G-actin guides p53 nuclear transport: potential contribution of monomeric actin in altered localization of mutant p53.
p53 preserves genomic integrity by restricting anomaly at the gene level. Till date, limited information is available for cytosol to nuclear shuttling of p53; except microtubule-based trafficking route, which utilizes minus-end directed motor dynein. The present study suggests that monomeric actin (G-actin) guides p53 traffic towards the nucleus. Histidine-tag pull-down assay using purified p53(1-393)-His and G-actin confirms direct physical association between p53 and monomeric G-actin. Co-immunoprecipitation data supports the same. Confocal imaging explores intense perinuclear colocalization between p53 and G-actin. To address atomistic details of the complex, constraint-based docked model of p53:G-actin complex was generated based on crystal structures. MD simulation reveals that p53 DNA-binding domain arrests very well the G-actin protein. Docking benchmark studies have been carried out for a known crystal structure, 1YCS (complex between p53DBD and BP2), which validates the docking protocol we adopted. Co-immunoprecipitation study using "hot-spot" p53 mutants suggested reduced G-actin association with cancer-associated p53 conformational mutants (R175H and R249S). Considering these findings, we hypothesized that point mutation in p53 structure, which diminishes p53:G-actin complexation results in mutant p53 altered subcellular localization. Our model suggests p53Arg249 form polar-contact with Arg357 of G-actin, which upon mutation, destabilizes p53:G-actin interaction and results in cytoplasmic retention of p53R249S.
Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata-700054, India.
Taniya
T
Saha
Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata-700054, India.
Deblina
D
Guha
Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata-700054, India.
Argha
A
Manna
Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata-700054, India.
Abir Kumar
AK
Panda
Division of Biophysics, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata-700054, India.
Jyotsna
J
Bhat
Division of Biophysics, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata-700054, India.
Subhrangsu
S
Chatterjee
Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata-700054, India.
Gaurisankar
G
Sa
D000199:Actins / Q000378:metabolism*; D000818:Animals; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000378:metabolism*; D058105:Polymerization / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011817:Rabbits; D013347:Subcellular Fractions / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D000199:Actins; D003432:Cross-Linking Reagents; D050505:Mutant Proteins; D016159:Tumor Suppressor Protein p53; D004317:Doxorubicin