tailieunhanh - Báo cáo khoa học: Role of phosphorylation in p53 acetylation and PAb421 epitope recognition in baculoviral and mammalian expressed proteins

Post-translational modifications, such as phosphorylation and acetylation of the tumour suppressor protein p53, elicit important effects on the func-tion and the stability of the resultant protein. However, as phosphorylation and acetylation are dynamic events subject to complex controls, elucidating the relationships between phosphorylation and acetylation is difficult. | ềFEBS Journal Role of phosphorylation in p53 acetylation and PAb421 epitope recognition in baculoviral and mammalian expressed proteins Lorna J. Warnock Sally A. Raines Trevor R. Mee and Jo Milner YCR p53 Research Group Department of Biology University of York York UK Keywords acetylation p53 protein PAb421 epitope phosphorylation Correspondence L. J. Warnock YCR p53 Research Group Department of Biology University of York York YO10 5 DD UK Fax 44 1904 328622 Tel 44 1904 328624 E-mail ljw7@ Received 1 December 2004 revised 24 January 2005 accepted 31 January 2005 doi Post-translational modifications such as phosphorylation and acetylation of the tumour suppressor protein p53 elicit important effects on the function and the stability of the resultant protein. However as phosphorylation and acetylation are dynamic events subject to complex controls elucidating the relationships between phosphorylation and acetylation is difficult. In the present study we sought to address this problem by comparing fulllength wild-type p53 with full-length p53 proteins mutated at specific phosphorylation targets. Recombinant murine p53 proteins were expressed in insect cells using the baculoviral expression vector system and in a mammalian in vitro transcription translation reticulocyte lysate system. In p53 proteins derived from baculoviral expression vectors S37A but not S37D was found to abrogate phosphorylation at S15. Lysine 382 K382 is constitutively acetylated and was shown to form part of the epitope recognized by PAb421. Lysine 373 K373 was only acetylated following substitutions at S315 S315A or S315D or at S378 S378A . Importantly in baculoviral expressed proteins PAb421 reactivity was independent of K373 acetylation status indicating that acetylation at K382 specifically determines the PAb421 epitope. In response to genotoxic stress the p53 tumour suppressor protein is transiently stabilized accumulates in the nucleus and acts as a