ARF function does not require p53 stabilization or Mdm2 relocalization.
It is generally accepted that the ARF tumor suppressor induces p53-dependent growth arrest by sequestering the p53 antagonist Mdm2 in the nucleolus. Previous mutagenic studies of murine ARF suggested that residues 1 through 14 and 26 through 37 were critical for Mdm2 binding, while the latter domain also governed ARF nucleolar localization. We show that mouse ARF residues 6 to 10 and 21 to 25 are required for ARF-induced growth arrest whereas residues 1 to 5 and 29 to 34 are dispensable. Deletion of the putative nucleolar localization signal (31)RRPR(34) did not prevent nucleolar localization. Surprisingly, unlike wild-type ARF, growth-inhibitory mutants D1-5 and D29-34 failed to stabilize p53 yet induced its transcriptional activation in reporter assays. This suggests that p53 stabilization is not essential for ARF-mediated activation of p53. Like wild-type ARF, both mutants also exhibited p53-independent function since they were able to arrest p53/Mdm2-null cells. Notably, other mutants lacking conserved residues 6 to 10 or 21 to 25 were unable to suppress growth in p53-positive cells despite nucleolar localization and the ability to import Mdm2. Those observations stood in apparent contrast to the ability of wild-type ARF to block growth in some cells without relocalizing endogenous Mdm2 to nucleoli. Together, these data show a lack of correlation between ARF activity and Mdm2 relocalization, suggesting that additional events other than Mdm2 import are required for ARF function.
Korgaonkar
Chandrashekhar
C
Department of Pharmacology. Molecular Biology Graduate Program, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Zhao
Lili
L
Modestou
Modestos
M
Quelle
Dawn E
DE
D016475:3T3 Cells; D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D002453:Cell Cycle / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D002466:Cell Nucleolus / Q000737:chemistry / Q000378:metabolism; D019941:Cyclin-Dependent Kinase Inhibitor p16; D005434:Flow Cytometry; D017930:Genes, Reporter; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009363:Neoplasm Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2; D025803:Tumor Suppressor Protein p14ARF / Q000737:chemistry / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
C417743:Cdkn2a protein, mouse; D019941:Cyclin-Dependent Kinase Inhibitor p16; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C497206:MDM2 protein, human; C497207:Mdm2 protein, mouse; D051736:Proto-Oncogene Proteins c-mdm2