Subcellular targeting of p33ING1b by phosphorylation-dependent 14-3-3 binding regulates p21WAF1 expression.
ING1 is a type II tumor suppressor that affects cell growth, stress signaling, apoptosis, and DNA repair by altering chromatin structure and regulating transcription. Decreased ING1 expression is seen in several human cancers, and mislocalization has been noted in diverse types of cancer cells. Aberrant targeting may, therefore, functionally inactivate ING1. Bioinformatics analysis identified a sequence between the nuclear localization sequence and plant homeodomain domains of ING1 that closely matched the binding motif of 14-3-3 proteins that target cargo proteins to specific subcellular locales. We find that the widely expressed p33(ING1b) splicing isoform of ING1 interacts with members of the 14-3-3 family of proteins and that this interaction is regulated by the phosphorylation status of ING1. 14-3-3 binding resulted in significant amounts of p33(ING1b) protein being tethered in the cytoplasm. As shown previously, ectopic expression of p33(ING1b) increased levels of the p21(Waf1) cyclin-dependent kinase inhibitor upon UV-induced DNA damage. Overexpression of 14-3-3 inhibited the up-regulation of p21(Waf1) by p33(ING1b), consistent with the idea that mislocalization blocks at least one of ING1's biological activities. These data support the idea that the 14-3-3 proteins play a crucial role in regulating the activity of p33(ING1b) by directing its subcellular localization.
Southern Alberta Cancer Research Institute, Dept. of Biochemistry, University of Calgary, #370 Heritage Medical Research Building, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada.
Wei
W
Gong
Michael
M
Russell
Keiko
K
Suzuki
Karl
K
Riabowol
D048948:14-3-3 Proteins / Q000378:metabolism*; D000409:Alanine / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D015153:Blotting, Western; D002458:Cell Fractionation; D002460:Cell Line; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D015972:Gene Expression Regulation, Neoplastic; D018390:Gene Targeting; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007211:Indoles; D000076023:Inhibitor of Growth Protein 1; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D048948:14-3-3 Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D005456:Fluorescent Dyes; C501503:ING1 protein, human; D007211:Indoles; D000076023:Inhibitor of Growth Protein 1; D047908:Intracellular Signaling Peptides and Proteins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; C007293:DAPI; D006639:Histidine; D005982:Glutathione Transferase; D000409:Alanine