Phosphatidic acid regulates the affinity of the murine phosphatidylinositol 4-phosphate 5-kinase-Ibeta for phosphatidylinositol-4-phosphate.
Type I phosphatidylinositol 4-phosphate 5-kinase (PI4P5K) catalyzes the phosphorylation of phosphatidylinositol 4 phosphate [PI(4)P] at carbon 5, producing phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2]. Phosphatidic acid (PA) activates PI4P5K in vitro and plays a central role in the activation of PIP5K pathways in vivo. This report demonstrates that actin fiber formation in murine fibroblasts involves PA activation of PIP5Ks and defines biochemical interactions between PA and the PIP5Ks. Inhibition of phospholipase D production of PA results in the loss of actin fibers. Overexpression of the beta isoform of the type I murine phosphatidylinositol 4-phosphate 5-kinase (mPIP5K-Ibeta) maintains actin fiber structure in the face of phospholipase D inhibition. PA activates mPIP5K-Ibeta by direct binding to mPIP5K-Ibeta through both electrostatic and hydrophobic interactions, with the fatty acid acyl chain length and degree of saturation acting as critical determinants of binding and activation. Furthermore, kinetic analysis suggests that phosphorylation of the PI(4)P substrate does not follow classical Michaelis-Menten kinetics. Instead, the kinetic data are consistent with a model in which mPIP5K-Ibeta initially binds to the lipid micelle and subsequently binds the PI(4)P substrate. In addition, the kinetics indicate substrate inhibition, suggesting that mPIP5K-Ibeta contains an inhibitory PI(4)P-binding site. These results suggest a model in which mPIP5K-Ibeta is surrounded by PI(4)P, but is unable to catalyze its conversion to PI(4,5)P2 unless PA is bound.
Jarquin-Pardo
Marta
M
Feist-Weiller Cancer Center and Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, Louisiana 71130-3932, USA.
Fitzpatrick
Abbie
A
Galiano
Floyd J
FJ
First
Eric A
EA
Davis
J Nathan
JN
D008841:Actin Cytoskeleton / Q000378:metabolism; D000818:Animals; D004789:Enzyme Activation; D007700:Kinetics; D051379:Mice; D041681:NIH 3T3 Cells; D010712:Phosphatidic Acids / Q000494:pharmacology*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011485:Protein Binding; D015398:Signal Transduction
D010712:Phosphatidic Acids; D018129:Phosphatidylinositol Phosphates; C037178:phosphatidylinositol 4-phosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D010739:Phospholipase D