A splicing isoform of LPP1, LPP1a, exhibits high phosphatase activity toward FTY720 phosphate

The sphingolipid metabolite sphingosine 1-phosphate (S1P) plays an essential function in the egress of T cells from the thymus and secondary lymphoid organs. The novel immunomodulating agent FTY720 is phosphorylated in vivo to the functional form FTY720 phosphate (FTY720-P), which is structurally similar to S1P. FTY720-P inhibits the S1P-mediated T cell egress as an agonist of S1P receptors. FTY720-P is not stable in plasma and is dephosphorylated to FTY720. In the present study, we investigated activities toward FTY720-P of LPP family members (LPP1, LPP1a, LPP2, and LPP3), which exhibit broad substrate specificity. Of the four, LPP1a, the splicing isoform of LPP1, had the highest activity toward FTY720-P, and the highest affinity. Among blood-facing cells tested, only endothelial cells displayed high phosphatase activity for FTY720-P. Significant levels of LPP1a expression were found in endothelial cells, suggesting that LPP1a is important for the dephosphorylation of FTY720-P in plasma.     

LPP,a LIM protein highly expressed in smooth muscle

An 80-kDa protein, prominently expressed in smooth muscle, was microsequenced and identified as LPP, the product of the lipoma-preferred partner gene (Petit MMR, Mols R, Schoenmakers EFPM, Mandahl N, and Van de Ven WJM. Genomics 36: 118–129, 1996). Using a specific anti-LPP antibody, we showed, in Western blots and with immunofluorescence microscopy, the selective expression of LPP in vascular and visceral smooth muscles (0.5–1 ng/µg total protein). In other mature (noncultured) tissues, including heart and skeletal muscle, the protein is present only in trace amounts and is closely correlated with the levels of the smooth muscle marker -actin. In freshly isolated guinea pig bladder smooth muscle cells, immunofluorescence images showed LPP as linear arrays of punctate, longitudinally oriented staining superimposed with vinculin staining on the plasma membrane surface. A corresponding pattern of periodic labeling at the membrane in transverse sections of bladder smooth muscle suggested an association of LPP with peripheral dense bodies. In cultured rat aortic smooth muscle cells, LPP colocalized with vinculin at focal adhesions but not with p120 catenin or -actinin. Overexpression of the protein increased EGF-stimulated migration of vascular smooth muscle cells in Transwell assays, suggesting the participation of LPP in cell motility. The Rho-kinase inhibitor Y-27632 dissociated focal adhesions and LPP staining at the cell periphery and enhanced the nuclear accumulation of LPP induced by leptomycin B, indicating that LPP has a potential for relocating to the nucleus through a shuttling mechanism that is sensitive to inhibition of Rho-kinase. LIM protein; dense plaque; Rho-kinase; nuclear transport; cell migration     

Emerging roles for LPP in metastatic cancer progression

LIM domain containing proteins are important regulators of diverse cellular processes, and play pivotal roles in regulating the actin cytoskeleton. Lipoma Preferred Partner (LPP) is a member of the zyxin family of LIM proteins that has long been characterized as a promoter of mesenchymal/fibroblast cell migration. More recently, LPP has emerged as a critical inducer of tumor cell migration, invasion and metastasis. LPP is thought to contribute to these malignant phenotypes by virtue of its ability to shuttle into the nucleus, localize to adhesions and, most recently, to promote invadopodia formation. In this review, we will examine the mechanisms through which LPP regulates the functions of adhesions and invadopodia, and discuss potential roles of LPP in mediating cellular responses to mechanical cues within these mechanosensory structures.     

Enzymological properties of the LPP1-encoded lipid phosphatase from Saccharomyces cerevisiae

The product of the LPP1 gene in Saccharomyces cerevisiae is a membrane-associated enzyme that catalyzes the Mg(2+)-independent dephosphorylation of phosphatidate (PA), diacylglycerol pyrophosphate (DGPP), and lysophosphatidate (LPA). The LPP1-encoded lipid phosphatase was overexpressed 681-fold in Sf-9 insect cells and used to examine the enzymological properties of the enzyme using PA, DGPP, and LPA as substrates. The optimum pH values for PA phosphatase, DGPP phosphatase, and LPA phosphatase activities were 7. 5, 7.0, and 7.0, respectively. Divalent cations (Mn(2+), Co(2+), and Ca(2+)), NaF, heavy metals, propranolol, phenylglyoxal, and N-ethylmaleimide inhibited the PA phosphatase, DGPP phosphatase, and LPA phosphatase activities of the enzyme. The inhibitory effects of N-ethylmaleimide and phenylglyoxal on the LPP1-encoded enzyme were novel properties when compared with other Mg(2+)-independent lipid phosphate phosphatases from S. cerevisiae and mammalian cells. The LPP1-encoded enzyme exhibited saturation kinetics with respect to the surface concentrations of PA (K(m)=0.05 mol%), DGPP (K(m)=0.07 mol%), and LPA (K(m)=0.08 mol%). Based on specificity constants (V(max)/K(m)LPA (1.3 units/mg/mol%). DGPP (K(i)=0.12 mol%) was a competitive inhibitor with respect to PA, and PA (K(i)=0.12 mol%) was a competitive inhibitor with respect to DGPP. This suggested that the binding sites for these substrates were the same. The enzymological properties of the LPP1-encoded enzyme differed significantly from those of the S. cerevisiae DPP1-encoded lipid phosphatase, a related enzyme that also utilizes PA, DGPP, and LPA as substrates.     

Lipid phosphate phosphatase (LPP3) and vascular development

Lipid phosphate phosphatases (LPP) are integral membrane proteins with broad substrate specificity that dephosphorylate lipid substrates including phosphatidic acid, lysophosphatidic acid, ceramide 1-phosphate, sphingosine 1-phosphate, and diacylglycerol pyrophosphate. Although the three mammalian enzymes (LPP1-3) demonstrate overlapping catalytic activities and substrate preferences in vitro, the phenotypes of mice with targeted inactivation of the Ppap2 genes encoding the LPP enzymes reveal nonredundant functions. A specific role for LPP3 in vascular development has emerged from studies of mice lacking Ppap2b. A meta-analysis of multiple, large genome-wide association studies identified a single nucleotide polymorphism in PPAP2B as a novel predictor of coronary artery disease. In this review, we will discuss the evidence that links LPP3 to vascular development and disease and evaluate potential molecular mechanisms. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

The focal adhesion and nuclear targeting capacity of the LIM-containing lipoma-preferred partner (LPP) protein

Targeting of proteins to a particular cellular compartment is a critical determinant for proper functioning. LPP (LIM-containing lipoma-preferred partner) is a LIM domain protein that is localized at sites of cell adhesion and transiently in the nucleus. In various benign and malignant tumors, LPP is present in a mutant form, which permanently localizes the LIM domains in the nucleus. Here, we have investigated which regions in LPP target the protein to its subcellular locations. We found that the LIM domains are the main focal adhesion targeting elements and that the proline-rich region of LPP, which harbors binding sites for alpha-actinin and vasodilator-stimulated phosphoprotein (VASP), has a weak targeting capacity. All of the LIM domains of LPP cooperate in order to provide robust targeting to focal adhesions, and the linker between LIM domains 1 and 2 plays a pivotal role in this targeting. When overexpressed in the cytoplasm of cells, the LIM domains of LPP can deplete endogenous LPP and vinculin from focal adhesions. The proline-rich region of LPP contains targeting sites for focal adhesions and stress fibers that are distinct from the alpha-actinin and VASP binding sites, and the LPP LIM domains are dispensable for targeting LPP to the nucleus. Our studies have defined novel functional domains in the LPP protein.     

Metabolic aspects of LPP cyanophage replication in the cyanobacterium Plectonema boryanum

Cyanophage LPP1G is reproduced at the same yield in heterotrophic conditions (dark, glucose) as in photoautotrophic conditions; aerobiosis is required for dark cyanophage replication. Exogenous glucose is not required for the cyanophage replication in the dark in heterotrophically grown cells. In photoautotrophically grown cells, the maximum burst size in dark and glucose is delayed for a period corresponding to glucose uptake induction. Cyanophage LPP2SPI replication occurs in conditions where only Photosystem I operates. Of photosynthesis parameters tested, only CO₂ photoassimilation is affected during cyanophage LPP1G infection under photoautotrophic conditions.     

Opposing roles of zyxin/LPP ACTA repeats and the LIM domain region in cell-cell adhesion

Cadherins mediate cell-cell adhesion by linking cell junctions to actin networks. Although several actin regulatory systems have been implicated in cell-cell adhesion, it remains unclear how such systems drive cadherin-actin network formation and how they are regulated to coincide with initiation of adhesion. Previous work implicated VASP in assembly of cell-cell junctions in keratinocytes and the VASP-binding protein zyxin colocalizes with VASP at cell-cell junctions. Here we examine how domains in zyxin and its relative LPP contribute to cell-cell junction assembly. Using a quantitative assay for cell-cell adhesion, we demonstrate that zyxin and LPP function to increase the rate of early cell-cell junction assembly through the VASP-binding ActA repeat region. We also identify the LIM region of zyxin and LPP to be a regulatory domain that blocks function of these proteins. Deletion of the LIM domains drives adhesion and increases VASP level in detergent insoluble cadherin-actin. Dominant-negative zyxin/LPP mutants reduce the rate of adhesion, lower VASP levels in detergent-insoluble cadherin-actin networks, and allow for the accumulation of capping protein at cell-cell contacts. These data implicate the LIM domains of zyxin and LPP in regulating cell-cell junction assembly through VASP.     

Transforming human blood stem and progenitor cells: A new way forward in leukemia modeling

MLL-AF9 (MA9) is a leukemia fusion gene formed upon translocation of the AF9 gene on chromosome 9 and the MLL gene on chromosome 11. MA9 is commonly found in acute myeloid leukemia (AML) and occasionally in acute lymphoid leukemia and is associated with intermediate to poor outcome. The specific signaling pathways downstream of MA9 are still poorly understood. We have recently described a model system whereby we expressed the MA9 fusion gene in human CD34+ Umbilical Cord Blood (UCB) cells and showed that these cells transformed to acute myeloid or lymphoid leukemia when injected into immunodeficient mice. The Mixed Lineage Leukemia oncogenes are unique in this model system in that they promote full transformation of primary human blood cells, while all other leukemia-associated oncogenes tested thus far have induced only partial phenotypes. Here we provide an update on the use of this system for modeling human leukemia and its potential application for therapeutic testing of novel compounds to treat the disease. We focus specifically on the Rho family of small guanosine triphosphatases (GTPases) as potential therapeutic targets, which we have implicated in the pathogenesis of AML associated with MA9 expression.     

TRIP6 and LPP,but not Zyxin,are present at a subset of telomeres in human cells

The protection of chromosome ends requires the inhibition of DNA damage responses at telomeres. This inhibition is exerted in great part by the shelterin complex, known to prevent inappropriate ATM and ATR activation. The molecular mechanisms by which shelterin protects telomeres are incompletely understood. Recently, we have implicated for the first time a class of molecules, LIM domain proteins, in telomere protection. This protection occurred through interaction with shelterin, possibly through POT1, and required the pair of LIM proteins TRIP6 and LPP, themselves part of the Zyxin family. The domain similarity between TRIP6, LPP and Zyxin led us to ask whether the latter also interacted with telomeres. Here, we show that there is specificity in the association of LIM proteins with telomeres: Zyxin, despite a high degree of similarity with TRIP6 and LPP, was not detected at telomeres, nor found in a complex with shelterin. TRIP6 and LPP, however, were detected by immunofluorescence at a small subset of telomeres, perhaps those that are critically short. We speculate that specific LIM proteins are part of complex events occurring in the context of the telomere dysfunction response and are possibly at play during the induction of senescence.Key words: telomere, LIM domain, shelterin, POT1, TRIP6, LPP, zyxin, DNA damage     

TRIP6 and LPP,but not Zyxin,are present at a subset of telomeres in human cells

The protection of chromosome ends requires the inhibition of DNA damage responses at telomeres. This inhibition is exerted in great part by the shelterin complex, known to prevent inappropriate ATM and ATR activation. The molecular mechanisms by which shelterin protects telomeres are incompletely understood. Recently, we have implicated for the first time a class of molecules, LIM domain proteins, in telomere protection. This protection occurred through interaction with shelterin, possibly through POT1, and required the pair of LIM proteins TRIP6 and LPP, themselves part of the Zyxin family. The domain similarity between TRIP6, LPP and Zyxin led us to ask whether the latter also interacted with telomeres. Here, we show that there is specificity in the association of LIM proteins with telomeres: Zyxin, despite a high degree of similarity with TRIP6 and LPP, was not detected at telomeres, nor found in a complex with shelterin. TRIP6 and LPP, however, were detected by immunofluorescence at a small subset of telomeres, perhaps those that are critically short. We speculate that specific LIM proteins are part of complex events occurring in the context of the telomere dysfunction response, and possibly at play during the induction of senescence.     

Effect of empathy trait on attention to various facial expressions: evidence from N170 and late positive potential (LPP)

Background

The present study sought to clarify the relationship between empathy trait and attention responses to happy, angry, surprised, afraid, and sad facial expressions. As indices of attention, we recorded event-related potentials (ERP) and focused on N170 and late positive potential (LPP) components.

Methods

Twenty-two participants (12 males, 10 females) discriminated facial expressions (happy, angry, surprised, afraid, and sad) from emotionally neutral faces under an oddball paradigm. The empathy trait of participants was measured using the Interpersonal Reactivity Index (IRI, J Pers Soc Psychol 44:113–126, 1983).

Results

Participants with higher IRI scores showed: 1) more negative amplitude of N170 (140 to 200 ms) in the right posterior temporal area elicited by happy, angry, surprised, and afraid faces; 2) more positive amplitude of early LPP (300 to 600 ms) in the parietal area elicited in response to angry and afraid faces; and 3) more positive amplitude of late LPP (600 to 800 ms) in the frontal area elicited in response to happy, angry, surprised, afraid, and sad faces, compared to participants with lower IRI scores.

Conclusions

These results suggest that individuals with high empathy pay attention to various facial expressions more than those with low empathy, from very-early stage (reflected in N170) to late-stage (reflected in LPP) processing of faces.