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Analysis of phosphorylated sites of P73 protein by pololike kinases 3
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Abstract:
Objective:To investigate the structural domains and sites of P73 which can be phosphorylated by polo like kinases 3 (Plk3), and to analyze the effect of Plk3 on P73mediated apoptosis. Methods: Coimmunoprecipitation experiment was used to examine the interaction between Plk3 and P73. Immunofluorescence was used to examine the localization of Plk3 and P73 in cells. Different deletion mutants of GSTP73 fusion protein were prepared. A sitemutation plasmid of GSTP73 (1130) was constructed by converting threonine86 to alanine86 (T86A) and was named GSTP73 (1130) (T86A). The phosphorylated domains and sites of P73 by Plk3 were determined by in vitro phosphorylation assay. The effect of Plk3 on P73mediated apoptosis of HeLa cells was examined by cleaved PARP detection. Results: Plk3 could interact with P73; Plk3 and P73 colocated in the cell nuclei. Different deletion mutants of GSTP73 fusion protein were successfully prepared, and Plk3 phosphorylated P73 at Nterminal 63113 amino residues. Point mutation (T86A) of GSTP73 (1130) fusion protein could not influence the phosphorylation status of P73 by Plk3. Furthermore, Plk3 inhibited P73mediated apoptosis in HeLa cells. Conclusion: Plk3 can interact with and phosphorylate P73 at Nterminal 63113 amino residues (but not at the 86 threonine), thereby inhibiting P73mediated apoptosis of HeLa cells.
Keywords:
pololike kinases 3（Plk3） ; P73 ; phosphorylation ; site ; apoptosis ; cervical cancer cell
Project Supported:
Project supported by the Scientific Research Foundation of Health Department of Hebei Province (No. 20090155)
