FIGURE 6.

Aurora A kinase phosphorylates Par3 at Ser⁹⁶². A, a schematic representation of Par3 and the sequence surrounding Ser⁹⁶² (amino acids 958–966) is shown. The numbers refer to the positions in the amino acid sequence. B, wild type (Wt) and mutant GFP-Par3-aPKCBD (Wt, S827A, S962A) and GFP-tagged Aurora A were expressed separately in HEK 293T cells and purified by immunoprecipitation (IP) with an anti-GFP antibody. Purified kinases and GFP-Par3-PKCBD were mixed and incubated in kinase buffer. Phosphorylated Par3-aPKCBD was detected by Western blotting (WB) using an anti-phosphoserine (p-Ser) antibody C, Myc-tagged wild type (wt) or mutant (S827A, S962A) full-length Par3 were immunoprecipitated with an anti-myc antibody from lysates of transfected HEK 293T cells after coexpression with GFP-tagged wild type (Aurora A) or kinase-dead (KD) Aurora A. Phosphorylated proteins and the expression of comparable amounts of myc-tagged and GFP-tagged proteins in lysates were analyzed by Western blotting using anti-phosphoserine, anti-myc and anti-GFP antibodies, respectively. Numbers indicate molecular mass in kDa.