Protein kinase Cα and integrin-linked kinase mediate the negative axon guidance effects of Sonic hedgehog.

In addition to its role as a morphogen, Sonic hedgehog (Shh) has also been shown to function as a guidance factor that directly acts on the growth cones of various types of axons. However, the noncanonical signaling pathways that mediate the guidance effects of Shh protein remain poorly understood. We demonstrate that a novel signaling pathway consisting of protein kinase Cα (PKCα) and integrin-linked kinase (ILK) mediates the negative guidance effects of high concentration of Shh on retinal ganglion cell (RGC) axons. Shh rapidly increased Ca(2+) level and activated PKCα and ILK in the growth cones of RGC axons. By in vitro kinase assay, PKCα was found to directly phosphorylate ILK on threonine-173 and -181. Inhibition of PKCα or expression of a mutant ILK with the PKCα phosphorylation sites mutated (ILK-DM), abolished the Shh-induced macropinocytosis, growth cone collapse and repulsive axon turning. In vivo, expression of a dominant negative PKCα or ILK-DM disrupted RGC axon pathfinding at the optic chiasm but not the projection toward the optic disk, supporting that this signaling pathway plays a specific role in Shh-mediated negative guidance effects.

Negative guidance factor-induced macropinocytosis in the growth cone plays a critical role in repulsive axon turning.

Macropinocytosis is a type of poorly characterized fluid-phase endocytosis that results in formation of relatively large vesicles. We report that Sonic hedgehog (Shh) protein induces macropinocytosis in the axons through activation of a noncanonical signaling pathway, including Rho GTPase and nonmuscle myosin II. Macropinocytosis induced by Shh is independent of clathrin-mediated endocytosis but dependent on dynamin, myosin II, and Rho GTPase activities. Inhibitors of macropinocytosis also abolished the negative effects of Shh on axonal growth, including growth cone collapse and chemorepulsive axon turning but not turning per se. Conversely, activation of myosin II or treatment of phorbol ester induces macropinocytosis in the axons and elicits growth cone collapse and repulsive axon turning. Furthermore, macropinocytosis is also induced by ephrin-A2, and inhibition of dynamin abolished repulsive axon turning induced by ephrin-A2. Macropinocytosis can be induced ex vivo by high Shh, correlating with axon retraction. These results demonstrate that macropinocytosis-mediated membrane trafficking is an important cellular mechanism involved in axon chemorepulsion induced by negative guidance factors.