Distinct Roles of PI(3,4,5)P3 during Chemoattractant Signaling in Dictyostelium: A Quantitative In Vivo Analysis by Inhibition of PI3-KinaseV

• Published in: Molecular biology of the cell Vol. 17; no. 4; pp. 1503 - 1513
• Main Authors: Loovers, Harriët M., Postma, Marten, Keizer-Gunnink, Ineke, Huang, Yi Elaine, Devreotes, Peter N., van Haastert, Peter J.M.
• Format: Journal Article
• Language: English
• Published: The American Society for Cell Biology 01-04-2006
• ISSN: 1059-1524
• DOI: 10.1091/mbc.E05-09-0825

The role of PI(3,4,5)P
3
in
Dictyostelium
signal transduction and chemotaxis was investigated using the PI3-kinase inhibitor LY294002 and
pi3k
-null cells. The increase of PI(3,4,5)P
3
levels after stimulation with the chemoattractant cAMP was blocked >95% by 60 μM LY294002 with half-maximal effect at 5 μM. This correlated well with the inhibition of the membrane translocation of the PH-domain protein, PHcracGFP. LY294002 did not reduce cAMP-mediated cGMP production, but significantly reduced the cAMP response up to 75% in wild type and completely in
pi3k
-null cells. LY294002-treated cells were round, not elongated as control cells. Interestingly, cAMP induced a time and dose-dependent recovery of cell elongation. These elongated LY294002-treated wild-type and
pi3k
-null cells exhibited chemotactic orientation toward cAMP that is statistically identical to chemotactic orientation of control cells. In control cells, PHcrac-GFP and F-actin colocalize upon cAMP stimulation. However, inhibition of PI3-kinases does not affect the first phase of the actin polymerization at a wide range of chemoattractant concentrations. Our data show that severe inhibition of cAMP-mediated PI(3,4,5)P
3
accumulation leads to inhibition of cAMP relay, cell elongation and cell aggregation, but has no detectable effect on chemotactic orientation, provided that cAMP had sufficient time to induce cell elongation.