Pools of Independently Cycling Inositol Phosphates Revealed by Pulse Labeling with 18O-Water.

Inositol phosphate kinases (IPKs) sequentially phosphorylate inositol phosphates (IPs) to yield a group of small signaling molecules involved in diverse cellular processes. IPK1 (inositol 1,3,4,5,6-pentakisphosphate 2-kinase) phosphorylates inositol 1,3,4,5,6-pentakisphosphate to inositol 1,2,3,4,5,6-hexakisphosphate; however, the mechanism of IP recognition employed by IPK1 is currently unresolved. We demonstrated previously that IPK1 possesses an unstable N-terminal 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 inositol 1,3,4,5,6-pentakisphosphate in IP binding and IPK1 activation. The 5- and 6-phosphate groups were the most important for IP binding to IPK1, and the 1- and 3-phosphate groups were more important for IPK1 activation than the others. Moreover, we demonstrate that there are three critical residues (Arg-130, Lys-170, and Lys-411) necessary for IPK1 activity. Arg-130 is the only substrate-binding N-terminal 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 C-terminal lobe, and subsequently, (ii) the interaction between the 1-phosphate and Arg-130 stabilizes the N-terminal 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.

Mammalian cells utilize multiple signaling mechanisms to protect against the osmotic stress that accompanies plasma membrane ion transport, solute uptake, and turnover of protein and carbohydrates (Schliess, F., and Haussinger, D. (2002) Biol. Chem. 383, 577-583). Recently, osmotic stress was found to increase synthesis of bisdiphosphoinositol tetrakisphosphate ((PP)2-InsP4), a high energy inositol pyrophosphate (Pesesse, X., Choi, K., Zhang, T., and Shears, S. B. (2004) J. Biol. Chem. 279, 43378-43381). Here, we describe the purification from rat brain of a diphosphoinositol pentakisphosphate kinase (PPIP5K) that synthesizes (PP)2-InsP4. Partial amino acid sequence, obtained by mass spectrometry, matched the sequence of a 160-kDa rat protein containing a putative ATP-grasp kinase domain. BLAST searches uncovered two human isoforms (PPIP5K1 (160 kDa) and PPIP5K2 (138 kDa)). Recombinant human PPIP5K1, expressed in Escherichia coli, was found to phosphorylate diphosphoinositol pentakisphosphate (PP-InsP5) to (PP)2-InsP4 (Vmax = 8.3 nmol/mg of protein/min; Km = 0.34 microM). Overexpression in human embryonic kidney cells of either PPIP5K1 or PPIP5K2 substantially increased levels of (PP)2-InsP4, whereas overexpression of a catalytically dead PPIP5K1(D332A) mutant had no effect. PPIP5K1 and PPIP5K2 were more active against PP-InsP5 than InsP6, both in vitro and in vivo. Analysis by confocal immunofluorescence showed PPIP5K1 to be distributed throughout the cytoplasm but excluded from the nucleus. Immunopurification of overexpressed PPIP5K1 from osmotically stressed HEK cells (0.2 M sorbitol; 30 min) revealed a persistent, 3.9 +/- 0.4-fold activation when compared with control cells. PPIP5Ks are likely to be important signaling enzymes.

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) converts inositol 1,3,4,5,6-pentakisphosphate(IP5) to inositol hexakisphosphate (IP6). IPK1 shares structural similarity with protein kinases and is suspected to employ a similar mechanism of activation. Previous studies revealed roles for the 1- and 3-phosphates of IP5 in IPK1 activation and revealed that the N-lobe of IPK1 is unstable in the absence of inositol phosphate (IP). Here, we demonstrate the link between IPK1 substrate specificity and the stability of its N-lobe. Limited proteolysis of IPK1 revealed that N-lobe stability is dependent on the presence of the 1-phosphate of the substrate, whereas overall stability of IPK1 was increased in ternary complexes with nucleotide and IPs possessing 1- and 3-phosphates that engage the N-lobe of IPK1. Thus, the 1- and 3-phosphates possess dual roles in both IPK1 activation and IPK1 stability. To test whether kinase stability directly contributed to substrate selectivity of the kinase, we engineered IPK1 mutants with disulfide bonds that artificially stabilized the N-lobe in an IP-independent manner thereby mimicking its substrate-bound state in the absence of IP. IPK1 E82C/S142C exhibited a DTT-sensitive 5-fold increase in kcat for 3,4,5,6-inositol tetrakisphosphate (3,4,5,6-IP4) as compared with wild-type IPK1. The crystal structure of the IPK1 E82C/S142C mutant confirmed the presence of the disulfide bond and revealed a small shift in the N-lobe. Finally, we determined that IPK1 E82C/S142C is substantially more stable than wild-type IPK1 under nonreducing conditions, revealing that increased stability of IPK1 E82C/S142C correlates with changes in substrate specificity by allowing IPs lacking the stabilizing 1-phosphate to be used. Taken together, our results show that IPK1 substrate selection is linked to the ability of each potential substrate to stabilize IPK1.

Eukaryotic cells have ubiquitously utilized the myo-inositol backbone to generate a diverse array of signalling molecules. This is achieved by arranging phosphate groups around the six-carbon inositol ring. There is virtually no biological process that does not take advantage of the uniquely variable architecture of phosphorylated inositol. In inositol biology, phosphates are able to form three distinct covalent bonds: phosphoester, phosphodiester and phosphoanhydride bonds, with each providing different properties. The phosphoester bond links phosphate groups to the inositol ring, the variable arrangement of which forms the basis of the signalling capacity of the inositol phosphates. Phosphate groups can also form the structural bridge between myo-inositol and diacylglycerol through the phosphodiester bond. The resulting lipid-bound inositol phosphates, or phosphoinositides, further expand the signalling potential of this family of molecules. Finally, inositol is also notable for its ability to host more phosphates than it has carbons. These unusual organic molecules are commonly referred to as the inositol pyrophosphates (PP-IPs), due to the presence of high-energy phosphoanhydride bonds (pyro- or diphospho-). PP-IPs themselves constitute a varied family of molecules with one or more pyrophosphate moiety/ies located around the inositol. Considering the relationship between phosphate and inositol, it is no surprise that members of the inositol phosphate family also regulate cellular phosphate homoeostasis. Notably, the PP-IPs play a fundamental role in controlling the metabolism of the ancient polymeric form of phosphate, inorganic polyphosphate (polyP). Here we explore the intimate links between phosphate, inositol phosphates and polyP, speculating on the evolution of these relationships.

Inositol phosphates are produced depending on the numbers of the phosphate group which is added to the inositol ring which is 6 membered ring derived from a component of a biological membrane. Inositol 1, 4, 5 trisphosphate (IP3) operates on IP3 receptor on the endoplasmic reticulum, and is related to a release of calcium in the cell. IP3 is associated with various brain functions and neurodegenerative disorders. Moreover, there are IP4, IP5, IP6 and IP7 such as inositol polyphosphates in mammals. Notably, inositol hexakisphoshate kinase (IP6) which phosphorylates IP6 to IP7 plays important roles in the pathophysiology of various neurodegenerative disorders.

Eukaryotes possess numerous inositol phosphate (IP) and diphosphoinositol phosphate (PP-IPs or inositol pyrophosphates) species that act as chemical codes important for intracellular signaling pathways. Production of IP and PP-IP molecules occurs through several classes of evolutionarily conserved inositol phosphate kinases. Here we report the characterization of a human inositol hexakisphosphate (IP6) and diphosphoinositol pentakisphosphate (PP-IP5 or IP7) kinase with similarity to the yeast enzyme Vip1, a recently identified IP6/IP7 kinase (Mulugu, S., Bai, W., Fridy, P. C., Bastidas, R. J., Otto, J. C., Dollins, D. E., Haystead, T. A., Ribeiro, A. A., and York, J. D. (2007) Science 316, 106-109). Recombinant human VIP1 exhibits in vitro IP6 and IP7 kinase activities and restores IP7 synthesis when expressed in mutant yeast. Expression of human VIP1 in HEK293T cells engineered to produce high levels of IP7 results in dramatic increases in bisdiphosphoinositol tetrakisphosphate (PP2-IP4 or IP8). Northern blot analysis indicates that human VIP1 is expressed in a variety of tissues and is enriched in skeletal muscle, heart, and brain. The subcellular distribution of tagged human VIP1 is indicative of a cytoplasmic non-membrane localization pattern. We also characterized human and mouse VIP2, an additional gene product with nearly 90% similarity to VIP1 in the kinase domain, and observed both IP6 and IP7 kinase activities. Our data demonstrate that human VIP1 and VIP2 function as IP6 and IP7 kinases that act along with the IP6K/Kcs1-class of kinases to convert IP6 to IP8 in mammalian cells, a process that has been found to occur in response to various stimuli and signaling events.

. Before describing thisresearch, however, I should say how that choice came about. While in graduate school at theUniversity of Wisconsin, I had had the good fortune to study under Karl Paul Link, who waswidely renowned for his discovery of dicumarol and the synthesis of related blood anticoagu-lants such as warfarin, work that was recognized with two Lasker Awards (1). On the side,however, Link remained a carbohydrate chemist at heart, a hobby that had grown out of hisstudies on plant polysaccharides and uronic acids while a student and then a young facultymember. In fact, Stanford Moore had completed his doctoral dissertation with Link on amethod for characterizing aldo-monosaccharides as benzimidazole derivatives (2).I arrived at Madison in the fall of 1946, fresh from a stint in the United States Navy, andI found Link’s laboratory bursting at the seams with about 15 ex-GIs, all hard at work tryingto make up for lost time. During earlier investigations on the structure-function relationshipof coumarin anticoagulants, an attempt to synthesize the glucoside of dicumarol had beenfrustrated because the acetylated intermediate was degraded in alkali under conditions usedfor deacetylation (3). Because glycosides are acetals, which are typically acid-labile andalkali-stable, I found the anomaly intriguing and decided to study a variety of syntheticcompounds in an effort to understand the structural basis for alkali sensitivity (4). Thisresearch formed the core of my doctoral dissertation, and although I failed to recognize it at thetime, the chance exposure to carbohydrate chemistry was to have a lasting influence on thedirection my career would take.I continued my indoctrination in sugar chemistry during a postdoctoral year in Edinburgh,Scotland, with E. G. V. Percival in the new Department of Chemistry at Kings Buildingsheaded by Edwin Hirst. This was a time of economic depression in Britain, which was stillsuffering the aftermath of the war, and I discovered that I had left a well equipped laboratoryin Madison to engage an unexpectedly primitive research environment. Wisely I did not letthis change in fortunes discourage me. Instead I undertook a project dealing with the structureof maple sapwood starch and did the best that I could with the available facilities (5). Myefforts were well rewarded because, in the process, I became adept at the uses of analytical andpreparative filter paper and cellulose column chromatography, skills that were to be extremelyvaluable in my later research. The greatest challenge to my ingenuity, however, was toconstruct an electric stirring device from a small board-mounted motor, a couple of woodenpulleys, a piece of string, and a glass rod. The speed of the motor was regulated by adjustinglight bulbs that were wired in series with the power cord to draw off electricity, a crude buteffective method of control. I have always enjoyed working with my hands, so this mundaneproject even took on a certain appeal.Living in a new environment always has its fringe benefits. While in Edinburgh, I developeda special affection for the Scots and a better understanding for the lingering resentment that

Many plasma membrane proteins destined for endocytosis are concentrated into clathrin-coated pits through the recognition of a tyrosine-based motif in their cytosolic domains by an adaptor (AP-2) complex. The mu2 subunit of isolated AP-2 complexes binds specifically, but rather weakly, to proteins bearing the tyrosine-based signal. We now demonstrate, using peptides with a photoreactive probe, that this binding is strengthened significantly when the AP-2 complex is present in clathrin coats, indicating that there is cooperativity between receptor-AP-2 interactions and coat formation. Phosphoinositides with a phosphate at the D-3 position of the inositol ring, but not other isomers, also increase the affinity of the AP-2 complex for the tyrosine-based motif. AP-2 is the first protein known (in any context) to interact with phosphatidylinositol 3-phosphate. Our findings indicate that receptor recruitment can be coupled to clathrin coat assembly and suggest a mechanism for regulation of membrane traffic by lipid products of phosphoinositide 3-kinases.

Protein kinase CK2 is a ubiquitous protein kinase that can phosphorylate various proteins involved in central cellular processes, such as signal transduction, cell division, and proliferation. We have shown that the human nucleolar phosphoprotein p140 (hNopp140) is able to regulate the catalytic activity of CK2. Unphosphorylated hNopp140 and phospho-hNopp140 bind to the regulatory and catalytic subunits of CK2, respectively, and the interaction between hNopp140 and CK2 was prevented by inositol hexakisphosphate (InsP(6)). Phosphorylation of alpha-casein, genimin, or human phosphatidylcholine transfer protein-like protein by CK2 was inhibited by hNopp140, and InsP(6) recovered the suppressed activity of CK2 by hNopp140. These observations indicated that hNopp140 serves as a negative regulator of CK2 and that InsP(6) stimulates the activity of CK2 by blocking the interaction between hNopp140 and CK2.

Inositol pyrophosphate diphosphoinositol pentakisphosphate is ubiquitously present in mammalian cells and contains highly energetic pyrophosphate bonds. We have previously reported that inositol hexakisphosphate kinase type 2 (InsP(6)K2), which converts inositol hexakisphosphate to inositol pyrophosphate diphosphoinositol pentakisphosphate, mediates apoptotic cell death via its translocation from the nucleus to the cytoplasm. Here, we report that InsP(6)K2 is localized mainly in the cytoplasm of lymphoblast cells from patients with Huntington disease (HD), whereas this enzyme is localized in the nucleus in control lymphoblast cells. The large number of autophagosomes detected in HD lymphoblast cells is consistent with the down-regulation of Akt in response to InsP(6)K2 activation. Consistent with these observations, the overexpression of InsP(6)Ks leads to the depletion of Akt phosphorylation and the induction of cell death. These results suggest that InsP(6)K2 activation is associated with the pathogenesis of HD.

ITPK1 is an InsP6/ADP phosphotransferase that controls phosphate signaling in Arabidopsis

Insulin Signaling: Inositol Phosphates Get into the Akt

The Role of Phosphatases in Inositol Signaling Reactions

Inositol Hexakisphosphate Kinase Products Contain Diphosphate and Triphosphate Groups

The endogenous cyclic adenosine monophosphate (AMP) antagonist, cyclic PIP, has been identified as a prostaglandylinositol cyclic phosphate. It inhibits protein kinase A 100% and activates protein serine phosphatase about sevenfold. It is biosynthesized by an enzyme of the plasma membrane when the assay mixture contains adenosine triphosphate (ATP), Mg2+, prostaglandin E and a novel inositol polyphosphate, which cannot be substituted by commercially available inositol phosphates. This novel inositol polyphosphate is a very labile compound. On anion exchange chromatography it elutes in the range of ATP, which may indicate the presence of three phosphate groups. It adsorbs on charcoal, which suggests the presence of a hydrophobic component, possibly a guanosine. Pyrophosphates obtained from inositol 1,4- and inositol 2,4-bisphosphate are accepted by cyclic PIP synthetase for the synthesis of cyclic PIP. The biosynthesis is characterized by enzyme kinetic parameters like dependence on time, enzyme and substrate concentration. The pH optimum of the enzyme is in the range 7.5-8. The enzyme functions optimally with prostaglandin E and poorly with prostaglandin A as the substrate. The presence of fluoride in the assay causes a three- to fourfold increase in cyclic PIP synthesis, which may be correlated with activation via G proteins. These data support previous reports on the chemical structure and action of cyclic PIP. With respect to the possible isomers of cyclic PIP, these indicate that it is most likely the C4-hydroxyl group of the inositol which binds the C15-hydroxyl group of prostaglandin E. A model of hormone-stimulated synthesis of cyclic PIP is proposed: phospholipase A2 and phospholipase C, activated by G proteins upon alpha-adrenergic stimulation, liberate either unsaturated fatty acids or inositol phosphates, which are transformed to prostaglandins and to novel inositol polyphosphate with an energy-rich bond. The cyclic PIP synthetase combines these two substrates to cyclic PIP.