John D. York

SAC1-like Domains of Yeast SAC1,INP52, and INP53 and of Human Synaptojanin Encode Polyphosphoinositide Phosphatases

The SAC1 gene product has been implicated in the regulation of actin cytoskeleton, secretion from the Golgi, and microsomal ATP transport; yet its function is unknown. Within SAC1 is an evolutionarily conserved 300-amino acid region, designated a SAC1-like domain, that is also present at the amino termini of the inositol polyphosphate 5-phosphatases, mammalian synaptojanin, and certain yeast INP5 gene products. Here we report that SAC1-like domains have intrinsic enzymatic activity that defines a new class of polyphosphoinositide phosphatase (PPIPase). Purified recombinantSAC1-like domains convert yeast lipids phosphatidylinositol (PI) 3-phosphate, PI 4-phosphate, and PI 3,5-bisphosphate to PI, whereas PI 4,5-bisphosphate is not a substrate. Yeast lacking Sac1p exhibit 10-, 2.5-, and 2-fold increases in the cellular levels of PI 4-phosphate, PI 3,5-bisphosphate, and PI 3-phosphate, respectively. The 5-phosphatase domains of synaptojanin, Inp52p, and Inp53p are also catalytic, thus representing the first examples of an inositol signaling protein with two distinct lipid phosphatase active sites within a single polypeptide chain. Together, our data provide a long sought mechanism as to how defects in Sac1p overcome certain actin mutants and bypass the requirement for yeast phosphatidylinositol/phosphatidylcholine transfer protein, Sec14p. We demonstrate that PPIPase activity is a key regulator of membrane trafficking and actin cytoskeleton organization and suggest signaling roles for phosphoinositides other than PI 4,5-bisphosphate in these processes. Additionally, the tethering of PPIPase and 5-phosphatase activities indicate a novel mechanism by which concerted phosphoinositide hydrolysis participates in membrane trafficking.

Tyrosine 1101 of Tie2 Is the Major Site of Association of p85 and Is Required for Activation of Phosphatidylinositol 3-Kinase and Akt

ABSTRACT Tie2 is an endothelium-specific receptor tyrosine kinase that is required for both normal embryonic vascular development and tumor angiogenesis and is thought to play a role in vascular maintenance. However, the signaling pathways responsible for the function of Tie2 remain unknown. In this report, we demonstrate that the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) associates with Tie2 and that this association confers functional lipid kinase activity. Mutation of tyrosine 1101 of Tie2 abrogated p85 association both in vitro and in vivo in yeast. Tie2 was found to activate PI3-kinase in vivo as demonstrated by direct measurement of increases in cellular phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate, by plasma membrane translocation of a green fluorescent protein-Akt pleckstrin homology domain fusion protein, and by downstream activation of the Akt kinase. Activation of PI3-kinase was abrogated in these assays by mutation of Y1101 to phenylalanine, consistent with a requirement for this residue for p85 association with Tie2. These results suggest that activation of PI3-kinase and Akt may in part account for Tie2’s role in both embryonic vascular development and pathologic angiogenesis, and they are consistent with a role for Tie2 in endothelial cell survival.

A new link between the c-Abl tyrosine kinase and phosphoinositide signalling through PLC-γ1

The c-Abl tyrosine (Tyr) kinase is activated after platelet-derived-growth factor receptor (PDGFR) stimulation in a manner that is partially dependent on Src kinase activity. However, the activity of Src kinases alone is not sufficient for activation of c-Abl by PDGFR. Here we show that functional phospholipase C-γ1 (PLC-γ1) is required for c-Abl activation by PDGFR. Decreasing cellular levels of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) by PLC-γ1-mediated hydrolysis or dephosphorylation by an inositol polyphosphate 5-phosphatase (Inp54) results in increased Abl kinase activity. c-Abl functions downstream of PLC-γ1, as expression of kinase-inactive c-Abl blocks PLC-γ1-induced chemotaxis towards PDGF-BB. PLC-γ1 and c-Abl form a complex in cells that is enhanced by PDGF stimulation. After activation, c-Abl phosphorylates PLC-γ1 and negatively modulates its function in vivo. These findings uncover a newly discovered functional interdependence between non-receptor Tyr kinase and lipid signalling pathways.

Specificity Determinants in Phosphoinositide Dephosphorylation: Crystal Structure of an Archetypal Inositol Polyphosphate 5-Phosphatase

Inositol polyphosphate 5-phosphatases are central to intracellular processes ranging from membrane trafficking to Ca(2+) signaling, and defects in this activity result in the human disease Lowe syndrome. The 1.8 resolution structure of the inositol polyphosphate 5-phosphatase domain of SPsynaptojanin bound to Ca(2+) and inositol (1,4)-bisphosphate reveals a fold and an active site His and Asp pair resembling those of several Mg(2+)-dependent nucleases. Additional loops mediate specific inositol polyphosphate contacts. The 4-phosphate of inositol (1,4)-bisphosphate is misoriented by 4.6 compared to the reactive geometry observed in the apurinic/apyrimidinic endonuclease 1, explaining the dephosphorylation site selectivity of the 5-phosphatases. Based on the structure, a series of mutants are described that exhibit altered substrate specificity providing general determinants for substrate recognition.

Structural Analysis and Detection of Biological Inositol Pyrophosphates Reveal That the Family of VIP/Diphosphoinositol Pentakisphosphate Kinases Are 1/3-Kinases

We have characterized the positional specificity of the mammalian and yeast VIP/diphosphoinositol pentakisphosphate kinase (PPIP5K) family of inositol phosphate kinases. We deployed a microscale metal dye detection protocol coupled to a high performance liquid chromatography system that was calibrated with synthetic and biologically synthesized standards of inositol pyrophosphates. In addition, we have directly analyzed the structures of biological inositol pyrophosphates using two-dimensional 1H-1H and 1H-31P nuclear magnetic resonance spectroscopy. Using these tools, we have determined that the mammalian and yeast VIP/PPIP5K family phosphorylates the 1/3-position of the inositol ring in vitro and in vivo. For example, the VIP/PPIP5K enzymes convert inositol hexakisphosphate to 1/3-diphosphoinositol pentakisphosphate. The latter compound has not previously been identified in any organism. We have also unequivocally determined that 1/3,5-(PP)2-IP4 is the isomeric structure of the bis-diphosphoinositol tetrakisphosphate that is synthesized by yeasts and mammals, through a collaboration between the inositol hexakisphosphate kinase and VIP/PPIP5K enzymes. These data uncover phylogenetic variability within the crown taxa in the structures of inositol pyrophosphates. For example, in the Dictyostelids, the major bis-diphosphoinositol tetrakisphosphate is 5,6-(PP)2-IP4 ( Laussmann, T., Eujen, R., Weisshuhn, C. M., Thiel, U., Falck, J. R., and Vogel, G. (1996) Biochem. J. 315, 715-725 ). Our study brings us closer to the goal of understanding the structure/function relationships that control specificity in the synthesis and biological actions of inositol pyrophosphates.

Roles of inositol phosphates and inositol pyrophosphates in development, cell signaling and nuclear processes.

Inositol phosphates and inositol pyrophosphates are small molecule metabolites that play important roles in nuclear processes such as transcription control, mRNA export and DNA repair. On this wonderful occasion of the fiftieth anniversary of the Advances in Enzyme Regulation conference, it is a privilege to participate through presenting recent developments in the area of inositol phosphate and pyrophosphate regulatory biology. This article summarizes recent advances in understanding IPs and PP-IPs biology in development and nuclear cell signaling. Data obtained from various model organisms hint at the emerging modes of mechanisms of these versatile molecules. Studies in model organisms revealed that IPs and PP-IPs are critical for development and signaling in plants, mice, zebrafish and slime mold. In plants, IP molecules are required for signaling in response to stress, hormone, and nutrient, by transcriptional regulation of the stimuli-responsive genes. In some cases, IPs act as regulatory molecules as the levels of IPKs/IPs are induced by those stimuli and a constitutively induction of downstream events can be accomplished by over-expressing the IPKs. Nutrient sensing is a recurring theme in IP biology as IPs and PP-IPs are also involved in amino acid and phosphate signaling in yeast and possible glucose sensing in pancreatic β cells, indicating it maybe acquired early during evolution. There are some newly discovered nuclear roles of IPs: 1) binding to and possibly regulation of the SCFTIR1 ubiquitin ligase complex, and 2) RNA editing by acting as structural co-factor of ADAR2 and ADAT1. On the other hand, IPs and PP-IPs are also implicated in some novel non-nuclear processes: 1) insulin secretion, 2) negative regulation of PIP3 signaling and 3) modulation of intracellular Ca2+ concentration. Following the discovery of new biological roles of IPs and PP-IPs, a few new receptors for IPs and PP-IPs were identified in the process. Three modes of receptor binding by IPs and PP-IPs can be summarized: 1) Direct binding to the receptor (e.g. TIR1), 2) displacement of a ligand (e.g. PIP3) already bound to the receptor (e.g. PH domain containing proteins, and 3) acting as a structural co-factor and possibly incorporated inside the receptor during protein folding (e.g. ADAR2). Hopefully, more experimentation will uncover more receptors for and biological roles of these versatile molecules.

Cloning and characterization of two human VIP1-like inositol hexakisphosphate and diphosphoinositol pentakisphosphate kinases.

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.

The endothelial receptor tyrosine kinase Tie1 activates phosphatidylinositol 3-kinase and Akt to inhibit apoptosis.

ABSTRACT Tie1 is an orphan receptor tyrosine kinase that is expressed almost exclusively in endothelial cells and that is required for normal embryonic vascular development. Genetic studies suggest that Tie1 promotes endothelial cell survival, but other studies have suggested that the Tie1 kinase has little to no activity, and Tie1-mediated signaling pathways are unknown. To begin to study Tie1 signaling, a recombinant glutathione S-transferase (GST)-Tie1 kinase fusion protein was produced in insect cells and found to be autophosphorylated in vitro. GST-Tie1 but not a kinase-inactive mutant associated with a recombinant p85 SH2 domain protein in vitro, suggesting that Tie1 might signal through phosphatidylinositol (PI) 3-kinase. To study Tie1 signaling in a cellular context, a c-fms-Tie1 chimeric receptor (fTie1) was expressed in NIH 3T3 cells. Ligand stimulation of fTie1 resulted in Tie1 autophosphorylation and downstream activation of PI 3-kinase and Akt. Stimulation of fTie1-expressing cells potently inhibited UV irradiation-induced apoptosis in a PI 3-kinase-dependent manner. Moreover, both Akt phosphorylation and inhibition of apoptosis were abrogated by mutation of tyrosine 1113 to phenylalanine, suggesting that this residue is an important PI 3-kinase binding site. These findings are the first biochemical demonstration of a signal transduction pathway and corresponding cellular function for Tie1, and the antiapoptotic effect of Tie1 is consistent with the results of previous genetic studies.

Cloning and Characterization of a Mammalian Lithium-sensitive Bisphosphate 3′-Nucleotidase Inhibited by Inositol 1,4-Bisphosphate

Discovery of a structurally conserved metal-dependent lithium-inhibited phosphomonoesterase protein family has identified several potential cellular targets of lithium as used to treat manic depression. Here we describe identification of a novel family member using a “computer cloning” strategy. Human and murine cDNA clones encoded proteins sharing 92% identity and were highly expressed in kidney. Native and recombinant protein harbored intrinsic magnesium-dependent bisphosphate nucleotidase activity (BPntase), which removed the 3′-phosphate from 3′-5′ bisphosphate nucleosides and 3′-phosphoadenosine 5′-phosphosulfate with K m andV max values of 0.5 μm and 40 μmol/min/mg. Lithium uncompetitively inhibited activity with aK i of 157 μm. Interestingly, BPntase was competitively inhibited by inositol 1,4-bisphosphate with aK i of 15 μm. Expression of mammalian BPntase complemented defects in hal2/met22 mutant yeast. These data suggest that BPntase’s physiologic role in nucleotide metabolism may be regulated by inositol signaling pathways. The presence of high levels of BPntase in the kidney are provocative in light of the roles of bisphosphorylated nucleotides in regulating salt tolerance, sulfur assimilation, detoxification, and lithium toxicity. We propose that inhibition of human BPntase may account for lithium-induced nephrotoxicity, which may be overcome by supplementation of current therapeutic regimes with inhibitors of nucleotide biosynthesis, such as methionine.

Inositol phosphate synthesis and the nuclear processes they affect.

Recent studies have implicated inositol phosphates, a highly charged family of lipid-derived metabolites, in a slue of cellular processes. However, it is their involvement in nuclear events that has attracted much attention. Several IP molecules have been linked to gene regulatory factors, chromatin-remodeling complexes, mRNA export, and DNA repair machinery, yet in many instances direct mechanistic roles remain elusive. The purpose of this review is to cover the latest data gathered regarding only the nuclear roles of the various inositol phosphates while simultaneously providing a step-by-step tour of IP synthesis in eukaryotes.