The Interaction of mPar3 with the Ubiquitin Ligase Smurf2 Is Required for the Establishment of Neuronal Polarity

Abstract

The Par polarity complex consisting of the evolutionarily conserved proteins mPar3, mPar6, and aPKC regulates cell polarity in many cell types including neurons. Here we show that mPar3 is required for the establishment of neuronal polarity and links the Smurf2 to Kinesin-2. The HECT domain E3 ubiquitin ligase Smurf2 ensures that neurons extend only a single axon by initiating the degradation of inactive Rap1B through the ubiquitin/ proteasome system. Its interaction with mPar3 is required to localize Smurf2 to growth cones and restrict Rap1B to the axon. Interfering with the binding of mPar3 to Kinesin-2 or Smurf2 to mPar3 and knockdown of mPar3 by RNAi disrupt the establishment of neuronal polarity through the failure to restrict Rap1B to a single neurite.During the differentiation of hippocampal neurons, unpolarized cells initially form several equivalent neurites, all of which have the potential to become an axon (stage 2) (1). Neuronal polarity is established when one of these is selected as the axon (stage 3) by a signaling pathway that includes PI3K, 4 the GTPases Rap1B, Cdc42, and Rac, the Par polarity complex, and GSK3␤ (2-8). The Par complex consisting of mPar3, mPar6, and aPKCs (PKC and PKC) plays an essential role in determining polarity in many cell types (9). mPar3 promotes axon extension downstream of Cdc42 through the STEF-dependent activation of Rac and the stimulation of aPKC. mPar6 interacts specifically with GTP-bound Cdc42 and Rac1 (10, 11). Binding of Cdc42 enhances the activity of mPar6-associated aPKCs (11,12). In addition, mPar3 interacts with the Rac-specific guanine nucleotide-exchange factors (GEFs) STEF and Tiam1 to control tight junction assembly and to mediate the activation of Rac by Cdc42 through the Par complex to promote axon growth (5,13,14). In neurons, mPar3 and mPar6 are present in all processes of non-polarized stage 2 neurons and become restricted to the axon of polarized stage 3 neurons (6, 7). Overexpression of mPar3 or mPar6 disrupts neuronal polarity and induces the extension of several long but Tau-1 negative processes instead of a single axon (4, 7). The mPar3 C terminus interacts with the Kinesin-2 subunit KIF3A (4). This interaction is essential for the transport of mPar3 and aPKCs into neurites and the establishment of neuronal polarity.We have shown that the sequential activity of the GTPases Rap1B and Cdc42 directs the establishment of polarity in hippocampal neurons downstream of PI3K (6). Initially, Rap1B is present at the tips of all neurites of unpolarized stage 2 neurons but, concomitant with the appearance of mPar3 in all processes, is restricted to a single neurite before the axon becomes distinguishable, and the Par complex is localized exclusively to the axon (6, 7). Rap1B is necessary and sufficient to specify axonal identity. Therefore, the restriction of Rap1B to a single neurite is an essential step in the establishment of neuronal polarity. This restriction is mediated by the selective degradation of Rap1B by the UPS and prevents the formation of ...

Phosphorylation of the Par Polarity Complex Protein Par3 at Serine 962 Is Mediated by Aurora A and Regulates Its Function in Neuronal Polarity

“…Briefly, the hippocampus from E18 rat embryos was dissected and dissociated, and neurons were plated onto glass coverslips coated with polyornithine (Sigma) at a density of 800,000 cells/coverslip in 24-well plates. Neurons were transfected 2 h after plating, using calcium phosphate coprecipitation (14). The cells were detached after the transfection and replated onto new coverslips at a lower density (40,000 -60,000 cells/coverslip in a 24-well plate).…”

“…Expression vectors for rat Aurora A (BC005425) and B (BC104676) were obtained from imaGenes (Berlin). Par3 deletion constructs comprising the CR1, PDZ1, PDZ2, PDZ3, and aPKC binding domain (aPKCBD) have been described before (14). The shRNA vector for the knockdown of Par3 was described previously (14).…”

“…Par3 deletion constructs comprising the CR1, PDZ1, PDZ2, PDZ3, and aPKC binding domain (aPKCBD) have been described before (14). The shRNA vector for the knockdown of Par3 was described previously (14). For the knockdown of Aurora A and Aurora B, the following target sequences were selected for the generation of shRNA vectors based on the pSHAG-1 or pSHAG-M2 plasmid (25,26): TAC TTG GCG AGG GAG AAG CAA AGC AAG TTC AT (Aurora A RNAi1), CTC ATT TCA AGA CTG TTA A (Aurora A RNAi2), TGC GGA CTT TGG CTG GTC TGT GCA TGC CCC AT (Aurora B RNAi1), CAG CCT TTC ACC ATT GAC A (Aurora B RNAi2), and GTA ATT CAC AGA GAC ATA A (Aurora B RNAi3).…”

“…Par3 and Par6 are present in all neurites of unpolarized neurons but become restricted to the axon in polarized stage 3 neurons, whereas active aPKC is found only at the tip of the axon (11,13,15). Par3 overexpression or suppression and aPKC inhibition result in defects in neuronal polarity and the loss of axons (14,15,(17)(18)(19). Par3 performs multiple function in neurons and mediates the transport of the Par complex and Smurf2 and the activation of Rac through Stef.…”

“…The Par polarity complex is required not only in Drosophila neuroblasts but also in rodent hippocampal neurons for the establishment of polarity, where it acts downstream of phosphoinositide 3-kinase and the GTPases Rap1B and Cdc42 (13)(14)(15). Cultured hippocampal neurons initially extend several short neurites (stage 2) (16).…”