Mutually inhibitory Rac-RhoA signaling has been observed in biological processes including cell polarization, where such crosstalk helps to spatially segregate of Rac and RhoA activity (40
). In this study, we found a signaling network dominated by strong mutual inhibition between components mediating actin comets (RhoA, ROCK, and endocytosis) and components mediating membrane ruffles (Rac and Cdc42). This crosstalk seems to function as a driving force to sharply define actin phenotypes based on differential input signals originating from membrane lipids in a substrate-product relationship.
Under physiological conditions, there are four main ways whereby extracellular stimuli regulate PI(4,5)P2
concentration. These are: i
) PLC-mediated conversion of PI(4,5)P2
into inositol phosphates and diacylglycerol, ii
) PI(4)P5K-mediated phosphorylation of PI(4)P, iii
) PI3K-mediated phosphorylation of PI(4,5)P2
, and iv
) masking and unmasking of PI(4,5)P2
by binding proteins such as myristoylated alanine-rich C-kinase substrate (5
). Each mode is associated with a unique combination of changes in the concentration of lipid products. Previous work has shown that decreased PI(4,5)P2
concentration in response to acetylcholine receptor stimulation (mode i
) deactivate ion channel activity (13
). Here, we manipulated PI(4,5)P2
synthetically to mimic modes ii
and found that PI(4)P and PI(4,5)P2
play distinct roles in remodeling of the actin cytoskeleton. Our findings indicate that the consequence of an increase in PI(4,5)P2
will depend on accompanying changes in different but closely related signaling molecules such as PI(4)P. Such a combinatorial regulation of downstream signals may at least partly explain the ability of a limited set of membrane lipids to exhibit such functional diversity.
Our model proposes that RhoA and ROCK signaling are activated downstream of PI(4)P. There seems to be no report of direct regulation of RhoA by PI(4)P. However, there are dozens of GEFs and GAPs for RhoA that contain lipid binding domains (41
), making them ideal candidates for PI4P-mediated RhoA regulation. Indeed, phospholipids have been reported to modulate activity of p190 RhoGAP, one of the Rho GAPs (42
). PI4P has long been viewed as a precursor of PI(4,5)P2
, and has only recently begun to be appreciated as a signaling molecule (18
). Our data provide support for such a role.
A Lowe syndrome is a genetic disease with defective 5-phosphatase activity for PI(4,5)P2
that causes various developmental defects including eye, kidney and brain problems. Of particular note, actin comets have been observed in fibroblasts from Lowe syndrome patients (43
). These fibroblasts also exhibited cell migration defects (44
). As membrane ruffles offer the driving force for the cell motility, it is possible that the cell migration defects of these fibroblasts are due to a lack of ruffles through the mutual inhibition between actin comets and ruffles. Our study provides not only a powerful technology for probing the complexity of phosphoinositide signaling, but also insights into the molecular mechanism of this currently incurable disease.