Importin alpha1 is involved in the nuclear localization of Zac1 and the induction of p21WAF1/CIP1 by Zac1.
Zac1, a novel seven-zinc-finger transcription factor, preferentially binds GC-rich DNA elements and has intrinsic transactivation activity. To date, the NLS (nuclear localization signal) of Zac1 has not been empirically determined. We generated a series of EGFP (enhanced green fluorescence protein)-tagged deletion mutants of Zac1 and examined their subcellular localization, from which we defined two NLSs within the DNA-binding (or zinc-finger) domain. Fusion proteins consisting of the two EGFP-tagged zinc-finger clusters (zinc finger motifs 1-3 and 4-7) were located exclusively in the nucleus, demonstrating that each of the zinc-finger clusters is sufficient for nuclear localization. Physical interactions between these two zinc-finger clusters and importin alpha1 were demonstrated using an in vitro glutathione S-transferase pull-down assay. Finally, our results indicate that the association of Zac1 with importin alpha1 is also involved in regulating the transactivation activity of Zac1 on the p21WAF1/CIP1 gene and protein expression.
Huang
Shih-Ming
SM
Department of Biochemistry and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan 114, Republic of China.
Huang
Sheng-Ping
SP
Wang
Sung-Ling
SL
Liu
Pei-Yao
PY
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers; D028901:alpha Karyopherins / Q000378:metabolism
D050759:Cyclin-Dependent Kinase Inhibitor p21; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D028901:alpha Karyopherins; C087978:karyopherin alpha 2