p250GAP Is a Novel Player in the Cdh1-APC/Smurf1 Pathway of Axon Growth Regulation

<div><p>Axon growth is an essential process during brain development. The E3 ubiquitin ligase Cdh1-APC has emerged as a critical regulator of intrinsic axon growth control. Here, we identified the RhoGAP p250GAP as a novel interactor of the E3 ubiquitin ligase Cdh1-APC and found that p250GAP promotes axon growth downstream of Cdh1-APC. We also report that p250GAP undergoes non-proteolytic ubiquitination and associates with the Cdh1 substrate Smurf1 to synergistically regulate axon growth. Finally, we found that <em>in vivo</em> knockdown of p250GAP in the developing cerebellar cortex results in impaired migration and axonal growth. Taken together, our data indicate that Cdh1-APC together with the RhoA regulators p250GAP and Smurf1 controls axon growth in the mammalian brain.</p> </div

Association of p250GAP with Smurf1 regulates axon growth. A.

<p>293T cells were transfected with control vector pCMV5 and Myc-p250GAP, or control pcDNA3 vector and HA-Smurf1, or both HA-Smurf1 and Myc-p250GAP plasmids and the lysates were subjected to immunoprecipitation with the Myc antibody followed by immunoblotting with the HA antibody. <b>B.</b> Reciprocal co-immunoprecipitation of <b>A</b>. <b>C.</b> Granule neurons transfected with control U6, U6 and Smurf1 RNAi, or U6 and p250GAP RNAi or both Smurf1 RNAi and p250GAP RNAi plasmids were analyzed at DIV 4 as described in <b>2B.</b> A total of 467 neurons were measured (ANOVA, ***p<0.0001, n.s. = not significant, values indicate mean+SEM). <b>D.</b> Granule neurons transfected with control U6, pCMV5 and p250GAP RNAi, or U6 and Smurf1 DBM3/4 or both p250GAP RNAi and Smurf1 DBM3/4 plasmids were subjected to axon growth assays at DIV 4 as described in <b>2B.</b> A total of 324 neurons were measured (ANOVA, ***p<0.0001, n.s. = not significant, values indicate mean+SEM). <b>E.</b> Representative images of transfected neurons in <b>D.</b> Scale bar equals 100 µm. Arrows indicate axons.</p

p250GAP is ubiquitinated but not degraded by the proteasome. A.

<p>293T cells were transfected with GFP-Cdh1 together with control vector pCMV-Myc, or plasmids encoding Myc-tagged full length, N- or C-terminal fragments of p250GAP (Myc-p250GAP, Myc-p250GAP-N or Myc-p250GAP-C) and the lysates were subjected to immunoprecipitation with the Myc antibody followed by immunoblotting with the GFP antibody. <b>B.</b> Granule neurons treated with vehicle or 5 µM lactacystin for 10 h following which lysates were subjected to immunoblotting using the p250GAP antibody. γ-tubulin served as the loading control. <b>C.</b> Lysates of granule neurons treated with vehicle or 10 nM bortezomib for 10 h were subjected to immunoblotting using the p250GAP or Smurf1 antibodies. γ-tubulin and 14-3-3β served as loading controls, respectively. <b>D.</b> Cerebellar lysates of postnatal day (P) 12 and week (W) 16 Cdh1^+/+ and Cdh1^+/− mice were immunoblotted using the p250GAP antibody. Erk1/2 served as loading control. <b>E.</b> 293T cells were transfected with control vector pCMVmyc, Myc-p250GAP-N or Myc-p250GAP-C plasmid and the lysates were immunoprecipitated using the Myc antibody followed by immunoblotting with ubiquitin antibody. Asterisk indicates IgG_H. <b>F.</b> Intensity of p250GAP ubiquitination in each condition in <b>B</b> was quantified and normalized to that of control using ImageJ software (ANOVA, **p<0.01, n.s. = not significant, values indicate mean+SEM). <b>G.</b> 293T cells were transfected with control vectors pCMV-Myc and pEGFP, Myc-p250GAP-N plasmid together with pEGFP or GFP-Cdh1 plasmid, or Myc-p250GAP-C plasmid together with pEGFP or GFP-Cdh1 plasmid and the lysates were immunoprecipitated using the Myc antibody followed by immunoblotting with ubiquitin antibody. Asterisk indicates IgG_H. <b>H.</b> Intensity of p250GAP ubiquitination in each condition in <b>D</b> was quantified and normalized to that of control using ImageJ software (ANOVA, *p<0.05, **p<0.01, values indicate mean+SEM).</p

p250GAP promotes axon growth. A.

<p>Lysates of 293T cells transfected with GFP-p250GAP plasmid together with control vector U6 or the p250GAP RNAi plasmid were subjected to immunoblotting using the GFP antibody. γ-tubulin served as the loading control. <b>B.</b> Cerebellar granule neurons were transfected at DIV 0 with control vector U6 or the p250GAP RNAi plasmid together with GFP and BCL_XL plasmids and maintained in conditioned media. At DIV 4, neurons were subjected to immunocytochemistry using the GFP antibody. Axonal length was measured in GFP-positive transfected neurons using ImageJ software. A total of 300 neurons were measured (t-test, ***p<0.0001, values indicate mean+SEM). <b>C.</b> Representative images of transfected neurons in <b>B.</b> Scale bar equals 100 µm. Arrows indicate axons. <b>D.</b> Lysates of 293T cells transfected with GFP-tagged mouse (m)p250GAP or human (h)p250GAP together with control vector U6 or rodent-specific p250GAP RNAi plasmid were immunoblotted with the GFP antibody. γ-tubulin served as the loading control. <b>E.</b> Granule neurons were transfected at DIV 0 with control vectors pcDNA3 and U6, or p250GAP RNAi together with pcDNA3 or human p250GAP plasmids. At DIV 4, neurons were analyzed as in <b>2B</b>. A total of 273 neurons were measured (t-test, ***p<0.0001, n.s. = not significant, values indicate mean+SEM). <b>F.</b> Representative images of transfected neurons in <b>E.</b> Scale bar equals 100 µm. Arrows indicate axons. <b>G.</b> Granule neurons were transfected at DIV 0 with control U6, U6 and Cdh1 RNAi, or U6 and p250GAP RNAi or both Cdh1 RNAi and p250GAP RNAi plasmids and were subjected to axon growth assays at DIV4 as described in <b>2B</b>. A total of 635 neurons were measured (ANOVA, ***p<0.0001, n.s. = not significant, values indicate mean+SEM). <b>H.</b> Representative images of transfected neurons in <b>G</b>. Scale bar equals 100 µm. Arrows indicate axons.</p

β-Arrestin1 and 2 differentially regulate PACAP-induced PAC1 receptor signaling and trafficking

<div><p>A pituitary adenylate cyclase-activating polypeptide (PACAP)-specific receptor, PAC1R, is coupled with multiple signal transduction pathways including stimulation of adenylate cyclase, phospholipase C and extracellular-signal regulated kinase (ERK)1/2. PAC1R has been shown to exert its long-lasting and potent signals via β-arrestin1 and β-arrestin2. However, the precise roles of the two β-arrestin isoforms in PACAP-PAC1R signaling remain unclear. Here we examined the interaction between the two β-arrestin isoforms and PAC1R, β-arrestin-dependent PAC1R subcellular localization and ERK1/2 activation. Upon PACAP stimulation, although PAC1R similarly interacted with β-arrestin1 and β-arrestin2 in HEK293T cells, the complex of PAC1R and β-arrestin2 was translocated from the cell surface into cytosol, but that of β-arrestin1 remained in the cell surface regions in HeLa cells and mouse primary cultured neurons. Silencing of β-arrestin2 blocked PACAP-induced PAC1R internalization and ERK1/2 phosphorylation, but silencing of β-arrestin1 increased ERK1/2 phosphorylation. These results show that β-arrestin1 and β-arrestin2 exert differential actions on PAC1R internalization and PAC1R-dependent ERK1/2 activation, and suggest that the two β-arrestin isoforms may be involved in fine and precise tuning of the PAC1R signaling pathways.</p></div

PAC1R and β-arrestin coupling and translocation in primary cultured cortical neurons.

<p>Primary cultured cortical neurons were infected with the indicated combinations of lentiviruses. <b>(A and B)</b> PACAP-dose dependent increase in NanoBiT luminescence accumulation intensity which was determined 60 min after PACAP addition. <b>(C and D)</b> Time course of changes in luminescence intensity for 60 min after addition of the indicated concentrations of PACAP. <b>(E and F)</b> Representative images of NanoBiT luminescence at 3, 15, 30 and 60 min after stimulation with 1 μM PACAP. <b>(G and H)</b> Representative time-dependent changes of line-scan images for 60 min after stimulation with 1 μM PACAP. Scale bars, 10 μm. β-arr1, β-arrestin1; β-arr2, β-arrestin2. Values are mean ± SEM <b>(A and B)</b> or mean <b>(C and D)</b> of three independent experiments each conducted in duplicate. *<i>P</i> < 0.05, **<i>p</i> < 0.001 and ***<i>p</i> < 0.001 vs. 0 nM PACAP, one-way ANOVA followed by Fisher-PLSD test. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196946#pone.0196946.s006" target="_blank">S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196946#pone.0196946.s007" target="_blank">S4</a> Movies.</p

Time-lapse cell imaging showing PAC1R and β-arrestin coupling and translocation in HeLa cells.

<p>HeLa cells were transfected with the indicated combinations of plasmid vectors. <b>(A and B)</b> Representative images of NanoBiT luminescence at 3, 15, 30 and 60 min after stimulation with 1 μM PACAP. <b>(C and D)</b> Representative time-dependent changes of line-scan images for 60 min after stimulation with 1 μM PACAP. <b>(E and F)</b> Time course of changes in luminescence intensity at the vicinity of the plasma membrane (membrane) and the cytoplasm for 60 min after stimulation with 1 μM PACAP in HeLa cells. Scale bars, 10 μm. β-arr1, β-arrestin1; β-arr2, β-arrestin2. Values are mean ± SEM (n = 3–5). *<i>p</i> < 0.05 vs. cytoplasm, repeated measure two-way ANOVA followed by Fisher-PLSD test. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196946#pone.0196946.s004" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196946#pone.0196946.s005" target="_blank">S2</a> Movies.</p

Differential effects of silencing of β-arrestin1 and β-arrestin2 on PACAP-induced ERK1/2 activation.

<p><b>(A)</b> Representative images of western blots for total and phosphorylated ERK1/2 in HEK293T cells transfected with the indicated siRNA and treated with 1 μM PACAP or saline for 3 or 25 min. <b>(B)</b> Quantification of ERK1/2 activation by normalizing phosphorylated ERK1/2 to total ERK1/2 levels analyzed by western blotting. β-arr1, β-arrestin1; β-arr2, β-arrestin2. Values are mean ± SEM of three independent experiments. *<i>p</i> < 0.05 and **<i>p</i> < 0.01, two-way ANOVA followed by Fisher-PLSD test.</p

Differential effects of β-arrestin1 and β-arrestin2 siRNA on PAC1R internalization.

<p><b>(A)</b> Knockdown of endogenous β-arrestin1 and β-arrestin2 in HEK293T cells. The upper images, representative images of western blots. Values are mean ± SEM of three independent experiments. **<i>p</i> < 0.01 and ***<i>p</i> < 0.001, two-way ANOVA followed by Fisher-PLSD test. <b>(B–E)</b> Representative images of HEK293T cells that were transfected with PAC1R-Halo and the indicated siRNA, labeled with Alexa Fluor 488 HaloTag ligand and treated with 1 μM PACAP or saline for 30 min. <b>(F)</b> Quantification of PAC1R-Halo internalization. Scale bar, 10 μm. β-arr1, β-arrestin1; β-arr2, β-arrestin2. Values are mean ± SEM of 60 cells obtained from three independent experiments. *<i>p</i> < 0.05 and **<i>p</i> < 0.01, two-way ANOVA followed by Fisher-PLSD test.</p