Phosphorylation and activation of phospholipase d1 by protein kinase C in vivo: Determination of multiple phosphorylation sites

Yong Kim, Jung Min Han, Jong Bae Park, Sang Do Lee, Yong Seok Oh, Churo Chung, Taehoon G. Lee, Jae Ho Kim, Seung Kiel Park, Jong Shin Yoo, Pann Ghill Suh, Sung Ho Ryu

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66 Citations (Scopus)

Abstract

Protein kinase C (PKC) is an important regulator of phospholipase D1 (PLD1). Currently there is some controversy about a phosphorylation-dependent or -independent mechanism of the activation of PLD1 by PKC. To solve this problem, we examined whether PLD1 is phosphorylated by PKC in vivo. For the first time, we have now identified multiple basal phophopeptides and multiple phorbol myristate acetate (PMA) induced phosphopeptides of endogenous PLD1 in 3Y1 cells as well as of transiently expressed PLD1 in COS-7 cells. Down regulation or inhibition of PKC greatly attenuated the PMA-induced phosphorylation as well as the activation of PLD1. In the presence of PMA, purified PLD1 from rat brain was also found to be phosphorylated by PKCα in vitro at multiple sites generating seven distinct tryptic phosphopeptides. Four phosphopeptides generated in vivo and in vitro correlated well with each other, suggesting direct phosphorylation of PLD1 by PKCα in the cells. Serine 2, threonine 147, and serine 561 were identified as phosphorylation sites, and by mutation of these residues to alanine these residues were proven to be specific phosphorylation sites in vivo. Interestingly, threonine 147 is located in the PX domain and serine 561 is in the negative regulatory 'loop' region of PLD1. Mutation of serine 2, threonine 147, or serine 561 significantly reduced PMA-induced PLD1 activity. These results strongly suggest that phosphorylation plays a pivotal role in PLD1 regulation in vivo.

Original languageEnglish
Pages (from-to)10344-10351
Number of pages8
JournalBiochemistry
Volume38
Issue number32
DOIs
Publication statusPublished - 1999 Aug 10

All Science Journal Classification (ASJC) codes

  • Biochemistry

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