Compartmentalization of phospholipase D regulation : (Localization of phospholipase D1 phosphorylation in caveolin-enriched membrane domain)

다국어 초록 (Multilingual Abstract)

Mammalian phsopholipase D(PLD) has been known as an important enzyme for the amplification of the signals for the proliferation, secretion and phagocytosis. cDNAs of the two isozymes, PLD1 and PLD2, have been cloned and the properties of the enzymes were studied. However, the molecular mechanism and functional significance of the regulation of each isozyme have not been well known yet.
Protein kinase C(PKC) is a major regulator of PLD. PLD1 has been considered as the isozyme avtivated by PKC. Serine 2, threonine 147, and serine 561 residues of phospholipase D1(PLD1) were determined as sites of phosphorylation by PKC(Kim et al.(1999) Biochemistry 38, 10344-10351). In our present study, a triple mutation of these phosphorylation sites diminished markedly PMA-induced PLD1 activity in COS-7 cells. Transiently expressed PLD1 revealed itself in multiple bands in SDS-PAGE, and this banding pattern was sensitive to protein phosphatase 1, suggesting that the multiple bands are due to phosphorylations at multiple sites. We looked at the location of the PLD1 phosphorylation by PKC by observing PMA-induced band shifts and by use of anti-phospho-PLD1 monoclonal antibody. The shifted PMA-induced proteins and the immunoreactivity of the anti-phospho-PLD1 antibody were mainly found in the caveolin-enriched membrane(CEM) fraction. Depletion of cellular cholesterol led to a loss of this compartmentalization of phosphorylated PLD1 in the CEM. Replacement of the cellular cholesterol led to the restoration of phosphorylated PLD1 in the CEM. Immunocytochemical studies of COS-7 cells revealed that PLD1 was localized in the plasma membrane as well as in the vesicular structures in the cytoplasm but the phosphorylation of PLD1 occurred only in the plasma membrane. Our results, therefore, show that phosphorylation and thereby activation of PLD1 by PKC occurs in the caveolin and cholesterol-enriched low-density domain of the plasma membrane in COS-7 cells.
Regulation of PLD2 by direct interaction with several cytoskeletal proteins and their implications in cellular functions may be presented and discussed, if time is available.