The activation of ezrin-radixin-moesin proteins is regulated by netrin-1 through Src kinase and RhoA/Rho kinase activities and mediates netrin-1-induced axon outgrowth.

The receptor Deleted in Colorectal Cancer (DCC) mediates the attractive response of axons to the guidance cue netrin-1 during development. On netrin-1 stimulation, DCC is phosphorylated and induces the assembly of signaling complexes within the growth cone, leading to activation of cytoskeleton regulators, namely the GTPases Rac1 and Cdc42. The molecular mechanisms that link netrin-1/DCC to the actin machinery remain unclear. In this study we seek to demonstrate that the actin-binding proteins ezrin-radixin-moesin (ERM) are effectors of netrin-1/DCC signaling in embryonic cortical neurons. We show that ezrin associates with DCC in a netrin-1-dependent manner. We demonstrate that netrin-1/DCC induces ERM phosphorylation and activation and that the phosphorylation of DCC is required in that context. Moreover, Src kinases and RhoA/Rho kinase activities mediate netrin-1-induced ERM phosphorylation in neurons. We also observed that phosphorylated ERM proteins accumulate in growth cone filopodia, where they colocalize with DCC upon netrin-1 stimulation. Finally, we show that loss of ezrin expression in cortical neurons significantly decreases axon outgrowth induced by netrin-1. Together, our findings demonstrate that netrin-1 induces the formation of an activated ERM/DCC complex in growth cone filopodia, which is required for netrin-1-dependent cortical axon outgrowth.

The UBL domain of PLIC-1 regulates aggresome formation.

Defects in protein folding and the proteasomal pathway have been linked with many neurodegenerative diseases. PLIC-1 (protein linking IAP to the cytoskeleton) is a ubiquitin-like protein that binds to the ubiquitin-interacting motif (UIM) of the proteasomal subunit S5a. Here, we show that PLIC-1 also binds to the UIM proteins ataxin 3--a deubiquitinating enzyme--HSJ1a--a co-chaperone--and EPS15 (epidermal growth factor substrate 15)--an endocytic protein. Using a polyglutamine (polyQ) disease model, we found that both endogenous PLIC-1 and EPS15 localize to perinuclear aggresomes, and that polyQ enhances their in vivo interaction. We show that knockdown of PLIC-1 and EPS15 by RNA interference reduces aggresome formation. In addition, PLIC-1(DeltaUBL) functions as a dominant-negative mutant, blocking both polyQ transport to aggresomes and the association of EPS15 with dispersed aggregates. We also show that PLIC-1 is upregulated by arsenite-induced protein misfolding. These results indicate a role for PLIC-1 in the protein aggregation-stress pathway, and we propose a novel function for the ubiquitin-like (UBL) domain--by means of UBL-UIM interactions--in transport to aggresomes.

The amino terminus of the glial glutamate transporter GLT-1 interacts with the LIM protein Ajuba.

We have identified a cytoplasmic LIM protein, Ajuba, which interacts with the amino terminus of GLT-1, the most abundant plasma membrane glutamate transporter in the brain. Ajuba has a cytoplasmic location when expressed alone in COS cells, but translocates to colocalize with GLT-1 at the plasma membrane when GLT-1 is coexpressed. Ajuba is expressed in cerebellum, cortex, hippocampus, and retina and also in organs outside the CNS. Ajuba is found with GLT-1 in astrocytes, cerebellar Bergmann glia and retinal neurons, and antibodies to Ajuba coimmunoprecipitate GLT-1 from brain. For GLT-1 expressed in COS cells, coexpression of Ajuba did not affect the transporter's K(m) or V(max) for glutamate. Since Ajuba is known to activate MAP kinase enzymes, and its homologue Zyxin binds to cytoskeletal proteins, we propose that Ajuba is a scaffolding protein allowing GLT-1 to regulate intracellular signaling or interact with the cytoskeleton.