Mauricio Di Fulvio

Phospholipase D2-derived phosphatidic acid binds to and activates ribosomal p70 S6 kinase independently of mTOR

The product of phospholipase D (PLD) enzymatic action in cell membranes, phosphatidic acid (PA), regulates kinases implicated in NADPH oxidase activation, as well as the mammalian target of rapamy‐cin (mTOR) kinase. However, other protein targets for this lipid second messenger must exist in order to explain other key PA‐mediated cellular functions. In this study, PA was found to specifically and saturably bind to and activate recombinant and immunoprecipitated endogenous ribosomal S6 kinase (S6K) with a stoichi‐ometry of 94:1 lipid/protein. Polyphosphoinositides PI4‐P and PI4, 5P2 and cardiolipin could also bind to and activate S6K, albeit with different kinetics. Conversely, PA with at least one acyl side chain saturated (10:0) was ineffective in binding or activating the enzyme. Transfection of COS‐7 cells with a wild‐type myc‐(pcDNA)‐PLD2 construct resulted in high PLD activity, concomitantly with an increase in ribosomal p70S6K enzyme activity and phosphorylation in T389 and T421/S424 as well as phosphorylation of p70S6Ks natural substrate S6 protein in S235/S236. Overexpression of a lipase inactive mutant (K758R), however, failed to induce an increase in both PLD and S6K activity or phosphorylation, indicating that the enzymatic activity of PLD2 (i.e., synthesis of PA) must be present to affect S6K. Neither inhibiting mTOR kinase activity with rapamycin nor silencing mTOR gene expression altered the augmentative effect of PLD2 exerted on p70S6K activity. This finding indicates that PA binds to and activates p70S6K, even in the absence of mTOR. Lastly, COS‐7 transfection with PLD2 changed the pattern of subcellular expression, and a colocalization of S6K and PLD2 was observed by immunofluorescence microscopy. These results show for the first time a direct (mTOR‐independent) participation of PLD in the p70S6K pathway and implicate PA as a nexus that brings together cell phospholipases and kinases.—Lehman, N., Ledford, B., Di Fulvio, M., Frondorf, K., McPhail, L. C., Gomez‐Cambronero, J. Phospholipase D2‐derived phosphatidic acid binds to and activates ribosomal p70 S6 kinase independently of mTOR. FASEB J. 21, 1075–1087 (2007)

Understanding phospholipase D (PLD) using leukocytes: PLD involvement in cell adhesion and chemotaxis

Phospholipase D (PLD) is an enzyme that catalyzes the conversion of membrane phosphatidylcholine to choline and phosphatidic acid (PA; a second messenger). PLD is expressed in nearly all types of leukocytes and has been associated with phagocytosis, degranulation, microbial killing, and leukocyte maturation. With the application of recently developed molecular tools (i.e., expression vectors, silencing RNA, and specific antibodies), the demonstration of a key role for PLD in those and related cellular actions has contributed to a better awareness of its importance. A case in point is the recent findings that RNA interference‐mediated depletion of PLD results in impaired leukocyte adhesion and chemotaxis toward a gradient of chemokines, implying that PLD is necessary for leukocyte movement. We forecast that based on results such as those, leukocytes may prove to be useful tools to unravel still‐unresolved mechanistic issues in the complex biology of PLD. Three such issues are considered here: first, whether the cellular actions of PLD are mediated entirely by PA (the product of its enzymatic reaction) or whether PLD by itself interacts with other protein signaling molecules; second, the current difficulty of defining a “PA consensus site” in the various intracellular protein targets of PA; and third, the resolution of specific PLD location (upstream or downstream) in a particular effector signaling cascade. There are reasons to expect that leukocytes and their leukemic cell line counterparts will continue yielding invaluable information to cell biologists to resolve standing molecular and functional issues concerning PLD.

Phospholipase D (PLD) gene expression in human neutrophils and HL-60 differentiation

Human neutrophils exhibit a regulated phospholipase D (PLD) activity that can be measured biochemically in vitro. However, the precise expression pattern of PLD isoforms and their specific biological role(s) are not well understood. Neutrophil mRNA is intrinsically difficult to isolate as a result of the extremely high content of lytic enzymes in the cell’s lysosomal granules. Reverse transcription coupled to polymerase chain reaction indicated that pure populations of human neutrophils had the CD16b+/CD115−/CD20−/CD3ζ−/interleukin‐5 receptor α− phenotype. These cells expressed the following splice variants of the PLD1 isoform: PLD1a, PLD1b, PLD1a2, and PLD1b2. As for the PLD2 isoform, neutrophils expressed the PLD2a but not the PLD2b mRNA variant. The relative amount of PLD1/PLD2 transcripts exists in an approximate 4:1 ratio. The expression of PLD isoforms varies during granulocytic differentiation, as demonstrated in the promyelocytic leukemia HL‐60 cell line. Further, the pattern of mRNA expression is dependent on the differentiation‐inducing agent, 1.25% dimethyl sulfoxide causes a dramatic increase in PLD2a and PLD1b transcripts, and 300 nM all‐trans‐retinoic acid induced PLD1a expression. These results demonstrate for the first time that human neutrophils express five PLD transcripts and that the PLD genes undergo qualitative changes in transcription regulation during granulocytic differentiation.

Tyrosine Phosphorylation of Grb2: Role in Prolactin/Epidermal Growth Factor Cross Talk in Mammary Epithelial Cell Growth and Differentiation∇

ABSTRACT Characterizing mechanisms regulating mammary cell growth and differentiation is vital, as they may contribute to breast carcinogenesis. Here, we examine a cross talk mechanism(s) downstream of prolactin (PRL), a primary differentiation hormone, and epidermal growth factor (EGF), an important proliferative factor, in mammary epithelial cell growth and differentiation. Our data indicate that EGF exerts inhibitory effects on PRL-induced cellular differentiation by interfering with Stat5a-mediated gene expression independent of the PRL-proximal signaling cascade. Additionally, our data show that PRL is a potent inhibitor of EGF-induced cell proliferation. We identify tyrosine phosphorylation of the growth factor receptor-bound protein 2 (Grb2) as a critical mechanism by which PRL antagonizes EGF-induced cell proliferation by attenuating the activation of the Ras/mitogen-activated protein kinase (MAPK) pathway. Together, our results define a novel negative cross-regulation between PRL and EGF involving the Jak2/Stat5a and Ras/MAPK pathways through tyrosine phosphorylation of Grb2.

PLD2 has both enzymatic and cell proliferation-inducing capabilities, that are differentially regulated by phosphorylation and dephosphorylation

Phospholipase D2 (PLD2) overexpression in mammalian cells results in cell transformation. We have hypothesized that this is due to an increase of de novo DNA synthesis. We show here that overexpression of PLD2-WT leads to an increased DNA synthesis, as measured by the expression levels of the proliferation markers PCNA, p27(KIP1) and phospho-histone-3. The enhancing effect was even higher with phosphorylation-deficient PLD2-Y179F and PLD2-Y511F mutants. The mechanism for this did not involve the enzymatic activity of the lipase, but, rather, the presence of the protein tyrosine phosphatase CD45, as silencing with siRNA for CD45 abrogated the effect. The two Y-->F mutants had in common a YxN consensus site that, in the phosphorylated counterparts, could be recognized by SH2-bearing proteins, such as Grb2. Even though Y179F and Y511F cannot bind Grb2, they could still find other protein partners, one of which, we have reasoned, could be CD45 itself. Affinity purified PLD2 is indeed activated by Grb2 and deactivated by CD45 in vitro. We concluded that phosphorylated PLD2, aided by Grb2, mediates lipase activity, whereas dephosphorylated PLD2 mediates an induction of cell proliferation, and the specific residues involved in this newly discovered regulation of PLD2 are Y(179) and Y(511).

Mammalian target of rapamycin (mTOR) and S6 kinase down-regulate phospholipase D2 basal expression and function.

The mammalian target of rapamycin (mTOR) and S6 kinase (S6K) pathway is essential for cell differentiation, growth, and survival. Phospholipase D2 (PLD2) plays a key role in mTOR/S6K mitogenic signaling. However, the impact of PLD on mTOR/S6K gene expression is not known. Here we show that interleukin-8 (IL-8) increases mRNA expression levels for PLD2, mTOR, and S6K, with PLD2 preceding mTOR/S6K in time. Silencing of PLD2 gene expression abrogated IL-8-induced mTOR/S6K mRNA expression, whereas silencing of mTOR or S6K gene expression resulted in large (>3-fold and >5-fold, respectively) increased levels of PLD2 RNA, which was paralleled by increases in protein expression and lipase activity. Treatment of cells with 0.5 nm rapamycin induced a similar trend. These results suggest that, under basal conditions, PLD2 expression and concomitant activity is negatively regulated by the mTOR/S6K signaling pathway. Down-regulation of PLD2 was confirmed in differentiated HL-60 leukocytes overexpressing an mTOR-wild type, but not an mTOR kinase-dead construct. At the cellular level, overexpression of mTOR-wild type resulted in lower basal cell migration, which was reversed by treatment with IL-8. We propose that IL-8 reverses an mTOR/S6K-led down-regulation of PLD2 expression and enables PLD2 to fully function as a facilitator for cell migration.

The NKCC and NCC Genes: An in Silico View

This chapter describes the structure, function and regulation of chloride transporters and channels. It also explains an in silico view of the NKCC and NCC genes. Genes, genomes, transcripts and protein sequences are organized in and maintained by several publicly available databases. The nomenclature of the electroneutral cation-chloride cotransporters (CCCs) gene family was proposed and approved by the Human Genome Organization (HUGO) Gene Nomenclature Committee (HGNC). The accepted nomenclature for membrane transporter proteins have the SLCnXm format where SLC stands for solute carrier, n is the number that represents the family, X is any letter representing the subfamily and m represents the actual member of the family. Each symbol is unique and identifies the gene and each gene has a unique accession number.Publisher Summary This chapter describes the structure, function and regulation of chloride transporters and channels. It also explains an in silico view of the NKCC and NCC genes. Genes, genomes, transcripts and protein sequences are organized in and maintained by several publicly available databases. The nomenclature of the electroneutral cation-chloride cotransporters (CCCs) gene family was proposed and approved by the Human Genome Organization (HUGO) Gene Nomenclature Committee (HGNC). The accepted nomenclature for membrane transporter proteins have the SLCnXm format where SLC stands for solute carrier, n is the number that represents the family, X is any letter representing the subfamily and m represents the actual member of the family. Each symbol is unique and identifies the gene and each gene has a unique accession number.

KCC2a Expression in a Human Fetal Lens Epithelial Cell Line

The fetal human lens epithelial cell (LEC) line (FHL124) possesses all four K+Cl- (KCC) cotransporter isoforms, KCC1-4, despite KCC2 being typically considered a neuronal isoform. Since at least two spliced variants, KCC2a and KCC2b, are co-expressed in cells of the central nervous system, this study sought to define the KCC2 expression profile in FHL124 cells. KCC2a, but not KCC2b transcripts were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Proteins of molecular weights ranging from 95 to 135 kDa were found by Western blotting using non-variant specific anti-KCC2 antibodies directed against two different regions of the KCC2 proteins, and by biotinylation suggesting membrane expression. Immunofluorescence revealed membrane and punctate cytoplasmic staining for KCC2. Low levels of cytosolic αA and αB crystallines, and neuron-specific enolase were also detected contrasting with the strong membrane immunofluorescence staining for the Na/K ATPase α1 subunit. Since the lack of neuron-specific expression of the KCC2b variant in non-neuronal tissues has been proposed under control of a neuron-restrictive silencing element in the KCC2 gene, we hypothesize that this control may be lifted for the KCC2a variant in the FHL124 epithelial cell culture, a non-neuronal tissue of ectodermal origin.

Enhanced insulin secretion and improved glucose tolerance in mice with homozygous inactivation of the Na+K+2Cl− co-transporter 1

The intracellular chloride concentration ([Cl(-)](i)) in β-cells plays an important role in glucose-stimulated plasma membrane depolarisation and insulin secretion. [Cl(-)](i) is maintained above equilibrium in β-cells by the action of Cl(-) co-transporters of the solute carrier family 12 group A (Slc12a). β-Cells express Slc12a1 and Slc12a2, which are known as the bumetanide (BTD)-sensitive Na(+)-dependent K(+)2Cl(-) co-transporters 2 and 1 respectively. We show that mice lacking functional alleles of the Slc12a2 gene exhibit better fasting glycaemia, increased insulin secretion in response to glucose, and improved glucose tolerance when compared with wild-type (WT). This phenomenon correlated with increased sensitivity of β-cells to glucose in vitro and with increased β-cell mass. Further, administration of low doses of BTD to mice deficient in Slc12a2 worsened their glucose tolerance, and low concentrations of BTD directly inhibited glucose-stimulated insulin secretion from β-cells deficient in Slc12a2 but expressing intact Slc12a1 genes. Together, our results suggest for the first time that the Slc12a2 gene is not necessary for insulin secretion and that its absence increases β-cell secretory capacity. Further, impairment of insulin secretion with BTD in vivo and in vitro in islets lacking Slc12a2 genes unmasks a potential new role for Slc12a1 in β-cell physiology.

Phospholipase D2 (PLD2) Shortens the Time Required for Myeloid Leukemic Cell Differentiation MECHANISM OF ACTION

Background: Leukemic cell differentiation is blocked in vivo but can be reversed in vitro. Results: A new signaling sequence involving specific kinases and phospholipases along the path to cell differentiation. Conclusion: PLD shortens the differentiation time of cells becoming mature neutrophils. Significance: Achieving a shortened time is highly beneficial for the induction therapy of acute myelocytic leukemia patients. Cell differentiation is compromised in acute leukemias. We report that mammalian target of rapamycin (mTOR) and S6 kinase (S6K) are highly expressed in the undifferentiated promyelomonocytic leukemic HL-60 cell line, whereas PLD2 expression is minimal. The expression ratio of PLD2 to mTOR (or to S6K) is gradually inverted upon in vitro induction of differentiation toward the neutrophilic phenotype. We present three ways that profoundly affect the kinetics of differentiation as follows: (i) simultaneous overexpression of mTOR (or S6K), (ii) silencing of mTOR via dsRNA-mediated interference or inhibition with rapamycin, and (iii) PLD2 overexpression. The last two methods shortened the time required for differentiation. By determining how PLD2 participates in cell differentiation, we found that PLD2 interacts with and activates the oncogene Fes/Fps, a protein-tyrosine kinase known to be involved in myeloid cell development. Fes activity is elevated with PLD2 overexpression, phosphatidic acid or phosphatidylinositol bisphosphate. Co-immunoprecipitation indicates a close PLD2-Fes physical interaction that is negated by a Fes-R483K mutant that incapacitates its Src homology 2 domain. All these suggest for the first time the following mechanism: mTOR/S6K down-regulation → PLD2 overexpression → PLD2/Fes association → phosphatidic acid-led activation of Fes kinase → granulocytic differentiation. Differentiation shortening could have a clinical impact on reducing the time of return to normalcy of the white cell counts after chemotherapy in patients with acute promyelocytic leukemia.