Distinct roles of PI(3,4,5)P3 during chemoattractant signaling in Dictyostelium:: A quantitative in vivo analysis by inhibition of PI3-kinase

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)P3 levels after stimulation with the chemoattractant cAMP was blocked > 95% by 60 mu M LY294002 with half-maximal effect at 5 mu M. 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.