To find out whether slower mobility of p73 was due to phosphorylation and whether Aurora A is directly involved in p73 phosphorylation, we treated cell extracts with lPPase, ATP-competitive HDAC inhibitor with or without Aurora A inhibitor. While inhibitor treatment alone resulted in small increase in mobility, lPPase treatment, both with or without Aurora A inhibitor, led to similar yet significantly faster migration in p73. These results suggest that slower flexibility was as a result of multiple phosphorylations, probably catalyzed by several kinases, including Aurora A. Aurora A inhibition alone triggered a small downward shift in gel mobility due to selective interference with Aurora A phosphorylation, however the faster moving kind was due to complete dephosphorylation with lPPase. We performed p73 immunoprecipitation, followed by immunoblotting with the anti phospho PKA substrate antibody, which acknowledges the Aurora A agreement phosphorylation theme in substrate proteins, to determine direct involvement of Aurora A in p73 phosphorylation in vivo. We observed clear phosphor Papillary thyroid cancer PKA signal in immunoprecipitated p73 from nocodazole addressed mitotic cells, which was diminished in inhibitortreated examples. In exponentially growing cells, the phosphorPKA indication changed little after treatment. These studies further confirmed the involvement of Aurora A in p73 phosphorylation in vivo. We next performed an in vitro kinase assay of p73, with or without wild type or kinase useless Aurora A, with the closely related paralog Aurora B as a control. Aurora A WT phosphorylated p73, but Aurora A KD did not. Total lack of phosphorylation signal on p73 with Aurora B further validated Aurora A as the bona fide kinase of p73. We next determined the specific Aurora A phosphorylated amino acid residue in p73 using site directed mutants in Aurora (-)-MK 801 kinase agreement phosphorylation motifs and submitting them to in vitro kinase assays. The serine 235alanine mutant ofp73 hadreduced phosphorylation than p73 WT, showing that S235 is phosphorylated by Aurora A. Wefurther established this phosphorylation having an anti phospho PKA substrate specific antibody. p73 WT phosphorylation was apparent in cells coexpressing Aurora A however not those expressing the empty vector. Phosphorylation was dramatically diminished in cells expressing the S235A mutant, displaying that serine 235 in p73 is phosphorylated by Aurora A. It is intriguing that transactivation defective DNp73 did actually join the WT and the phosphor mimetic mutant of p73 with similar performance and showed minimal loss of phosphorylation in the SA mutant of the conserved motif. We identified in vivo relationship between Aurora A and p73 by immunoprecipitation of 293T cells cotransfected with FlagAurora A and GFP p73.