Monita P. Wilson

The Activation Loop of Phosphatidylinositol Phosphate Kinases Determines Signaling Specificity

Phosphatidylinositol-4,5-bisphosphate plays a pivotal role in the regulation of cell proliferation and survival, cytoskeletal reorganization, and membrane trafficking. However, little is known about the temporal and spatial regulation of its synthesis. Higher eukaryotic cells have the potential to use two distinct pathways for the generation of phosphatidylinositol-4,5-bisphosphate. These pathways require two classes of phosphatidylinositol phosphate kinases, termed type I and type II PIP kinases. While highly related by sequence, these kinases localize to different subcellular compartments, phosphorylate distinct substrates, and are functionally nonredundant. Here, we show that a 20- to 25-amino acid loop spanning the catalytic site, termed the activation loop, determines both enzymatic specificity and subcellular targeting of PIP kinases. Therefore, the activation loop controls signaling specificity and PIP kinase function at multiple levels.

The synthesis of inositol hexakisphosphate. Characterization of human inositol 1,3,4,5,6-pentakisphosphate 2-kinase.

The enzyme(s) responsible for the production of inositol hexakisphosphate (InsP6) in vertebrate cells are unknown. In fungal cells, a 2-kinase designated Ipk1 is responsible for synthesis of InsP6 by phosphorylation of inositol 1,3,4,5,6-pentakisphosphate (InsP5). Based on limited conserved sequence motifs among five Ipk1 proteins from different fungal species, we have identified a human genomic DNA sequence on chromosome 9 that encodes human inositol 1,3,4,5,6-pentakisphosphate 2-kinase (InsP5 2-kinase). Recombinant human enzyme was produced in Sf21 cells, purified, and shown to catalyze the synthesis of InsP6 or phytic acid in vitro. The recombinant protein converted 31 nmol of InsP5 to InsP6/min/mg of protein (V max). The Michaelis-Menten constant for InsP5 was 0.4 μm and for ATP was 21 μm. Saccharomyces cerevisiae lackingIPK1 do not produce InsP6 and show lethality in combination with a gle1 mutant allele. Here we show that expression of the human InsP5 2-kinase in a yeastipk1 null strain restored the synthesis of InsP6 and rescued the gle1–2 ipk1–4 lethal phenotype. Northern analysis on human tissues showed expression of the human InsP5 2-kinase mRNA predominantly in brain, heart, placenta, and testis. The isolation of the gene responsible for InsP6 synthesis in mammalian cells will allow for further studies of the InsP6 signaling functions.

Isolation of inositol 1,3,4-trisphosphate 5/6-kinase, cDNA cloning and expression of the recombinant enzyme.

Inositol 1,3,4-trisphosphate 5/6-kinase was purified 12,900-fold from calf brain using chromatography on heparin-agarose and affinity elution with inositol hexakisphosphate. The final preparation contained proteins of 48 and 36-38 kDa. All of these proteins had the same amino-terminal sequence and were enzymatically active. The smaller species represent proteolysis products with carboxyl-terminal truncation. The Kof the enzyme for inositol 1,3,4-trisphosphate was 80 nM with a V of 60 nmol of product/min/mg of protein. The amino acid sequence of the tryptic peptide HSKLLARPAGGLVGERTCNAXP matched the protein sequence encoded by a human expressed sequence tag clone (GB T09063) at 16 of 22 residues. The expressed sequence tag clone was used to screen a human fetal brain cDNA library to obtain a cDNA clone of 1991 base pairs (bp) that predicts a protein of 46 kDa. The clone encodes the amino-terminal amino acid sequence obtained from the purified calf brain preparation, suggesting that it represents its human homologue. The cDNA was expressed as a fusion protein in Escherichia coli and was found to have inositol 1,3,4-trisphosphate 5/6-kinase activity. Remarkably, both the purified calf brain and recombinant proteins produced both inositol 1,3,4,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate as products in a ratio of 2.3-5:1. This finding proves that a single kinase phosphorylates inositol in both the D5 and D6 positions. Northern blot analysis identified a transcript of 3.6 kilobases in all tissues with the highest levels in brain. The composite cDNA isolated contains 3054 bp with a poly(A) tail, suggesting that 500-600 bp of 5′ sequence remains to be identified.

Type I phosphatidylinositol-4,5-bisphosphate 4-phosphatase regulates stress-induced apoptosis

A recently discovered phosphatidylinositol monophosphate, phosphatidylinositol 5-phosphate (PtdIns-5-P), plays an important role in nuclear signaling by influencing p53-dependent apoptosis. It interacts with a plant homeodomain finger of inhibitor of growth protein-2, causing an increase in the acetylation and stability of p53. Here we show that type I phosphatidylinositol-4,5-bisphosphate 4-phosphatase (type I 4-phosphatase), an enzyme that dephosphorylates phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2), forming PtdIns-5-P in vitro, can increase the cellular levels of PtdIns-5-P. When HeLa cells were treated with the DNA-damaging agents etoposide or doxorubicin, type I 4-phosphatase translocated to the nucleus and nuclear levels of PtdIns-5-P increased. This action resulted in increased p53 acetylation, which stabilized p53, leading to increased apoptosis. Overexpression of type I 4-phosphatase increased apoptosis, whereas RNAi of the enzyme diminished it. The half-life of p53 was shortened from 7 h to 1.8 h upon RNAi of type I 4-phosphatase. This enzyme therefore controls nuclear levels of PtdIns-5-P and thereby p53-dependent apoptosis.

Myotubularin-related protein (MTMR) 9 determines the enzymatic activity, substrate specificity, and role in autophagy of MTMR8

The myotubularins are a large family of inositol polyphosphate 3-phosphatases that, despite having common substrates, subsume unique functions in cells that are disparate. The myotubularin family consists of 16 different proteins, 9 members of which possess catalytic activity, dephosphorylating phosphatidylinositol 3-phosphate [PtdIns(3)P] and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] at the D-3 position. Seven members are inactive because they lack the conserved cysteine residue in the CX5R motif required for activity. We studied a subfamily of homologous myotubularins, including myotubularin-related protein 6 (MTMR6), MTMR7, and MTMR8, all of which dimerize with the catalytically inactive MTMR9. Complex formation between the active myotubularins and MTMR9 increases their catalytic activity and alters their substrate specificity, wherein the MTMR6/R9 complex prefers PtdIns(3,5)P2 as substrate; the MTMR8/R9 complex prefers PtdIns(3)P. MTMR9 increased the enzymatic activity of MTMR6 toward PtdIns(3,5)P2 by over 30-fold, and enhanced the activity toward PtdIns(3)P by only 2-fold. In contrast, MTMR9 increased the activity of MTMR8 by 1.4-fold and 4-fold toward PtdIns(3,5)P2 and PtdIns(3)P, respectively. In cells, the MTMR6/R9 complex significantly increases the cellular levels of PtdIns(5)P, the product of PI(3,5)P2 dephosphorylation, whereas the MTMR8/R9 complex reduces cellular PtdIns(3)P levels. Consequentially, the MTMR6/R9 complex serves to inhibit stress-induced apoptosis and the MTMR8/R9 complex inhibits autophagy.

Neural tube defects in mice with reduced levels of inositol 1,3,4-trisphosphate 5/6-kinase

Inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) is a key regulatory enzyme at the branch point for the synthesis of inositol hexakisphosphate (IP6), an intracellular signaling molecule implicated in the regulation of ion channels, endocytosis, exocytosis, transcription, DNA repair, and RNA export from the nucleus. IP6 also has been shown to be an integral structural component of several proteins. We have generated a mouse strain harboring a β-galactosidase (βgal) gene trap cassette in the second intron of the Itpk1 gene. Animals homozygous for this gene trap are viable, fertile, and produce less ITPK1 protein than wild-type and heterozygous animals. Thus, the gene trap represents a hypomorphic rather than a null allele. Using a combination of immunohistochemistry, in situ hybridization, and βgal staining of mice heterozygous for the hypomorphic allele, we found high expression of Itpk1 in the developing central and peripheral nervous systems and in the paraxial mesoderm. Examination of embryos resulting from homozygous matings uncovered neural tube defects (NTDs) in some animals and axial skeletal defects or growth retardation in others. On a C57BL/6 × 129(P2)Ola background, 12% of mid-gestation embryos had spina bifida and/or exencephaly, whereas wild-type animals of the same genetic background had no NTDs. We conclude that ITPK1 is required for proper development of the neural tube and axial mesoderm.

The role of inositol signaling in the control of apoptosis

Inositol signaling reactions are very broad in scope affecting many cellular functions. In this report we describe experiments showing that two distinct parts of this system play pivotal roles in an important cellular event, namely apoptosis. Apoptosis is important for organ development and also for controlling cell survival after various stresses which include DNA damage and other proapoptotic stimuli such as tumor necrosis factor α. We show that the inositol phosphate InsP6 or one of its pyrophosphate metabolites determines the extent of apoptosis following tumor necrosis factor α treatment whereby increased cellular levels of InsP6 protect from apoptosis and decreased levels promote it. Cellular levels of InsP6 are determined by the activity of 5/6-kinase since this is the rate limiting enzyme in production of the highly phosphorylated inositol phosphates including InsP6. A lipid inositol metabolite PtdIns5P is also critical in regulating the activity of p53-dependent apoptosis. This phospholipid is formed in cells by the action of type I 4-phosphatase on PtdIns(4,5)P2. PtdIns5P stabilizes p53 by promoting its acetylation in complex with the nuclear factor ING2. Upon genotoxic stress type I 4-phosphatase migrates to the nucleus where it catalyzes the formation of PtdIns5P to stabilize p53 and increase apoptosis.

Regulation of inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) by reversible lysine acetylation

The enzyme inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) catalyzes the rate-limiting step in the formation of higher phosphorylated forms of inositol in mammalian cells. Because it sits at a key regulatory point in the inositol metabolic pathway, its activity is likely to be regulated. We have previously shown that ITPK1 is phosphorylated, a posttranslational modification used by cells to regulate enzyme activity. We show here that ITPK1 is modified by acetylation of internal lysine residues. The acetylation sites, as determined by mass spectrometry, were found to be lysines 340, 383, and 410, which are all located on the surface of this protein. Overexpression of the acetyltransferases CREB-binding protein or p300 resulted in the acetylation of ITPK1, whereas overexpression of mammalian silent information regulator 2 resulted in the deacetylation of ITPK1. Functionally, ITPK1 acetylation regulates its stability. CREB-binding protein dramatically decreased the half-life of ITPK1. We further found that ITPK1 acetylation down-regulated its enzyme activity. HEK293 cells stably expressing acetylated ITPK1 had reduced levels of the higher phosphorylated forms of inositol, compared with the levels seen in cells expressing unacetylated ITPK1. These results demonstrate that lysine acetylation alters both the stability as well as the activity of ITPK1 in cells.

Expression of inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) and its role in neural tube defects

ITPK1 is the rate limiting enzyme in the pathway leading to formation of the highly phosphorylated inositol phosphates including IP6 and the inositol pyrophosphates. One or more of these metabolites are essential for life as deletion of either of the kinases that form IP5 or IP6 in mice results in embryonic lethality. We have produced mice harboring a hypomorphic allele for Itpk1, and mice homozygous for this gene trap allele produce low but detectable levels of active enzyme. We have studied the expression of Itpk1 in various tissues and found that the enzyme is highly expressed in smooth muscle of vessels and other tissues. In addition, these mice have neural tube defects in 12% of homozygous embryos. Since the levels of enzyme expression vary greatly in homozygous animals, we speculate that relative deficiency of one or more inositol phosphates accounts for these defects. We plan to feed an inositol deficient diet or one with supplemental inositol to animals to demonstrate altered prevalence of neural tube defects.

The role of inositol polyphosphate 4-phosphatase 1 in platelet function using a weeble mouse model

Platelet activation plays an important role in the development and course of cardiovascular disease. It is triggered by the interaction of subendothelial matrix-bound and/or soluble agonists with platelet surface receptors causing a series of morphological and biochemical changes leading to the recruitment of additional platelets and formation of stable platelet aggregates. In addition to events causing initial activation and recruitment of platelets, signaling continues post-aggregation that promotes stability of the thrombus (Brass et al., 2004). Platelet levels of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) increase dramatically in response to agonist stimulation in an aggregation-dependent manner (Kucera et al., 1990; Nolan et al., 1990; Sultan et al., 1990). Furthermore the increase in platelet PtdIns(3,4)P2 occurs late in platelet aggregation and correlates with the irreversible phase of platelet aggregation (Sorisky et al., 1992; Sultan et al., 1991; Trumel et al., 1999), suggesting that PtdIns(3,4)P2 mediates the stabilization of platelet aggregates. However little is known about the regulation of PtdIns(3,4)P2 in platelets. PtdIns(3,4)P2 can be formed by three different routes 1) by direct phosphorylation of PtdIns(4)P by phosphatidylinositol 3-kinase (EC2.7.1.153) (PI 3-K) 2) by PI 3-K using PtdIns(4,5)P2 as a substrate followed by dephosphorylation by a 5-phosphatase (EC3.1.3.56) and, 3) by the action of type I PtdIns(4)P phosphate kinase (EC2.7.1.68) using PI 3-K as substrate (Zhang et al., 1998), shown diagramatically in figure 1. It is well documented that different PI 3-K isoforms play important roles in both early and later stages of platelet aggregation (Jackson et al., 2006). It has been shown that that pharmacologic inhibition of PI 3-K prevents agonist-induced formation of PtdIns(3,4)P2 (Kovacsovics et al., 1995; Schoenwaelder et al., 2007). Furthermore addition of PI 3-K inhibitors after the onset of platelet aggregation induces a decline in PtdIns(3,4)P2 and disaggregation of platelets, supporting a role for PtdIns(3,4)P2 in the stabilization of aggregates. However, platelet agonist-dependent activation of PI 3-K mediates an increase in both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 making approaches using pharmacologic inhibition or genetic disruption of PI 3-K unsuitable to distinguish the contributions of the individual 3-phosphorylated phosphoinositides to platelet signaling. The levels of inositol lipids in cells and their distribution in discrete cellular compartments are regulated by the balance of the enzymatic activities of kinases and phosphatases. Figure 1 Pathways for the formation of PtdIns (3,4)P2. The major route of PtdIns(3,4)P2 hydrolysis is the removal of the D4 phosphate by the enzymes cloned and characterized in our lab (Norris et al., 1997; Norris et al., 1995), inositol polyphosphate 4-phosphatase type I (EC3.1.3.40) and type II (EC3.1.3.66) that are magnesium-independent phosphatases. Over their entire sequence type I and type II 4-phosphatases are 37% identical (Norris et al., 1997). The active site region is more highly conserved, and contains a consensus sequence found in other magnesium-independent phosphatases (Zhang et al., 1994). The consensus sequence CX5 RT/S, is conserved throughout 4-ptases from C. elegans to humans as shown in figure 2. These enzymes do not catalyze the hydrolysis of lipids other than PtdIns(3,4)P2 and therefore provide unique means for the study of this lipid in platelet activation. We have shown that 4-ptase I forms a complex with PI 3-K in platelets which localizes the complex to sites of PtdIns(3,4)P2 production (Munday et al., 1999). Figure 2 Alignment of active sites of 4ptases. We postulate that PtdIns(3,4)P2 is important for platelet function and will study this using a mouse model. We previously showed that an antibody that reacts with 4-phosphatases immunoprecipitates all of the PtdIns(3,4)P2 hydrolyzing activity from human platelets (Munday et al., 1999). An early indication that PtdIns(3,4)P2 was important for platelet function was the work of Norris (Norris et al., 1997). It was shown that calpain caused degradation of recombinant 4-phosphatase I in vitro thereby inactivating it. It was also shown that activation of human platelets with either calcium ionophore or thrombin led to proteolysis of endogenous platelet 4-phosphatase I. If calpeptin, a cell-permeable inhibitor of calpain, was included in these experiments no proteolysis was seen. The levels of PtdIns(3,4)P2 in platelets were lower when calpeptin was included, indicating that 4-phosphatase I was important for controlling the levels of PtdIns(3,4)P2 during platelet activation. A naturally occurring mutation in type I 4-phosphatase is a single nucleotide deletion which is found in the weeble mouse. These animals suffer from severe neurodegeneration and die within the first weeks of life. Therefore such mutant mice cannot be used to study platelet function. We circumvented this problem by creating chimeric mice by bone marrow transplantation of weeble fetal liver cells into lethally irradiated wild type mice. These mice lack 4-phosphatase in bone marrow derived cells including platelets. The mice are viable, but lack platelet 4-phosphatase I.