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KMID : 1161520130170030186
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Animal Cells and Systems
2013 Volume.17 No. 3 p.186 ~ p.195
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Phosphorylation of human chromosome maintenance 1 mediates association with 14-3-3 proteins
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Kang Sang-Sun
Shin Sung-Hwa
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Abstract
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Human chromosome maintenance 1 (CRM1) was originally cloned based on homology to a yeast gene. CRM1, which belongs to the family of importin β-related nuclear transport receptors, directly and specifically associates with nuclear export signals (NESs) and mediates nuclear export of proteins containing leucine-rich NESs. We present evidence that CRM1 associates with a 22-kDa 14-3-3 scaffolding protein that is a principal structural and regulatory component of Human embryonic kidney (HEK 293) cells. We found a potential 14-3-3-binding motif (1049KHKRQMSVPG1058) in the CRM1 C-terminal domain that depended on serine 1055 phosphorylation by Protein Kinase A (PKA). We demonstrated that CRM1 is a PKA substrate using an in vitro assay. Using a pull-down approach and co-immunoprecipitation, we found that CRM1 interacted with the 14-3-3 motif in vivo and in vitro. We also detected colocalization of CRM1 and 14-3-3 proteins using confocal microscopy. Nuclear pore localization of CRM1 was disrupted by treatment with a PKA activator or inhibitor or by a S1055D/S1055A mutation in the CRM1 14-3-3-binding motif. Transient transfection assays showed that the apoptosis rate of cells with the S1055D construct was twice that of cells with wild type (WT) or S1055A construct. Our observations indicated that phosphorylation on the serine 1055 residue of CRM1 by PKA promoted 14-3-3 binding and cytoplasmic localization, resulting in enhancement of cell apoptosis.
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KEYWORD
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14-3-3, CRM1, PKA, phosphorylation, protein-protein interaction
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