Mutational analysis of the carboxy-terminal portion of p53 using both yeast and mammalian cell assays in vivo.
Increasing evidence indicates that p53 is a transcriptional trans-activator through its sequence-specific DNA binding domain. Tumor-derived p53 mutations disrupt the trans-activation ability mainly due to loss of its sequence-specific DNA binding. Using both yeast and mammalian cell assays, the effect of p53 mutations in the carboxy terminal portion was investigated in order to address how p53 mutations outside of the DNA binding domain affect p53 function. The p53 cDNA in the carboxy-terminus was randomly mutagenized by error-prone polymerase chain reactions and the amplified cDNA was screened for the ability to trans-activate using a yeast assay. Four p53 mutations, including two missense and two nonsense mutations located in the carboxy-terminal oligomerization domain, were further analysed for trans-activation, cell cycle arrest and colony formation in a human osteosarcoma cell line, Saos-2. These functional properties of p53 were disrupted by the missense mutations. Surprisingly, one of the nonsense mutations disrupts the trans-activation function and the ability to G1 arrest but shows a strong inhibition of colony formation. These results confirm that mutations in the oligomerization domain can inactivate p53 function and also indicate that p53-mediated cell growth inhibition does not necessarily depend on the ability to arrest cell cycle.
Ishioka
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C
Division of Molecular Genetics, Massachusetts General Hospital Cancer Center, Charlestown, USA.
Englert
C
C
Winge
P
P
Yan
Y X
YX
Engelstein
M
M
Friend
S H
SH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle; D002455:Cell Division; D018076:DNA, Complementary / Q000032:analysis; D008969:Molecular Sequence Data; D009154:Mutation; D013329:Structure-Activity Relationship; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000502:physiology*; D015003:Yeasts / Q000235:genetics
D018076:DNA, Complementary; D016159:Tumor Suppressor Protein p53