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M A Frohman

Publications and source records attributed to M A Frohman.

At least 37 records · Page 2Linked to original sources
PubMed1999

Structural analysis of human phospholipase D1.

Activation of phosphatidylcholine-specific phospholipase D (PLD) has been proposed to play roles in numerous cellular pathways including signal transduction and membrane vesicular trafficking. We previously reported the cloning of two mammalian genes, PLD1 and PLD2, that encode PLD activities. We additionally reported that PLD1 is activated in a synergistic manner by protein kinase c-alpha (PKC-alpha), ADP-ribosylation factor 1 (ARF1), and Rho family members. We describe here molecular analysis of PLD1 using a combination of domain deletion and mutagenesis. We show that the amino-terminal 325 amino acids are required for PKC-alpha activation of PLD1 but not for activation by ARF1 and RhoA. This region does not contain the sole PKC-alpha interaction site and additionally functions to inhibit basal PLD activity in vivo. Second, a region of sequence unique to PLD1 (as compared with other PLDs) known as the "loop" region had been proposed to serve as an effector regulatory region but is shown here only to mediate inhibition of PLD1. Finally, we show that modification of the amino terminus, but not of the carboxyl terminus, is compatible with PLD enzymatic function and propose a simple model for PLD activation.

ADP-Ribosylation Factor 1↗
PubMed1999

Phospholipase D and its product, phosphatidic acid, mediate agonist-dependent raf-1 translocation to the plasma membrane and the activation of the mitogen-activated protein kinase pathway.

The primary known function of phospholipase D (PLD) is to generate phosphatidic acid (PA) via the hydrolysis of phosphatidylcholine. However, the functional role of PA is not well understood. We report here evidence that links the activation of PLD by insulin and the subsequent generation of PA to the activation of the Raf-1-mitogen-activated protein kinase (MAPK) cascade. Brefeldin A (BFA), an inhibitor of the activation of ADP-ribosylation factor proteins, inhibited insulin-dependent production of PA and MAPK phosphorylation. The addition of PA reversed the inhibition of MAPK activation by BFA. Overexpression of a catalytically inactive variant of PLD2, but not PLD1, blocked insulin-dependent activation of PLD and phosphorylation of MAPK. Real time imaging analysis showed that insulin induced Raf-1 translocation to cell membranes by a process that was inhibited by BFA. PA addition reversed the effects of BFA on Raf-1 translocation. However, PA did not activate Raf-1 in vitro or in vivo, suggesting that the primary function of PA is to enhance the recruitment of Raf-1 to the plasma membrane where other factors may activate it. Finally, we found that the recruitment of Raf-1 to the plasma membrane was transient, but Raf-1 remained bound to endocytic vesicles.

Animals↗
PubMed1999

Molecular analysis of mammalian phospholipase D2.

The mammalian phosphatidylcholine-specific phospholipase D (PLD) enzymes PLD1 and PLD2 have been proposed to play roles in signal transduction and membrane vesicular trafficking in distinct subcellular compartments. PLD1 is activated in a synergistic manner in vitro by protein kinase C-alpha, ADP-ribosylation factor 1 (ARF1), and Rho family members. In contrast, PLD2 is constitutively active in vitro. We describe here molecular analysis of PLD2. We show that the NH2-terminal 308 amino acids are required for PLD2's characteristic high basal activity. Unexpectedly, PLD2 lacking this region becomes highly responsive to ARF proteins and displays a modest preference for activation by ARF5. Chimeric analysis of PLD1 and PLD2 suggests that the ARF-responsive region is in the PLD carboxyl terminus. We also inserted into PLD2 a region of sequence unique to PLD1 known as the "loop" region, which had been proposed initially to mediate effector stimulation but that subsequently was shown instead to be required in part for the very low basal activity characteristic of PLD1. The insertion decreased PLD2 activity, consistent with the latter finding. Finally, we show that the critical role undertaken by the conserved carboxyl terminus is unlikely to involve promoting PLD association with membrane surfaces.

ADP-Ribosylation Factor 1↗
PubMed1999

Phospholipase D structure and regulation.

The recent identification of cDNA clones for phospholipase D has opened the door to new types of investigations into its structure and regulation. PLD activity has been found to be encoded by at least two different genes that contain catalytic domains that relate their mechanism of action to phosphodiesterases. In vivo roles for PLD suggest that it may be important for multiples steps in regulated secretion and membrane biogenesis.

Amino Acid Sequence↗
PubMed1998

PLD2 complexes with the EGF receptor and undergoes tyrosine phosphorylation at a single site upon agonist stimulation.

Mammalian phospholipase D (PLD) activity becomes up-regulated when cells are stimulated by a variety of hormones, growth factors, and other extracellular signals. Two distinct PLDs, PLD1 and PLD2, have been identified. The mechanism through which each PLD is activated, however, is poorly understood. Using transiently transfected human embryonic kidney fibroblasts (HEK293), we demonstrate here that PLD1 activity, and to a lesser extent PLD2 activity, is stimulated in response to epidermal growth factor (EGF). PLD2, but not PLD1, associates with the EGF receptor in a ligand-independent manner and becomes tyrosine-phosphorylated upon EGF receptor activation. Tyrosine 11 (Tyr-11) of PLD2 was identified as the specific phosphorylation site. Mutation of this residue to phenylalanine enhanced basal activity almost 2-fold, but did not alter the magnitude of the EGF-mediated increase in PLD2 activity. In conclusion, we show here for the first time agonist-stimulated activation of both PLD1 and PLD2 in vivo and provide evidence of a distinct type of interaction for each isoform with the EGF receptor. Moreover, our results suggest that agonist-induced tyrosine phosphorylation plays a role in PLD2 regulation.

Amino Acid Sequence↗
PubMed1998

Genomic analysis of murine phospholipase D1 and comparison to phospholipase D2 reveals an unusual difference in gene size.

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid and choline. In mammals, PLD activity is encoded by two different genes, PLD1 and PLD2. cDNAs for human, mouse and rat PLD1 and PLD2 and the mouse PLD2 genomic organization have recently been reported. In this article, we describe the genomic organization of mouse PLD1. Mouse PLD1 (mPLD1) contains 28 exons and spans approximately 147 kb. This is much larger than the closely related mouse PLD2 (mPLD2) gene (17 kb) and indicates that a rapid 20-fold extension/contraction of the set of introns for one of the genes has occurred. This change is not apparently due to PLD1-specific repetitive element mediated expansion. Similar to mPLD2, most PLD1 exons are less than 200 bp in length. Only the final exon, containing in part the 3' UTR, is significantly longer (881 bp). Comparison of the mPLD1 and mPLD2 exon-intron boundaries revealed that almost all of the sites are conserved. The mPLD1 5' UTR is interrupted by two large introns, 16 and 30 kb in length. A PLD1-specific sequence known as the 'loop' region is encoded by two non-conserved exons. The boundary between the loop-encoding exons matches precisely the site at which an alternately spliced isoform had been proposed to be initiated.

Alternative Splicing↗
PubMed1998

Organization and alternative splicing of the murine phospholipase D2 gene.

Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine, generating phosphatidic acid and choline. Mammalian PLD activity derives from a family of membrane-associated enzymes that are activated by a wide variety of signal transduction events. cDNA species encoding human, mouse and rat PLD1 and PLD2 have recently been reported. In this study we undertook to determine the organization of the mouse PLD2 gene. We report that the gene spans 17.1 kb and contains 25 exons. Mouse PLD2 is notable for a relatively GC-rich and large 5' untranslated region. Proximal promoter sequences upstream of the first exon contain several consensus SP1 sequences (GGGCGG) but lack TATA and CAAT boxes. Finally, alternatively spliced cDNA species identified for PLD1 and PLD2 are discussed in the context of the PLD2 genomic organization.

Alternative Splicing↗
PubMed1998

Increased mRNA expression of phospholipase D (PLD) isozymes during granulocytic differentiation of HL60 cells.

In response to dibutyryl cyclic AMP (dbcAMP) and all-trans retinoic acid (ATRA), HL60 cells differentiate into granulocyte-like cells. Membrane-associated phospholipase D (PLD) activity in response to guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or phorbol myristate acetate (PMA) was upregulated by these treatments. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses revealed that both hPLD1a and hPLD1b mRNAs were expressed in HL60 cells and that their expression levels increased during differentiation. hPLD2 mRNA levels rose dramatically during differentiation. These results suggest that the PLD genes undergo changes in transcriptional regulation during granulocytic differentiation of HL60 cells.

Bucladesine↗
PubMed1997

Different domains of mammalian ADP-ribosylation factor 1 mediate interaction with selected target proteins.

Mammalian ADP-ribosylation factor 1 (mARF1) is a small GTP-binding protein that is activated by a Golgi guanine nucleotide exchange factor. Once bound to the Golgi membranes in the GTP form, mARF1 initiates the recruitment of the adaptor protein 1 (AP-1) complex and coatomer (COPI) onto these membranes and activates phospholipase D1 (PLD1). To map the domains of mARF1 that are important for these activities, we constructed chimeras between mARF1 and Saccharomyces cerevisiae ARF2, which functions poorly in all of these processes except COPI recruitment. The carboxyl half of mARF1 (amino acids 95-181) was essential for activation by the Golgi guanine nucleotide exchange factor, whereas a separate domain (residues 35-94) was required to effectively activate PLD1 and to promote efficient AP-1 recruitment. Since residues 35-94 of mARF1 are critical for optimal activity in both PLD1 activation and AP-1 recruitment, we hypothesize that this region of ARF contains residues that interact with effector molecules.

ADP-Ribosylation Factors↗
PubMed1997

Measurement of phospholipase D activity.

Phosphodiesteric cleavage of phosphatidylcholine by members of a growing family of phospholipases D produces choline and phosphatidic acid. These enzymes can also catalyse a transphosphatidylation reaction in which the aliphatic chain of a primary alcohol is transferred to the phosphatidyl moiety of the phosphatidic acid product. PLD enzymes are found in a variety of organisms including bacteria, yeast, plants, and vertebrates. In mammalian systems, biochemical and cell biological approaches have identified phosphatidic acid as a mediator (or progenitor of mediators) that play important roles in the transduction of extracellular signals. Phosphatidic acid or its metabolites may be regulators of key cellular processes such as the control of intracellular protein trafficking, secretion, and alterations in cell morphology and motility. This review discusses methods for the determination of PLD activity both in vitro and in intact cells.

Animals↗
PubMed1997

Cloning and expression analysis of murine phospholipase D1.

Activation of phosphatidylcholine-specific phospholipase D(PLD) occurs as part of the complex signal-transduction cascade initiated by agonist stimulation of tyrosine kinase and G-protein-coupled receptors. A variety of mammalian PLD activities have been described, and cDNAs for two PLDs recently reported (human PLD1 and murine PLD2). We describe here the cloning and chromosomal localization of murine PLD1. Northern-blot hybridization and RNase protection analyses were used to examine the expression of murine PLD1 and PLD2 ina variety of cell lines and tissues. PLD1 and PLD2 were expressed in all RNA samples examined, although the absolute expression of each isoform varied, as well as the ratio of PLD1 to PLD2. Moreover, in situ hybridization of adult brain and murine embryo sections revealed high levels of expression of individual PLDs in some cell types and no detectable expression in others. Thus the two PLDs probably carry out distinct roles in restricted subsets of cells rather than ubiquitous roles in all cells.

Amino Acid Sequence↗
PubMed1997

Phospholipase D stimulates release of nascent secretory vesicles from the trans-Golgi network.

Phospholipase D (PLD) is a phospholipid hydrolyzing enzyme whose activation has been implicated in mediating signal transduction pathways, cell growth, and membrane trafficking in mammalian cells. Several laboratories have demonstrated that small GTP-binding proteins including ADP-ribosylation factor (ARF) can stimulate PLD activity in vitro and an ARF-activated PLD activity has been found in Golgi membranes. Since ARF-1 has also been shown to enhance release of nascent secretory vesicles from the TGN of endocrine cells, we hypothesized that this reaction occurred via PLD activation. Using a permeabilized cell system derived from growth hormone and prolactin-secreting pituitary GH3 cells, we demonstrate that immunoaffinity-purified human PLD1 stimulated nascent secretory vesicle budding from the TGN approximately twofold. In contrast, a similarly purified but enzymatically inactive mutant form of PLD1, designated Lys898Arg, had no effect on vesicle budding when added to the permeabilized cells. The release of nascent secretory vesicles from the TGN was sensitive to 1% 1-butanol, a concentration that inhibited PLD-catalyzed formation of phosphatidic acid. Furthermore, ARF-1 stimulated endogenous PLD activity in Golgi membranes approximately threefold and this activation correlated with its enhancement of vesicle budding. Our results suggest that ARF regulation of PLD activity plays an important role in the release of nascent secretory vesicles from the TGN.

1-Butanol↗
PubMed1997

Mutagenesis of phospholipase D defines a superfamily including a trans-Golgi viral protein required for poxvirus pathogenicity.

Phospholipase D (PLD) genes are members of a superfamily that is defined by several highly conserved motifs. PLD in mammals has been proposed to play a role in membrane vesicular trafficking and signal transduction. Using site-directed mutagenesis, 25 point mutants have been made in human PLD1 (hPLD1) and characterized. We find that a motif (HxKxxxxD) and a serine/threonine conserved in all members of the PLD superfamily are critical for PLD biochemical activity, suggesting a possible catalytic mechanism. Functional analysis of catalytically inactive point mutants for yeast PLD demonstrates that the meiotic phenotype ensuing from PLD deficiency in yeast derives from a loss of enzymatic activity. Finally, mutation of an HxKxxxxD motif found in a vaccinia viral protein expressed in the Golgi complex results in loss of efficient vaccinia virus cell-to-cell spreading, implicating the viral protein as a member of the superfamily and suggesting that it encodes a lipid modifying or binding activity. The results suggest that vaccinia virus and hPLD1 may act through analogous mechanisms to effect viral cellular egress and vesicular trafficking, respectively.

Amino Acid Sequence↗
PubMed1997

RalA interacts directly with the Arf-responsive, PIP2-dependent phospholipase D1.

RalA GTPase associates with a phospholipase D (PLD) that is activated in v-Src- and v-Ras-transformed cells. Two mammalian PLDs were recently cloned: PLD1, which is activated by Arf family GTPases and dependent upon phosphatidylinositol-4,5-bisphosphate (PIP2), and PLD2, which is also dependent upon PIP2, but not stimulated by Arf. Another PLD has been described that is stimulated by oleate. Evidence is provided that the RalA-assiciated PLD is PLD1. First, the PLD precipitated by RalA from murine fibroblasts was stimulated by Arf, dependent upon PIP2, and inhibited by oleate. Second, immobilized RalA precipitated PLD1 from sf9 insect cells overexpressing PLD1. Third, a series of RalA mutants precipitated PLD activity from both PLD1-expressing insect cells and murine fibroblasts with the same efficiency. And finally, immobilized RalA precipitated PLD1 from a purified PLD1 preparation. These data argue that RalA associates directly with the Arf-responsive, PIP2-dependent PLD1.

3T3 Cells↗
PubMed1997

Phospholipase D2, a distinct phospholipase D isoform with novel regulatory properties that provokes cytoskeletal reorganization.

BACKGROUND: Activation of phospholipase D (PLD) is an important but poorly understood component of receptor-mediated signal transduction responses and regulated secretion. We recently reported the cloning of the human gene encoding PLD1; this enzyme has low basal activity and is activated by protein kinase C and the small GTP-binding proteins, ADP-ribosylation factor (ARF), Rho, Rac and Cdc42. Biochemical and cell biological studies suggest, however, that additional and distinct PLD activities exist in cells, so a search was carried out for novel mammalian genes related to PLD1. RESULTS: We have cloned the gene for a second PLD family member and characterized the protein product, which appears to be regulated differently from PLD1: PLD2 is constitutively active and may be modulated in vivo by inhibition. Unexpectedly, PLD2 localizes primarily to the plasma membrane, in contrast to PLD1 which localizes solely to peri-nuclear regions (the endoplasmic reticulum, Golgi apparatus and late endosomes), where PLD activity has been shown to promote ARF-mediated coated-vesicle formation. PLD2 provokes cortical reorganization and undergoes redistribution in serum-stimulated cells, suggesting that it may have a role in signal-induced cytoskeletal regulation and/or endocytosis. CONCLUSIONS: PLD2 is a newly identified mammalian PLD isoform with novel regulatory properties. Our findings suggest that regulated secretion and morphological reorganization, the two most frequently proposed biological roles for PLD, are likely to be effected separately by PLD1 and PLD2.

Amino Acid Sequence↗
PubMed1997

Characterization of two alternately spliced forms of phospholipase D1. Activation of the purified enzymes by phosphatidylinositol 4,5-bisphosphate, ADP-ribosylation factor, and Rho family monomeric GTP-binding proteins and protein kinase C-alpha.

We previously reported the cloning of a cDNA encoding human phosphatidylcholine-specific phospholipase D1 (PLD1), an ADP-ribosylation factor (ARF)-activated phosphatidylcholine-specific phospholipase D (Hammond, S. M., Tsung, S., Autschuller, Y., Rudge, S. A., Rose, K., Engebrecht, J., Morris, A. J., and Frohman, M. A. (1995) J. Biol. Chem. 270, 29640-29643). We have now identified an evolutionarily conserved shorter splice variant of PLD1 lacking 38 amino acids (residues 585-624) that arises from regulated splicing of an alternate exon. Both forms of PLD1 (PLD1a and 1b) have been expressed in Sf9 cells using baculovirus vectors and purified to homogeneity by detergent extraction and immunoaffinity chromatography. PLD1a and 1b have very similar properties. PLD1a and 1b activity is Mg2+dependent but insensitive to changes in free Ca2+ concentration. Phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate activate PLD1a and 1b but a range of other acidic phospholipids are ineffective. PLD1a and 1b are highly responsive to activation by GTP-gammaS-liganded ADP-ribosylation factor-1 (ARF-1) and can also be activated to a lesser extent by three purified RHO family monomeric GTP-binding proteins, RHO A, RAC-1, and CDC42. Activation of PLD1a and 1b by the RHO family monomeric GTP-binding proteins is GTP-dependent and synergistic with ARF-1. Purified protein kinase C-alpha activates PLD1a and 1b in a manner that is stimulated by phorbol esters and does not require ATP. Activation of PLD1a and 1b by protein kinase C-alpha is synergistic with ARF and with the RHO family monomeric GTP-binding proteins, suggesting that these three classes of regulators interact with different sites on the enzyme.

Adenosine Diphosphate Ribose↗