Many proteins associated with the phenotype microcephaly have been localized to the centrosome or linked to it functionally. demonstrated that the loss of in mouse neuroepithelium alters the orientation of cleavage plane in the progenitor cells, resulting in an increase in asymmetric divisions and reduction in the neuronal progenitor pool [14]. Bioinformatic analysis has predicted the following domains and motifs in ASPM: an N-terminal microtubule binding domain, two calponin homology domains, 81 IQ (isoleucine-glutamine) repeat motifs, and a C-terminal region with a conserved armadillo-like repeat domain [12]. A recent study demonstrated that the IQ motifs in ASPM-1 associate with calmodulin to affect ASPM-1’s centrosomal localization [15]. Apart from IQ motifs, the rest of the domains remain to be characterized functionally. Interestingly, the loss of the last 149 amino acids from the C-terminal region of ASPM is sufficient to cause MCPH [9], indicating that it is indispensable for the function of ASPM and directly involved in neurogenesis. The purpose of this study was to investigate the novel protein interactions mediated by the C-terminal region of ASPM. Results Yeast two-hybrid (Y2H) analysis with a C-terminal region of ASPM To find novel interacting partners for the C-terminal region (CTR) of ASPM, we cloned this region corresponding to amino acids 3,276C3,477 in the Y2H DNA binding domain vector pGBKT7 (Figure 1A). The clone (pGBKT7-CTR) was subsequently used as a bait to screen a human fetal brain cDNA library cloned in the Y2H activation domain vector pACT2. The screen identified eight transformants which were further tested for growth and blue color on plates with quadruple dropout medium (SD/-His/-Ade/-Leu/-Trp) and X–gal. DNA sequence analysis of the pACT2 plasmid from one of the 67526-95-8 supplier transformants showed that it harbours a 714 bp long fragment of UBE3A (ubiquitin protein ligase E3A) corresponding to amino acids 639C875. The 714 bp fragment of UBE3A was recloned in pACT2 (pACT2-UBE3A) and co-transformed with 67526-95-8 supplier the bait pGBKT7-CTR in yeast cells. The transformant was re-assessed by nutritional selection and X–gal plate assay as described above. The growth of the transformant and blue color suggested that ASPM interacts with UBE3A (Figure 1B). The region of UBE3A from amino acids 639C875 overlaps with the HECT domain (homologous to the E6AP carboxyl terminus) of UBE3A which is known to be involved in ubiquitination of its target proteins (Figure 1C). Figure 1 Identification of interaction between ASPM and UBE3A by Y2H analysis. ASPM interacts with UBE3A using human fetal kidney lysate. Due to the limited availability of human fetal brain tissue, we used the fetal kidney tissue for co-immunopreciptation studies as it was readily available. Immunoprecipitation using anti-ASPM antibody followed by immunoblot analysis using anti-UBE3A-sc-8926 antibody detected a 100 kDa band corresponding to the expected size of UBE3A (Figure 2D). Similarly, immunoprecipitation with anti-UBE3A antibody pulled Rabbit Polyclonal to TK (phospho-Ser13) down ASPM (Figure 2D). Pre-immune serum (PIS/IgG) and protein A/G beads (P A/G) did not pull down either of the proteins (Figure 67526-95-8 supplier 2D). UBE3A localizes to the centrosome throughout mitosis An analysis of UBE3A immunofluorescence staining with anti-UBE3A-sc-8926 antibody in HEK293 cells by confocal microscopy revealed that it stains the centrosome throughout the mitotic progression from prophase to telophase and colocalizes with ASPM (Figure 3A). Identical results were obtained in A549 cell line (Figure 3B). In interphase cells, UBE3A staining observed at the centrosome was weak (Figure 3A). A similar result was obtained using a different anti-UBE3A antibody (anti-UBE3A-sc-12380), confirming the centrosomal localization of UBE3A (Figure 4). Henceforth, 67526-95-8 supplier we have used the anti-UBE3A-sc-8926 antibody for all the experiments. Figure 3 UBE3A colocalizes with ASPM at the centrosome. Figure 4 Confirmation of colocalization of UBE3A with ASPM at the centrosome using a different UBE3A antibody. UBE3A is a cell cycle dependent protein and its overexpression does not degrade ASPM We investigated the role of UBE3A in cell cycle further by studying its cell cycle dependence. For this, we synchronized HEK293 cells at three different stages- G1/S.