Regulation of Cdc42 activity by Nudel. (1) Environmental stimuli for cell migration activate both GEFs and Erk. GEFs activate Cdc42. (2) Erk phosphorylates Nudel and facilitates Nudel's leading-edge localization. (3) Nudel and active Cdc42 compete against each other for binding Cdc42GAP to regulate regional activation of Cdc42. (4) Active Cdc42 triggers downstream effects.

Image courtesy of Dr Xueliang Zhu, Institute of Biochemistry and Cell Biology, Shanghai, China.

Activation of Cdc42 at the leading edge is crucial for cell movement. Like other small GTPases, Cdc42 cycles between active GTP-bound and inactive GDP-bound states. It is activated by guanine nucleotide exchange factors (GEFs) and inactivated by the GTPase-activating protein (GAP) Cdc42GAP. Nudel is a known regulator of the microtubule minus-end directed motor dynein. Xueliang Zhu and colleagues now describe a new role for Nudel in the modulation of Cdc42 activity through its association with Cdc42GAP.

The authors first observed that silencing of Nudel reduced cell motility and migration in wounded NIH3T3 monolayers. Nudel-depleted cells displayed defective protrusion and had a randomly localized centrosome rather than one positioned between the nucleus and the wound edge. Thus, Nudel depletion impairs two critical aspects of migration: centrosome polarization and protrusion. Centrosome polarization in moving cells depends upon backward nuclear movement and centrosome maintenance at the cell centroid. Whereas both events require Cdc42 activity, only dynein affects centrosome maintenance. In contrast, Nudel knockdown abolished nuclear translocation, suggesting that it may regulate migration independently of dynein.

To understand how Nudel may affect cell migration, Xueliang Zhu and colleagues searched for proteins that associate with Nudel and isolated Cdc42GAP. Cdc42GAP interacted with either Cdc42 or Nudel, but interestingly never concurrently. Furthermore, Nudel decreased the amount of Cdc42GAP that coimmunoprecipitated with active Cdc42, suggesting that Nudel sequesters Cdc42GAP from active Cdc42 in vitro. Active Cdc42 levels correlated with levels of Nudel, showing that Nudel activates Cdc42 by sequestering Cdc42Gap.

In a scratch assay, Nudel knockdown reduced Cdc42 activation, and Cdc42 effectors, such as protein kinase C and the MT plus-end-binding protein APC (Adenomatous Polyposis Coli), failed to localize at the leading edge. Together, these results show that Nudel functions in vivo to regionally activate Cdc42 in migrating cells.

Nudel is a substrate of Erk (extracellular signal-regulated protein kinase), whose activation in response to various external stimuli is an important upstream event in cell migration. Nudel enrichment at the leading edge was Erk-dependent in wounded cell monolayers. Moreover, overexpression of a Nudel phosphorylation-dead mutant led to random centrosome positioning and inhibited cell migration, showing that Nudel phosphorylation is important for both its localization and function at the cells' leading edge.

This work unravels a new role for Nudel that is independent of dynein - Nudel increases the levels of active Cdc42 at the cells' leading edge by sequestering its negative regulator Cdc42GAP. Nudel accumulation at the leading edge depends upon phosphorylation, but further studies are needed to uncover how phosphorylated Nudel is recruited to the leading edge.

Author: Kim Baumann