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)P(3) levels after stimulation with the chemoattractant cAMP was blocked >95% by 60 microM LY294002 with half-maximal effect at 5 microM. 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.
Loovers
Harriët M
HM
Department of Molecular Cell Biology, University of Groningen, 9751 NN Haren, The Netherlands.
Postma
Marten
M
Keizer-Gunnink
Ineke
I
Huang
Yi Elaine
YE
Devreotes
Peter N
PN
van Haastert
Peter J M
PJ
D000199:Actins / Q000378:metabolism; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002449:Cell Aggregation / Q000187:drug effects; D049108:Cell Enlargement / Q000187:drug effects; D016764:Cell Polarity; D048430:Cell Shape / Q000187:drug effects; D002630:Chemotactic Factors / Q000494:pharmacology*; D002633:Chemotaxis / Q000187:drug effects; D002867:Chromones / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology*; D004023:Dictyostelium / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics; D010716:Phosphatidylinositols / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D015800:Protozoan Proteins / Q000032:analysis / Q000235:genetics; D015398:Signal Transduction; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines
D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002630:Chemotactic Factors; D002867:Chromones; D000242:Cyclic AMP; D004791:Enzyme Inhibitors; D049452:Green Fluorescent Proteins; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D010716:Phosphatidylinositols; D000081082:Phosphoinositide-3 Kinase Inhibitors; D015800:Protozoan Proteins; D013844:Thiazoles; D053778:Thiazolidines; C089420:dagA protein, Dictyostelium; C118303:phosphoinositide-3,4,5-triphosphate; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C037067:latrunculin A