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ATP-induced focal adhesion kinase activity is negatively modulated by phospholipase D2 in PC12 cells
1Division of Molecular and Life Sciences, Pohang University of Science and Technology, Korea.
Abstract:
Extracellular ATP has been known to modulate various cellular responses including mitogenesis, secretion and morphogenic activity in neuronal cells. In the ATP-induced morphogenic activity, focal adhesion kinase(s) such as Fak have been suggested to play a critical role. Binding of ATP to its specific cell surface receptor in PC12 cells induces phospholipase D (PLD) activity. However, the role of PLD on ATP-induced Fak activation in PC12 cells remains unclear. In this study, we investigated the role of PLD on the ATP-induced Fak activation and paxillin phosphorylation using two established cell lines: wild type PLD2- and lipase-inactive mutant PLD2-inducible PC12 cells. Stimulation of cells with ATP caused PLD2 activation via classical protein kinase C activation. ATP also induced Fak activation, and paxillin phosphorylation, and were dramatically reduced by wild type PLD2 overexpression but not by lipase-inactive mutant PLD2 overexpression. When the PC12 cells were pretreated with propranolol, a specific inhibitor for phosphatidic acid phosphohydrolase resulting in the accumulation of PA, ATP-induced Fak activation and paxillin phosphorylation were also reduced. We found that inhibition of tyrosine phosphatases by pervanadate completely blocked PLD2-dependent Fak and paxillin dephosphorylation. Taken together, we suggest that PLD2 activity might play a negative role in ATP-induced Fak and paxillin phosphorylation possibly through tyrosine phosphatases.
Insights
Extracellular ATP triggers cell changes, but phospholipase D2 (PLD2) appears to negatively regulate focal adhesion kinase (Fak) and paxillin phosphorylation in PC12 cells, potentially via tyrosine phosphatases.
Area of Science:
- Cell Biology
- Molecular Signaling
- Neuroscience
Background:
- Extracellular ATP (adenosine triphosphate) influences neuronal cell functions like mitogenesis and morphogenic activity.
- Focal adhesion kinase (Fak) is implicated in ATP-induced morphogenic activity.
- Phospholipase D (PLD) activation by ATP in PC12 cells is known, but its role in Fak activation is unclear.
Purpose of the Study:
- To investigate the role of PLD in ATP-induced Fak activation and paxillin phosphorylation in PC12 cells.
- To elucidate the mechanism by which PLD influences these signaling pathways.
Main Methods:
- Utilized wild type and lipase-inactive mutant PLD2-inducible PC12 cell lines.
- Stimulated cells with ATP and observed effects on PLD2, Fak, and paxillin.
- Employed propranolol (phosphatidic acid phosphohydrolase inhibitor) and pervanadate (tyrosine phosphatase inhibitor).
Main Results:
- ATP stimulation activated PLD2 via protein kinase C.
- Overexpression of wild type PLD2, but not inactive mutant PLD2, significantly reduced ATP-induced Fak activation and paxillin phosphorylation.
- Inhibition of phosphatidic acid phosphohydrolase with propranolol reduced ATP-induced Fak and paxillin phosphorylation.
- Inhibition of tyrosine phosphatases blocked PLD2-dependent dephosphorylation of Fak and paxillin.
Conclusions:
- PLD2 activity appears to negatively regulate ATP-induced Fak and paxillin phosphorylation in PC12 cells.
- This regulation may occur through the modulation of tyrosine phosphatases.