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 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 full-length 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.
Warnock
Lorna J
LJ
YCR p53 Research Group, Department of Biology, University of York, York, UK. ljw7@york.ac.uk
Raines
Sally A
SA
Mee
Trevor R
TR
Milner
Jo
J
D000107:Acetylation; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D016367:Baculoviridae; D000939:Epitopes / Q000276:immunology*; D005822:Genetic Vectors; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D000906:Antibodies; D000939:Epitopes; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D012694:Serine; D008239:Lysine