Marina V. Kisseleva

The role of phosphatases in inositol signaling reactions.

The phosphatidylinositol signaling pathway employs a host of kinases and phosphatases that form and degrade the many signaling molecules that act in this system. The complexity and robustness of the system are indicated by the fact that there are six inositol phospholipids and more than 20 soluble inositol phosphates that have been found in mammalian cells (1). The system is present in all eukaryotic cells in one form or another. This review will focus on a few of the phosphatases as they exist in mammalian cells. In general, the inositol phosphatases are analogous to protein phosphatases in that they both tend to inhibit or terminate signaling reactions. There are specific exceptions to this general rule in both systems. Numerous inositol phosphatases have been discovered and characterized in the past 15 years. Surprisingly, in many cases the same enzymes hydrolyze phosphate from both water-soluble inositol phosphates and the corresponding lipids with the same arrangement of phosphate groups.

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.

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.

The LIM Protein Ajuba Regulates Phosphatidylinositol 4,5-Bisphosphate Levels in Migrating Cells through an Interaction with and Activation of PIPKIα

ABSTRACT The phosphoinositide phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] regulates the activity of many actin-binding proteins and as such is an important modulator of cytoskeleton organization during cell migration, for example. In migrating cells actin remodeling is tightly regulated and localized; therefore, how the PI(4,5)P2 level is spatially and temporally regulated is crucial to understanding how it controls cell migration. Here we show that the LIM protein Ajuba contributes to the cellular regulation of PI(4,5)P2 levels by interacting with and activating the enzymatic activity of the PI(4)P 5-kinase (PIPKIα), the predominant enzyme in the synthesis of PI(4,5)P2, in a migration stimulus-regulated manner. In migrating primary mouse embryonic fibroblasts (MEFs) from Ajuba−/− mice the level of PI(4,5)P2 was decreased with a corresponding increase in the level of the substrate PI(4)P. Reintroduction of Ajuba into these cells normalized PI(4,5)P2 levels. Localization of PI(4,5)P2 synthesis and PIPKIα in the leading lamellipodia and membrane ruffles, respectively, of migrating Ajuba−/− MEFs was impaired. In vitro, Ajuba dramatically activated the enzymatic activity of PIPKIα while inhibiting the activity of PIPKIIβ. Thus, in addition to its effects upon Rac activity Ajuba can also influence cell migration through regulation of PI(4,5)P2 synthesis through direct activation of PIPKIα enzyme activity.

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.

Phospholipase C Gamma 2 Is Critical for Development of a Murine Model of Inflammatory Arthritis by Affecting Actin Dynamics in Dendritic Cells

Background Dendritic cells (DCs) are highly specialized cells, which capture antigen in peripheral tissues and migrate to lymph nodes, where they dynamically interact with and activate T cells. Both migration and formation of DC-T cell contacts depend on cytoskeleton plasticity. However, the molecular bases governing these events have not been completely defined. Methodology/Principal Findings Utilizing a T cell-dependent model of arthritis, we find that PLCγ2−/− mice are protected from local inflammation and bone erosion. PLCγ2 controls actin remodeling in dendritic cells, thereby affecting their capacity to prime T cells. DCs from PLCγ2−/− mice mature normally, however they lack podosomes, typical actin structures of motile cells. Absence of PLCγ2 impacts both DC trafficking to the lymph nodes and migration towards CCL21. The interaction with T cells is also affected by PLCγ2 deficiency. Mechanistically, PLCγ2 is activated by CCL21 and modulates Rac activation. Rac1/2−/− DCs also lack podosomes and do not respond to CCL21. Finally, antigen pulsed PLCγ2−/− DCs fail to promote T cell activation and induce inflammation in vivo when injected into WT mice. Conversely, injection of WT DCs into PLCγ2−/− mice rescues the inflammatory response but not focal osteolysis, confirming the importance of PLCγ2 both in immune and bone systems. Conclusions/Significance This study demonstrates a critical role for PLCγ2 in eliciting inflammatory responses by regulating actin dynamics in DCs and positions the PLCγ2 pathway as a common orchestrator of bone and immune cell functions during arthritis.

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.