Regulation of Phospholipase D in HL60 Cells

The activity of phospholipase D (PLD) is regulated by a variety of hormonal stimuli and provides a mechanistic pathway for response of cells to extracellular stimuli. The two identified mammalian PLD enzymes possess highly homologous C termini, which are required for catalytic activity. Mutational analysis of PLD1 and PLD2 reveals that modification of as little as the Cterminal threonine or the addition of a single alanine attenuates activity of the enzyme. Protein folding appears to be intact because mutant enzymes express to similar levels in Sf9 cells and addition of peptides representing the C-terminal amino acids, including the simple hexamer PMEVWT, restores partial activity to several of the mutants. Analysis of several mutants suggests a requirement for the hydrophobic reside at the ؊2-position but not an absolute requirement for the hydroxyl side chain of threonine at the C terminus. The inability of peptides amidated at their C termini to effect restoration of activity indicates the involvement of the C-terminal ␣ carboxyl group in functional activity of these enzymes. The ability of peptides to restore activity to PLD enzymes mutated at the C terminus suggests a flexible interaction of this portion of the molecule with a catalytic core constructed on conserved HKD motifs. Participation of these C termini residues in either stabilization of the catalytic site or the enzymatic reaction itself remains to be determined. This requirement for the C terminus provides an excellent potential site for interaction with regulatory proteins that may either enhance or down-regulate the activity of these enzymes in vitro.Phosphatidic acid plays a prominent role in phospholipid metabolism (1) and signal transduction. As a signaling molecule, phosphatidic acid is produced through the action of phospholipase D (PLD) 1 in response to a variety of cellular stimuli and acts directly as a second messenger or as a precursor for the formation of another second messenger, diacylglycerol (see Refs. 2-5 for reviews).Understanding of mechanisms by which mammalian PLD enzymes are regulated has been greatly accelerated by the development of a sensitive in vitro assay for the activity (6, 7) and identification of two mammalian genes (8 -10). The regulation of PLD activity in vitro allowed the identification of four distinct mechanisms for stimulation of the enzymes. Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) was the first activator and a key element in establishing assays for the enzymes (6, 11-13). This was followed by the elucidation of activation by members of both the Arf (6, 14) and Rho (15-19) families of small monomeric GTPases and by classical isoforms of protein kinase C (PKC) via a phosphorylation independent pathway (20, 21). Subsequent studies with recombinant proteins in vitro have shown that PLD1 responds to all of these activators (22), while PLD2 is only stimulated by PIP 2 and Arf (9,23,24). Elucidation of these regulatory mechanisms and investigations to determine which of these pathways are utilized in the ce...

Section: Discussionmentioning

confidence: 54%

The C Terminus of Mammalian Phospholipase D Is Required for Catalytic Activity

Liu

Gutowski²,

Sternweis

2001

“…HL60 cells were purchased from ATCC and maintained as described (17). COS7 cells were purchased from ATCC and grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 50 units/ml penicillin, and 50 g/ml streptomycin at 37°C in a humidified atmosphere of 10% CO 2 and 90% air.…”

Section: Yeast Strain and Cell Lines-mentioning

confidence: 99%

“…Miscellaneous Procedures-SDS-PAGE and Western blotting were performed as described (17) except that an ECL kit (Amersham Corp.) was used for detection. Protein concentrations were determined using the Coomassie Plus protein assay reagent (Pierce) with bovine serum albumin as a standard.…”

Section: The Nucleotide Sequence(s) Reported In This Paper Has Been Smentioning

confidence: 99%

Arfaptin 1, a Putative Cytosolic Target Protein of ADP-ribosylation Factor, Is Recruited to Golgi Membranes

Kanoh

Williger

Exton

1997

Self Cite

111

101

ADP-ribosylation factors (ARFs) have been implicated in vesicle transport in the Golgi complex. Employing yeast two-hybrid screening of an HL60 cDNA library using a constitutively active mutant of ARF3 (ARF3⅐Q71L), as a probe, we have identified a cDNA encoding a novel protein with a calculated molecular mass of 38.6 kDa, which we have named arfaptin 1. The mRNA of arfaptin 1 was ubiquitously expressed, and recombinant arfaptin 1 bound preferentially to class I ARFs, especially ARF1, but only in the GTP-bound form. The interactions were independent of myristoylation of ARF. Arfaptin 1 in cytosol was recruited to Golgi membranes by ARF in a guanosine 5 -O-(3-thiotriphosphate)-dependent and brefeldin A-sensitive manner. When expressed in COS cells, arfaptin 1 was localized to the Golgi complex. The yeast two-hybrid system yielded another clone, which encoded a putative protein, which we have named arfaptin 2. This consisted of the same number of amino acids as arfaptin 1 and was 60% identical to it. Arfaptin 2 was also ubiquitously expressed and bound to the GTP-, but not GDP-liganded form of class I ARFs, especially ARF1. These results suggest that arfaptins 1 and 2 may be direct target proteins of class 1 ARFs. Arfaptin 1 may be involved in Golgi function along with ARF1.

“…In addition, there may be multiple forms of small G protein-dependent PLD including ARF-sensitive, RhoA-sensitive, and ARF-, RhoA-sensitive PLDs. Siddiqi et al (14) reported that the cytosolic fraction of HL-60 cells contained a soluble PLD activated by ARF, but not RhoA. Malcolm et al (8) showed rat liver plasma membrane PLD to be sensitive to RhoA, but not to ARF.…”

mentioning

confidence: 99%

Cloning, Expression, and Characterization of a Novel Phospholipase D Complementary DNA from Rat Brain

Kodaki

Yamashita

1997

219

165

Here we report the cloning and characterization of cDNA for a different type of PLD (rat PLD2 (rPLD2)) from rat brain. We synthesized highly degenerate amplimers corresponding to the conserved regions of eukaryote PLDs and performed polymerase chain reaction on a rat brain cDNA library. Using the amplified sequence as the probe, we cloned a rat brain cDNA clone that contained an open reading frame of 933 amino acids with an M r of 105,992. The deduced amino acid sequence showed significant similarity to hPLD1 with a large deletion in the middle of the sequence. When the sequence was expressed in the fission yeast Schizosaccharomyces pombe, PLD activity was greatly increased. The activity was markedly stimulated by phosphatidylinositol 4,5-bisphosphate, but not by ADPribosylation factor 1 and RhoA. Rat brain cytosol known to stimulate small GTP-binding protein-dependent PLD did not stimulate rPLD2 expressed in S. pombe. The transcript was detected at significant levels in brain, lung, heart, kidney, stomach, small intestine, colon, and testis, but at low levels in thymus, liver, and muscle. Only a negligible level was found in spleen and pancreas. Thus rPLD2 is a novel type of PLD dependent on phosphatidylinositol 4,5-bisphosphate, but not on the small GTP-binding proteins ADP-ribosylation factor 1 and RhoA. Phospholipase D (PLD)1 catalyzes the hydrolysis of phosphatidylcholine (PC) to phosphatidic acid and choline (1). A variety of signal molecules such as hormones, neurotransmitters, and growth factors are known to induce the activation of PLD in a wide range of cell types. Hence PLD is implicated in a broad spectrum of physiological processes and diseases, including metabolic regulation, inflammation, secretion, mitogenesis, oncogenesis, neural and cardiac stimulation, diabetes, and senescence (for reviews, see Ref.2). Despite its crucial importance in signal transduction, the molecular structure and characteristics of PLD enzyme are only poorly understood.Multiple PLD isoforms exist in mammalian tissues. Several factors were reported to stimulate PLD activity in vitro, including unsaturated fatty acid (3), phosphatidylinositol 4,5-bisphosphate (PIP 2 ) (4), monomeric GTP-binding proteins (G proteins) such as ADP-ribosylation factor 1 (ARF1) (5, 6) and RhoA (7, 8), protein kinase C (9), and calmodulin (10). Massenburg et al. (11) showed that two major forms of PLD activity in rat brain membranes can be separated into ARF-dependent and oleate-dependent enzymes, clearly indicating that these are distinct isoforms. Both oleate-dependent and ARF-dependent types of PLD were recently highly purified from pig lung and brain, respectively (12, 13). In addition, there may be multiple forms of small G protein-dependent PLD including ARF-sensitive, RhoA-sensitive, and ARF-, RhoA-sensitive PLDs. Siddiqi et al. (14) reported that the cytosolic fraction of HL-60 cells contained a soluble PLD activated by ARF, but not RhoA. Malcolm et al. (8) showed rat liver plasma membrane PLD to be sensitive to RhoA, but not to ARF. PL...