|Budget Amount *help
¥13,300,000 (Direct Cost : ¥13,300,000)
Fiscal Year 2001 : ¥3,500,000 (Direct Cost : ¥3,500,000)
Fiscal Year 2000 : ¥3,500,000 (Direct Cost : ¥3,500,000)
Fiscal Year 1999 : ¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 1998 : ¥3,300,000 (Direct Cost : ¥3,300,000)
Mammalian phospholipase D (PLD) is a novel signal transducing enzyme, which is believed to play roles in a wide variety of cell functions. Two mammalian PLD isozymes, PLD1 and PLD2, have been identified. However, activation mechanisms and physiological functions of each PLD isozymes still remains to be clarified.
(1) Identification of the RhoA binding site of PLD1a : We have previously identified RhoA as the PLD1a activating factor. I clarified a RhoA binding site of PLDla using east two-hybrid and in vitro interaction systems. PLD1a comprises of 1074 amino acid residues, and its C-terminal region of 712-1074 amino acids was found to be the region of RhoA binding site.
(2) Investigation of downstream signaling molecules of PLD : PLD transmits signals to downstream molecules through its product phosphatidic acid (PA). However, target molecules of PA have not yet been identified. I found that the PLD product PA synergistically activates phosphatidylinositol 4-phosphate 5-kinase (PI4P 5-kin
ase) with the small G protein ARF. In addition, protein kinase Cε was found to be strongly activated by PA than by phosphatidylserine, indicating that protein kinase Cε and PI4P 5-kinase are downstream signaling molecules of PLD.
(3) Investigation of physiological functions of PLD2 : Although PLD1 has been suggested to play roles in membrane trafficking, secretion, and O_<2><-> production in neutrophils, physiological functions of PLD2 have not yet been thoroughly examined. In the present study, I found that overexpressed PLD2, but not PLD1, translocates to ruffling membranes produced by EGF stimulation of HeLa cells, suggesting that PLD2 is involved in membrane raffle formation. I also obtained results indicating that PLD2 plays a crucial role in neurite remodeling. Overexpressed PLD2, but not PLD1, extremely extended neurites produced by NGF-stimulated PC12 cells. When PLD2 was overexpressed in cerebellar granule neurons, axon produced during cell culture were significantly extended. Furthermore, It was found that stimulation of axon extension by L1 stimulation of cerebellar granule neurons was completely blocked by primary alcohols, which suppress PLD-catalyzed PA production. From these results, it is concluded that PLD2 is a critical signaling molecule in signal transduction of membrane dynamics.
(4) Activation mechanisms of PLD2 : Although activating factors of PLD1 have been identified, activation mechanisms of PLD2 remains to be clarified. Here I found that PLD2 is activated by ERK and p38 MAP kinases. In PC12 cells where PLD2 was overexpressed, PLD activity was stimulated by NGF, and the activation was suppressed by the ERK inhibitor PD98059 and the p38 inhibitor SB203580.Furthermore, active forms of MEK and MKK6 stimulated PLD2 activity overexpressed in PC12 cells. PLD activation was also observed in cerebellar granule neurons, in which only PLD2 is expressed, by L1 stimulation, and this L1-stimulated PLD activation was inhibited by the MEK inhibitor U0126. Less