TY - JOUR
T1 - Regulation of DNA binding and transactivation in p53 by nuclear localization and phosphorylation
AU - Martinez, J. D.
AU - Craven, M. T.
AU - Joseloff, E.
AU - Milczarek, G.
AU - Bowden, G. T.
N1 - Funding Information:
We are grateful to Donna George for providing the murine mdm2 cDNA, to Konrad Huppi for providing the p21waf1 cDNA and to Jamil Momand for providing the 2A10 antibody. These studies were supported in part by a minority supplement to NIH grant CA40584 which was awarded to JDM, R29-CA64842 and a Cancer Center Core Grant CA23074.
PY - 1997
Y1 - 1997
N2 - Compelling evidence indicates that p53 acts as a transcription factor and that this activity is regulated by several factors including subcellular localization and phosphorylation status of the protein. To learn more about how these two processes determine whether p53 becomes activated, we studied the temperature sensitive murine p53, p53(val135). At nonpermissive temperatures, p53(val135) remains sequestered in the cytoplasm of cells which express it. Electrophoretic mobility shift assays demonstrated that, under these conditions, the protein lacked DNA binding activity. However, by shifting to the permissive temperature, p53(val135) became concentrated in the nucleus, hyperphosphorylated, and had acquired the ability to bind DNA in a sequence specific manner. This was accompanied by the induction of two p53 regulated genes, mdm2 and p21(waf1), which indicated that p53(val135) had become an active transcription factor. Two dimensional gel electrophoresis and tryptic peptide mapping showed that entry into the nucleus resulted in the appearance of new phosphorylated isoforms and that the protein had become extensively phosphorylation at the N-terminus. Notably, phosphorylation at the N-terminus occurred only in the nucleus, whereas phosphorylation at the C-terminus could occur in both the cytoplasm and the nucleus. Based on these observations, we suggest that phosphorylation of p53's N-terminus is compartmentally restricted.
AB - Compelling evidence indicates that p53 acts as a transcription factor and that this activity is regulated by several factors including subcellular localization and phosphorylation status of the protein. To learn more about how these two processes determine whether p53 becomes activated, we studied the temperature sensitive murine p53, p53(val135). At nonpermissive temperatures, p53(val135) remains sequestered in the cytoplasm of cells which express it. Electrophoretic mobility shift assays demonstrated that, under these conditions, the protein lacked DNA binding activity. However, by shifting to the permissive temperature, p53(val135) became concentrated in the nucleus, hyperphosphorylated, and had acquired the ability to bind DNA in a sequence specific manner. This was accompanied by the induction of two p53 regulated genes, mdm2 and p21(waf1), which indicated that p53(val135) had become an active transcription factor. Two dimensional gel electrophoresis and tryptic peptide mapping showed that entry into the nucleus resulted in the appearance of new phosphorylated isoforms and that the protein had become extensively phosphorylation at the N-terminus. Notably, phosphorylation at the N-terminus occurred only in the nucleus, whereas phosphorylation at the C-terminus could occur in both the cytoplasm and the nucleus. Based on these observations, we suggest that phosphorylation of p53's N-terminus is compartmentally restricted.
KW - DNA binding
KW - Nuclear localization
KW - P53
KW - Phosphorylation
KW - Transactivation
UR - http://www.scopus.com/inward/record.url?scp=0030942625&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0030942625&partnerID=8YFLogxK
U2 - 10.1038/sj.onc.1201095
DO - 10.1038/sj.onc.1201095
M3 - Article
C2 - 9191051
AN - SCOPUS:0030942625
SN - 0950-9232
VL - 14
SP - 2511
EP - 2520
JO - Oncogene
JF - Oncogene
IS - 21
ER -