Rab35 Functions in Axon Elongation Are Regulated by P53-Related Protein Kinase in a Mechanism That Involves Rab35 Protein Degradation and the Microtubule-Associated Protein 1B

Abstract

Rab35 is a key protein for cargo loading in the recycling endosome. In neuronal immortalized cells, Rab35 promotes neurite differentiation. Here we describe that Rab35 favors axon elongation in rat primary neurons in an activity-dependent manner. In addition, we show that the p53-related protein kinase (PRPK) negatively regulates axonal elongation by reducing Rab35 protein levels through the ubiquitin-proteasome degradation pathway. PRPK-induced Rab35 degradation is regulated by its interaction with microtubuleassociated protein 1B (MAP1B), a microtubule stabilizing binding protein essential for axon elongation. Consistently, axon defects found in MAP1B knock-out neurons were reversed by Rab35 overexpression or PRPK inactivation suggesting an epistatic relationship among these proteins. These results define a novel mechanism to support axonal elongation, by which MAP1B prevents PRPK-induced Rab35 degradation. Such a mechanism allows Rab35-mediated axonal elongation and connects the regulation of actin dynamics with membrane trafficking. In addition, our study reveals for the first time that the ubiquitin-proteasome degradation pathway regulates a Rab GTPase.

Emerging roles of MICAL family proteins – from actin oxidation to membrane trafficking during cytokinesis

“…As a newly discovered Rab35 effector, MICAL1 could potentially be involved in other processes in which Rab35 and actin remodelling have been implicated (reviewed in , such as endocytic recycling (Cauvin et al, 2016), phagocytosis (Egami et al, 2011), neurite outgrowth (Kobayashi and Fukuda, 2012;Villarroel-Campos et al, 2016), immunological synapse formation (Patino-Lopez et al, 2008) or the establishment of apical polarity in 3D organoids . The fact that each MICAL protein interacts with multiple Rabs suggests that Rabs other than Rab35 could potentially activate and thus locally regulate the actin-depolymerizing activity of MICAL family members in the cell.…”

Tuba activates Cdc42 during neuronal polarization downstream of the small GTPase Rab8a

“…The CRIB domain for Cdc42 activity was purified as described previously (Villarroel-Campos et. al, 2016b).…”

“…On other hand, it has been recently discovered that exocytic (secretory) and endocytic pathways are critical for neuronal differentiation and axonal elongation (Villarroel-Campos et al, 2016a;Villarroel-Campos et al, 2014;Villarroel-Campos et al, 2016b). In the secretory pathway, the small Rab GTPase Rab8 regulates the transport of exocytic vesicles from the trans-Golgi network (TGN) to the plasma membrane (Stenmark, 2009).…”

Regulation of plasma membrane expansion during axon formation

“…The trafficking of membrane components is an essential step for axon growth and differentiation. It involves organelles of the exocytic pathway, such as the rough endoplasmic reticulum (RER) and the Golgi apparatus (Bentley & Banker, ; Wojnacki and Galli, ), as well as endosomes (Villarroel‐Campos et al, ; Wojnacki and Galli, ). At the trans Golgi network (TGN), or eventually at recycling endosomes (RE), axonal membrane proteins are sorted and packed into appropriate carriers.…”

“…Together, these results suggest a common mechanism involving the exocyst for the insertion of PPVs and RE-derived vesicles. Rab35, another resident recycling endosomes GTPase has been proposed to promote neurite outgrowth in PC12 cells and axon elongation in cultured hippocampal neurons by regulating Cdc42 activity (Chevallier et al, 2009, Kobayashi and Fukuda, 2012, Villarroel-Campos et al, 2016b. By contrast, the RE resident GTPase, Arf6 negatively regulates axon outgrowth and trafficking of b1-integrin-containing RE (Hern andez- Deviez et al, 2004;Eva et al, 2012).…”