Athanasios A Panagiotopoulos1, Chara Polioudaki2, Sotirios G Ntallis3, Dimitris Dellis4, George Notas1, Christos A Panagiotidis3, Panayiotis A Theodoropoulos2, Elias Castanas5, Marilena Kampa6. 1. Laboratory of Experimental Endocrinology, School of Medicine, University of Crete, 71013, Greece. 2. Laboratory of Biochemistry, School of Medicine, University of Crete, 71013, Greece. 3. Laboratory of Pharmacology, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece. 4. Greek Research & Technology Network, Athens 11523, Greece. 5. Laboratory of Experimental Endocrinology, School of Medicine, University of Crete, 71013, Greece. Electronic address: castanas@uoc.gr. 6. Laboratory of Experimental Endocrinology, School of Medicine, University of Crete, 71013, Greece. Electronic address: kampam@uoc.gr.
Abstract
BACKGROUND: Nuclear translocation of large proteins is mediated through specific protein carriers, collectively named karyopherins (importins, exportins and adaptor proteins). Cargo proteins are recognized by importins through specific motifs, known as nuclear localization signals (NLS). However, only the NLS recognized by importin α and transportin (M9 NLS) have been identified so far METHODS: An unsupervised in silico approach was used, followed by experimental validation. RESULTS: We identified the sequence EKRKI(E/R)(K/L/R/S/T) as an NLS signal for importin 7 recognition. This sequence was validated in the breast cancer cell line T47D, which expresses importin 7. Finally, we verified that importin 7-mediated nuclear protein transport is affected by cargo protein phosphorylation. CONCLUSIONS: The NLS sequence for importin 7 was identified and we propose this approach as an identification method of novel specific NLS sequences for β-karyopherin family members. GENERAL SIGNIFICANCE: Elucidating the complex relationships of the nuclear transporters and their cargo proteins may help in laying the foundation for the development of novel therapeutics, targeting specific importins, with an immediate translational impact.
BACKGROUND: Nuclear translocation of large proteins is mediated through specific protein carriers, collectively named karyopherins (importins, exportins and adaptor proteins). Cargo proteins are recognized by importins through specific motifs, known as nuclear localization signals (NLS). However, only the NLS recognized by importin α and transportin (M9 NLS) have been identified so far METHODS: An unsupervised in silico approach was used, followed by experimental validation. RESULTS: We identified the sequence EKRKI(E/R)(K/L/R/S/T) as an NLS signal for importin 7 recognition. This sequence was validated in the breast cancer cell line T47D, which expresses importin 7. Finally, we verified that importin 7-mediated nuclear protein transport is affected by cargo protein phosphorylation. CONCLUSIONS: The NLS sequence for importin 7 was identified and we propose this approach as an identification method of novel specific NLS sequences for β-karyopherin family members. GENERAL SIGNIFICANCE: Elucidating the complex relationships of the nuclear transporters and their cargo proteins may help in laying the foundation for the development of novel therapeutics, targeting specific importins, with an immediate translational impact.