Huntingtin-associated protein 1 interacts with breakpoint cluster region protein to regulate neuronal differentiation

Abstract

Alterations in microtubule-dependent trafficking and certain signaling pathways in neuronal cells represent critical pathogenesis in neurodegenerative diseases. Huntingtin (Htt)-associated protein-1 (Hap1) is a brain-enriched protein and plays a key role in the trafficking of neuronal surviving and differentiating cargos. Lack of Hap1 reduces signaling through tropomyosin-related kinases including extracellular signal regulated kinase (ERK), resulting in inhibition of neurite outgrowth, hypothalamic dysfunction and postnatal lethality in mice. To examine how Hap1 is involved in microtubule-dependent trafficking and neuronal differentiation, we performed a proteomic analysis using taxol-precipitated microtubules from Hap1-null and wild-type mouse brains. Breakpoint cluster region protein (Bcr), a Rho GTPase regulator, was identified as a Hap1-interacting partner. Bcr was co-immunoprecipitated with Hap1 from transfected neuro-2a cells and co-localized with Hap1A isoform more in the differentiated than in the nondifferentiated cells. The Bcr downstream effectors, namely ERK and p38, were significantly less activated in Hap1-null than in wild-type mouse hypothalamus. In conclusion, Hap1 interacts with Bcr on microtubules to regulate neuronal differentiation.

Fig 1. Association of Bcr with Hap1 on microtubules in mouse brains.

(A) The relative amount of a microtubule subunit (β-tubulin), a small GTPase (RhoA) and a GAP/GEF (Bcr) in wild-type (WT) and Hap1-null newborn mouse brains normalized to isotope-labeled counterparts in wild type adult mouse brain. (B) Western blotting showing Hap1 isoforms, Bcr, RhoA and α-Tubulin in microtubule pellets precipitated by taxol-GTP treatment from Hap1-null and WT mouse brains. Input is the supernatant of brain lysates after centrifugation at 18,000 × g for 20 min (S2). The nonspecific protein species reacting to Hap1 antibody in the inputs (arrow) was not precipitated with microtubules.

Fig 2. Co-immunoprecipitation of Hap1 and Bcr from neuro-2A cells.

(A) The protein levels of Hap1A and Hap1B in the mouse neuroblastoma neuro-2a cells and total brain lysates from wild-type (WT) and Hap1-null mice. The nonspecific protein in each lane implicated an equal loading of the samples. (B) Immunoprecipitation (IP) of neuro-2a cells transfected with GFP, GFP-Hap1A, or GFP-Hap1B with Bcr-Myc. The cell lysates were precipitated with anti-GFP antibody. The input and precipitates were analyzed using SDS-PAGE and immunoblotted (IB) with anti-Bcr and anti-GFP antibodies. (C) Quantitative analysis demonstrating the binding of Bcr to Hap1. The Bcr abundance was normalized with the bait (GFP, Hap1A-GFP or Hap1B-GFP), and the result in GFP was set as 1. Four independent experiments were performed. * p < 0.05.

Fig 3. Co-localization of Bcr and Hap1 in differentiated neuro-2A cells.

(A) Confocal imaging of differentiated neuro-2a cells transfected with GFP-Hap1A or GFP-Hap1B (green) with Bcr-myc (red). Scale bar, 10 μm. (B) Statistical analysis showing the percentage of GFP, GFP-Hap1A and GFP-Hap1B co-localized with Bcr in differentiated cells. Five or more sets of image were analyzed. * p < 0.05; ** p < 0.01; *** p < 0.001.

Fig 4. Lack of Hap1 inhibits Bcr signaling in mouse hypothalamus.

(A) Western blotting analysis of Bcr and downstream signaling molecules including p38, ERK1/2, PAK, JNK and their phosphorylated forms from the WT and Hap1-null mouse hypothalamic (left panel) and non-hypothalamic regions (right panel). (B) Quantitative and statistical analysis of the changes of Bcr and its downstream signaling molecules in the hypothalamic and non-hypothalamic regions. The presented value was the ratio of the phosphorylated protein level to the total protein level and normalized with the result in WT mouse hypothalamic or non-hypothalamic region, which was set as 1. Three independent experiments were performed for statistical analysis. * p < 0.05; ** p < 0.01.