Functional implications of post-translational modifications of phospholipases D1 and D2

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Abstract

Our previous studies showed that truncation of the N-terminal 168 amino acids of rat brain phospholipase D1 (rPLD1) abolishes its response to protein kinase C (PKC) and greatly diminishes its palmitoylation and Ser/Thr phosphorylation. In this study, we show that the response to PKC as well as the palmitoylation and Ser/Thr phosphorylation were restored when the truncated rPLD1 mutant (rPLD1(ΔN168)) was coexpressed with a fragment containing the N-terminal 168 amino acids. Immunoprecipitation experiments showed that the N-terminal fragment associated with rPLD1(ΔN168) when coexpressed in COS 7 cells and that palmitoylation of Cys240 and Cys241 was not necessary for the association. In addition, we found that rat PLD2 (rPLD2) was palmitoylated on Cys223 and Cys224 in COS 7 cells. Mutation of both these cysteines reduced the basal activity of rPLD2, however its response to PMA stimulation in vivo was retained. As in the case of rPLD1, loss of palmitoylation weakened membrane association of rPLD2. In summary, the N-terminal 168-amino-acid fragment of rPLD1 can associate with truncated rPLD1(ΔN168) to restore its palmitoylation, Ser/Thr phosphorylation and PKC response. Although rPLD2 differs from rPLD1 in many properties, it is palmitoylated at the corresponding conserved cysteine residues in COS 7 cells.

Introduction

Phospholipase D (PLD) is a ubiquitous enzyme that hydrolyzes phosphatidylcholine to phosphatidic acid and choline [1]. Phosphatidic acid is generally recognized as the signaling product of PLD action, but it can also be converted to diacylglycerol or lysophosphatidic acid. Diacylglycerol is a well characterized activator of protein kinase C (PKC), while lysophosphatidic acid is a major extracellular signal. PLD also catalyzes a phosphatidyl transfer reaction using primary alcohols as nucleophilic acceptors to produce phosphatidylalcohols. This reaction is used as a specific measure of PLD activity.

PLD is highly regulated and responds to many growth factors, hormones and other agonists [2]. Although the physiological role of PLD remains unclear, multiple functions have been proposed based on the wide involvement of PLD in signaling pathways and the actions of phosphatidic acid and its products [2], [3]. Two isoforms of mammalian PLD (PLD1 and PLD2) have been cloned so far. Although these share about 50% amino acid similarity, they exhibit very different regulatory properties. PLD1 exhibits a low basal activity and responds strongly to PKC and to members of the Rho and ARF families of small G proteins [4], [5], [6], [7]. On the other hand, PLD2 exhibits a high basal activity [8], [9], [10] and is generally less responsive to activators that stimulate PLD1, and may be regulated differently [10], [11], [12], [13].

PLD belongs to a superfamily defined by the motif, HxK(x)4D, denoted ‘HKD’ [14], [15], [16], [17], which is essential for enzymatic activity. PLD contains two copies of the HKD motif located in the N- and C-terminal halves of the molecule. Our studies on the rat PLD1 (rPLD1) isoform showed that the enzyme could be split into two halves and that PLD activity could be restored when the two fragments were coexpressed in COS 7 cells. Coimmunoprecipitation experiments showed that the N- and C-terminal fragments could physically associate [18], and it was proposed that the association brought the two HKD domains together to form a catalytic center. Conserved hydrophobic amino acids in the HKD domains were found to be important for the interdomain association, and the association was essential for catalysis of the enzyme [19]. In addition, conserved C-terminal amino acids in PLD1 and PLD2 play essential roles for catalytic activity of both enzymes [20], [21].

Our recent studies showed that rPLD1 is palmitoylated and Ser/Thr phosphorylated in vivo [22]. Both these modifications require association between the N- and C-terminal halves of rPLD1 [22]. Although neither palmitoylation nor phosphorylation was found to be essential for the catalysis of rPLD1 [22], they influenced its association with membranes [22] and other studies have shown that palmitoylation specifies the cellular location of hPLD1 [23]. The N-terminal sequence of rPLD1 was also required for palmitoylation and Ser/Thr phosphorylation of rPLD1 [22]. Truncation of the first 168 amino acids diminished both of these modifications, as well as activation by PKC [18], [24], [25]. In present study, we found that the 1–168 N-terminal fragment can associate with the truncated rPLD1 mutant to restore multiple properties of the enzyme. We also found that rPLD2 was not detectably phosphorylated on Ser/Thr residues. However, it was shown to be palmitoylated and we examined the effect of this modification on its catalytic activity.

Section snippets

Materials

4β-Phorbol 12-myristate 13-acetate, bovine serum albumin, Triton X-100 and microcystin were from Sigma. Phosphatidylbutanol (PtdBut) standard was from Avanti Polar Lipids. [3H]Myristic acid and [3H]palmitic acid were from NEN Life Science Products. Protein A-agarose beads, Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin and fetal bovine serum (FBS) were from Life Technologies. The transfection reagent FuGENE6 and the protease inhibitor cocktail were from Roche Molecular

Restoration of the PKC response and Ser/Thr phosphorylation of an N-terminal truncation mutant of rPLD1

The N-terminus of mammalian PLD1 is important for PKC activation of the enzyme [18], [24], [25]. Compared with wild-type enzyme, rPLD1 that lacks the first 168 amino acids (rPLD1(ΔN168)) has increased basal activity, but no response to PMA in COS 7 cells. In addition, truncation of the first 168 amino acids of rPLD1 diminishes palmitoylation and Ser/Thr phosphorylation of the enzyme [22]. Thus, the N-terminal 168 amino acids of rPLD1 are important for many properties of the enzyme. Therefore,

Discussion

Our previous characterization of rPLD1 showed that the first 168 amino acids are very important for many properties of the enzyme [18]. Truncating these amino acids increases the basal activity of the enzyme, but renders it unresponsive to activation by PKC [18], [24], [25]. In addition, both palmitoylation and Ser/Thr phosphorylation are diminished when the N-terminal 168 amino acids are deleted [22]. In this study, we found that multiple properties of rPLD1(ΔN168) were restored when this

Acknowledgements

We wish to thank Judy Nixon for typing the manuscript.

References (32)

  • J.H. Exton

    Biochim. Biophys. Acta

    (1998)
  • Y. Zhang et al.

    J. Biol. Chem.

    (2000)
  • S.M. Hammond et al.

    J. Biol. Chem.

    (1995)
  • S.M. Hammond et al.

    J. Biol. Chem.

    (1997)
  • T. Kodaki et al.

    J. Biol. Chem.

    (1997)
  • W.C. Colley et al.

    Curr. Biol.

    (1997)
  • I. Lopez et al.

    J. Biol. Chem.

    (1998)
  • J.B. Park et al.

    J. Biol. Chem.

    (2000)
  • H. Watanabe et al.

    Biochim. Biophys. Acta

    (2000)
  • R. Slaaby et al.

    J. Biol. Chem.

    (1998)
  • A.J. Morris et al.

    Trends Pharmacol. Sci.

    (1996)
  • E.G. Koonin

    Trends Biochem. Sci.

    (1996)
  • Z. Xie et al.

    J. Biol. Chem.

    (1998)
  • Z. Xie et al.

    J. Biol. Chem.

    (2000)
  • Z. Xie et al.

    J. Biol. Chem.

    (2001)
  • J.M. Sugars et al.

    J. Biol. Chem.

    (1999)
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