Regulation of phospholipase D isoenzymes by transforming Ras and atypical protein kinase C-iota

The activation of phospholipase D (PLD) by transforming Ras is well documen ted. Although two distinct PLD isoforms, PLD1 and PLD2, have been cloned fr om mammalian cells, it has remained unclear whether both isoenzymes are act ivated by Ras and, if this is the case, whether they are stimulated by a co mmon mechanism. In the present study we show that expression of transformin g Ras in HCH mouse mammary epithelia] cells enhanced the activity of endoge nous PLD. Co-expression of Ras with either PLD1 b or PLD2 resulted in eleva ted activities of both PLD isoenzymes in HC11 cells, indicating that transf orming Ras was capable of activating both PLD isoforms in vivo. Ras-induced activation of PLD was resistant to the protein kinase C (PKC) inhibitor GF 109203X, which preferentially affects conventional- and novel-type PKCs, bu t sensitive to Ro-31-8220, which inhibits atypical PKCs more effectively. C o-transfection of atypical PKC-iota with either PLD1b or PLD2 led to a sele ctive activation of PLD2 by PKC-iota, whereas PLD1b was not affected. PLD1b , however, was found to be a potent activator of PKC-iota. whereas PLD2 was less effective in this respect. The data suggest that PKC-iota acts upstre am of PLD2 and that PLD1b is implicated in the activation of PKC-iota. The data are discussed as indicating a putative signalling cascade comprising R as-->PLD1b-->PKC-iota --> PLD2. Evidence for the implication of this pathwa y in the transcriptional regulation of cyclin D1 is also presented.