl and 50% glycerol using a sonicator. The supernatant was separated from lysate using centrifugation at 45000xg. The supernatant was then diluted 5-fold using 20 mM Tris-HCl (pH 8.0) and 500 mM NaCl, and 25 mM imidazole was added. IPK1 was purified using NiNTA beads (Thermo Scientific) in a gravity column using 4 mL dry beads per 250 mL of culture. Beads were washed with 20-column volumes of 50 mM KPO4 (pH 8.0), 800 mM NaCl, 1% Triton X-100, 1.7 mM β-mercaptoethanol. Protein was eluted using 250 mM imidazole in 20 mM Tris-HCl (pH 8.0), 300 mM NaCl buffer and then 2 mM DTT was added to the eluate. Protein concentration was determined by Bradford assay (Thermo Scientific) using BSA as a standard. Protein was stored at 4oC and used within 72 h. Isothermal titration calorimetry (ITC) – Experiments were performed on a MicroCal iTC200 titration calorimeter (GE Healthcare). Wild-type IPK1 was purified and dialyzed into ITC buffer, which contained 50 mM HEPES (pH 7.5), 6 mM MgCl2, 150 mM NaCl, 1 mM TCEP (pH 7.0). After dialysis of protein was complete, dialysis buffer was used to dissolve the ligands, IP and AMPPNP (Jena Bioscience). Titration experiments were performed at 25 oC with 100 μM IPK1 and 1 mM AMPPNP in the cell and 1-2 mM of IP in the syringe to ensure a final IP:IPK1 molar ratio of at least 2:1. Titration experiments were performed at least twice for each IP and one set was chosen to represent data. Calorimetric data was analyzed using Origin 7.0 (MicroCal). Data was fitted using a one-site model using Equation