The 7-amino-acid site in the proline-rich region of the N-terminal domain of p53 is involved in the interaction with FAK and is critical for p53 functioning.
It is known that p53 alterations are commonly found in tumour cells. Another marker of tumorigenesis is FAK (focal adhesion kinase), a non-receptor kinase that is overexpressed in many types of tumours. Previously we determined that the N-terminal domain of FAK physically interacted with the N-terminal domain of p53. In the present study, using phage display, sitedirected mutagenesis, pulldown and immunoprecipitation assays we localized the site of FAK binding to a 7-amino-acid region(amino acids 65-71) in the N-terminal proline-rich domain of human p53. Mutation of the binding site in p53 reversed the suppressive effect of FAK on p53-mediated transactivation ofp21, BAX (Bcl-2-associated X protein) and Mdm2 (murine double minute 2) promoters. In addition, to functionally test this p53 site, we conjugated p53 peptides [wild-type (containing the wild-type binding site) and mutant (with a mutated 7-aminoacid binding site)] to a TAT peptide sequence to penetrate the cells, and demonstrated that the wild-type p53 peptide disrupted binding of FAK and p53 proteins and significantly inhibited cell viability of HCT116 p53+/+ cells compared with the control mutant peptide and HCT116 p53-/- cells. Furthermore, the TAT-p53 peptide decreased the viability of MCF-7 cells, whereas the mutant peptide did not cause this effect. Normal fibroblast p53+/+ and p53-/- MEF (murine embryonic fibroblast) cells and breast MCF10A cells were not sensitive to p53 peptide. Thus, for the first time, we have identified the binding site of the p53 andFAK interaction and have demonstrated that mutating this site and targeting the site with peptides affects p53 functioning and viability in the cells.
Golubovskaya
Vita M
VM
Department of Surgery, University of Florida, Gainesville, FL 32610, USA. golubvi@surgery.ufl.edu
Finch
Richard
R
Zheng
Min
M
Kurenova
Elena V
EV
Cance
William G
WG
D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D011392:Proline; D011485:Protein Binding; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology
D016159:Tumor Suppressor Protein p53; D011392:Proline; D051417:Focal Adhesion Kinase 1