Shh-mediated degradation of Hhip allows cell autonomous and non-cell autonomous Shh signalling.

The distribution of Sonic Hedgehog (Shh) is a highly regulated and critical process for development. Several negative feedback mechanisms are in place, including the Shh-induced upregulation of Hedgehog-interacting protein (Hhip). Hhip sequesters Shh, leading to a non-cell autonomous inhibition of the pathway. Hhip overexpression has a severe effect on neural tube development, raising the question why normal sites of Hhip expression have a seemingly unimpaired response to Shh. Here we show that although Hhip is able to leave its sites of synthesis to inhibit Shh non-cell autonomously, activation of Smoothened (Smo) drastically increases Hhip internalization and degradation cell autonomously. Although Hhip is unable to cell autonomously inhibit the consequences of Smo activation, it can inhibit the Shh response non-cell autonomously. Our data provide a mechanism by which the Shh ligand can activate the response and negate cell autonomous effects of Hhip, while Hhip can still induce non-cell autonomous inhibition.

Ptch2 mediates the Shh response in Ptch1-/- cells.

The Hedgehog (Hh) signaling response is regulated by the interaction of three key components that include the sonic hedgehog (Shh) ligand, its receptor patched 1 (Ptch1) and the pathway activator smoothened (Smo). Under the prevailing model of Shh pathway activation, the binding of Shh to Ptch1 (the key Shh receptor) results in the release of Ptch1-mediated inhibition of Smo, leading to Smo activation and subsequent cell-autonomous activation of the Shh response. Consistent with this model, Ptch1(-/-) cells show a strong upregulation of the Shh response. Our finding that this response can be inhibited by the Shh-blocking antibody 5E1 indicates that the Shh response in Ptch1(-/-) cells remains ligand dependent. Furthermore, we find that Shh induces a strong response in Ptch1(-/-);Shh(-/-) cells, and that Ptch1(-/-) fibroblasts retain their ability to migrate towards Shh, demonstrating that Ptch1(-/-) cells remain sensitive to Shh. Expression of a dominant-negative Ptch1 mutant in the developing chick neural tube had no effect on Shh-mediated patterning, but expression of a dominant-negative form of patched 2 (Ptch2) caused an activation of the Shh response. This indicates that, at early developmental stages, Ptch2 functions to suppress Shh signaling. We found that Ptch1(-/-);Ptch2(-/-) cells cannot further activate the Shh response, demonstrating that Ptch2 mediates the response to Shh in the absence of Ptch1.

R-Ras controls membrane protrusion and cell migration through the spatial regulation of Rac and Rho.

Although it is known that the spatial coordination of Rac and Rho activity is essential for cell migration, the molecular mechanisms regulating these GTPases during migration are unknown. We found that the expression of constitutively activated R-Ras (38V) blocked membrane protrusion and random migration. In contrast, expression of dominant negative R-Ras (41A) enhanced migrational persistence and membrane protrusion. Endogenous R-Ras is necessary for cell migration, as cells that were transfected with siRNA for R-Ras did not migrate. Expression of R-Ras (38V) decreased Rac activity and increased Rho activity around the entire cell periphery, whereas expression of dominant negative R-Ras (41A) showed the converse, suggesting that R-Ras can spatially activate Rho and inactivate Rac. Consistent with this role, endogenous R-Ras localized and was preferentially activated at the leading edge of migratory cells in response to adhesion. The effects of R-Ras on cell migration are mediated by PI3-Kinase, as an effector mutant that uncouples PI3-Kinase binding from R-Ras (38V) rescued migration. From these data, we hypothesize that R-Ras plays a key role in cell migration by locally regulating the switch from Rac to Rho activity after membrane protrusion and adhesion.

Focal adhesion regulation of cell behavior.

Focal adhesions lie at the convergence of integrin adhesion, signaling and the actin cytoskeleton. Cells modify focal adhesions in response to changes in the molecular composition, two-dimensional (2D) vs. three-dimensional (3D) structure, and physical forces present in their extracellular matrix environment. We consider here how cells use focal adhesions to regulate signaling complexes and integrin function. Furthermore, we examine how this regulation controls complex cellular behaviors in response to matrices of diverse physical and biochemical properties. One event regulated by the physical structure of the ECM is phosphorylation of focal adhesion kinase (FAK) at Y397, which couples FAK to several signaling pathways that regulate cell proliferation, survival, migration, and invasion.

R-Ras promotes focal adhesion formation through focal adhesion kinase and p130(Cas) by a novel mechanism that differs from integrins.

R-Ras regulates integrin function, but its effects on integrin signaling pathways have not been well described. We demonstrate that activation of R-Ras promoted focal adhesion formation and altered localization of the alpha2beta1 integrin from cell-cell to cell-matrix adhesions in breast epithelial cells. Constitutively activated R-Ras(38V) dramatically enhanced focal adhesion kinase (FAK) and p130(Cas) phosphorylation upon collagen stimulation or clustering of the alpha2beta1 integrin, even in the absence of increased ligand binding. Signaling events downstream of R-Ras differed from integrins and K-Ras, since pharmacological inhibition of Src or disruption of actin inhibited integrin-mediated FAK and p130(Cas) phosphorylation, focal adhesion formation, and migration in control and K-Ras(12V)-expressing cells but had minimal effect in cells expressing R-Ras(38V). Therefore, signaling from R-Ras to FAK and p130(Cas) has a component that is Src independent and not through classic integrin signaling pathways and a component that is Src dependent. R-Ras effector domain mutants and pharmacological inhibition suggest a partial role for phosphatidylinositol 3-kinase (PI3K), but not Raf, in R-Ras signaling to FAK and p130(Cas). However, PI3K cannot account for the Src-independent pathway, since simultaneous inhibition of both PI3K and Src did not completely block effects of R-Ras on FAK phosphorylation. Our results suggest that R-Ras promotes focal adhesion formation by signaling to FAK and p130(Cas) through a novel mechanism that differs from but synergizes with the alpha2beta1 integrin.