Beteiligung der Phospholipase D an der Regulation der Synthese von Phosphatidylinositol-4,5-bisphosphat durch Arf- und Rho-GTPasen
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vor 20 Jahren
The reaction product of phospholipase D (PLD), phosphatidic acid
(PA), was found to stimulate
phosphatidylinositol-4-phosphate-5-kinase (PIP-5-kinase) activity
in vitro. In the present study, we have examined wether PLD affects
the synthesis of phosphatidylinositol 4,5-bisphosphate (PIP2) by
PIP-5-kinase. Overexpression of PLD isoforms in HEK-293 cells led
to an increase in PIP-5-kinase activity and to elevated PIP2 levels
in intact cells. As both PLD and PIP-5-kinase are stimulated by the
GTPases Arf1 and RhoA, we investigated in the following, if PLD is
involved in the regulation of PIP2 synthesis by these GTPases. Both
PLD1- and PLD2-induced PIP2 synthesis was completely blocked by
coexpression of catalytically inactive Arf1 T31N. Reversely, the
effect of constitutive active Arf1 Q71L was fully inhibited by
catalytically inactive PLD constructs. Whereas the effects of Arf1
Q71L and wild-type PLD2 were additive, coexpression of Arf1 Q71L
with wild-type PLD1 led to a synergistic increase in PIP-5-kinase
activity. Previously, we have shown that RhoA regulates the
activity of PLD and PIP-5-kinase by its downstream effector
Rho-kinase. Expression of small amounts of inactive PLD1, but not
of PLD2, nearly completely abolished Rho-kinase-stimulated
PIP-5-kinase activity. Also expression of a non-phosphorylatable
mutant of cofilin, which participates in the signalling cascade
from RhoA via Rho-kinase and LIM-kinase to PLD1, suppressed the
stimulating effect of Rho-kinase on PIP2 synthesis. These findings
suggest that PLD1 is involved in the stimulation of PIP-5-kinase by
Arf1 as well as by RhoA and Rho-kinase. After sucrose density
gradient centrifugation of HEK-293 cell lysates, we isolated two
separate PIP2 pools. PLD1 and Arf1 selectively control the
non-caveolar PIP2 pool in the high density fraction, whereas PLD2
affected PIP2 in both pools. In summary, these data suggest that
particularly PLD1, apparently by the production of PA, functions as
a physiological regulator of PIP-5-kinase that controls the
synthesis of cellular PIP2 downstream to Arf1 and RhoA.
(PA), was found to stimulate
phosphatidylinositol-4-phosphate-5-kinase (PIP-5-kinase) activity
in vitro. In the present study, we have examined wether PLD affects
the synthesis of phosphatidylinositol 4,5-bisphosphate (PIP2) by
PIP-5-kinase. Overexpression of PLD isoforms in HEK-293 cells led
to an increase in PIP-5-kinase activity and to elevated PIP2 levels
in intact cells. As both PLD and PIP-5-kinase are stimulated by the
GTPases Arf1 and RhoA, we investigated in the following, if PLD is
involved in the regulation of PIP2 synthesis by these GTPases. Both
PLD1- and PLD2-induced PIP2 synthesis was completely blocked by
coexpression of catalytically inactive Arf1 T31N. Reversely, the
effect of constitutive active Arf1 Q71L was fully inhibited by
catalytically inactive PLD constructs. Whereas the effects of Arf1
Q71L and wild-type PLD2 were additive, coexpression of Arf1 Q71L
with wild-type PLD1 led to a synergistic increase in PIP-5-kinase
activity. Previously, we have shown that RhoA regulates the
activity of PLD and PIP-5-kinase by its downstream effector
Rho-kinase. Expression of small amounts of inactive PLD1, but not
of PLD2, nearly completely abolished Rho-kinase-stimulated
PIP-5-kinase activity. Also expression of a non-phosphorylatable
mutant of cofilin, which participates in the signalling cascade
from RhoA via Rho-kinase and LIM-kinase to PLD1, suppressed the
stimulating effect of Rho-kinase on PIP2 synthesis. These findings
suggest that PLD1 is involved in the stimulation of PIP-5-kinase by
Arf1 as well as by RhoA and Rho-kinase. After sucrose density
gradient centrifugation of HEK-293 cell lysates, we isolated two
separate PIP2 pools. PLD1 and Arf1 selectively control the
non-caveolar PIP2 pool in the high density fraction, whereas PLD2
affected PIP2 in both pools. In summary, these data suggest that
particularly PLD1, apparently by the production of PA, functions as
a physiological regulator of PIP-5-kinase that controls the
synthesis of cellular PIP2 downstream to Arf1 and RhoA.
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