Characterization of Nuclear Localization Signal in the N Terminus of Integrin-linked Kinase-associated Phosphatase (ILKAP) and Its Essential Role in the Down-regulation of RSK2 Protein Signaling

• Published in: The Journal of biological chemistry 2013-03, Vol.288 (9), p.6259-6271
• Main Authors: Zhou, Wang, Cao, Hao, Yang, Xinghai, Cong, Kan, Wang, Wei, Chen, Tianrui, Yin, Huabin, Wu, Zhipeng, Cai, Xiaopan, Liu, Tielong, Xiao, Jianru
• Format: Article
• Language: English
• Subjects: Apoptosis; Apoptosis - physiology; Cell Biology; Cell Cycle; Down-Regulation - physiology; Gene Expression Regulation, Enzymologic - physiology; Hep G2 Cells; Humans; ILKAP; Karyopherins - genetics; Karyopherins - metabolism; Nuclear Localization Signals - genetics; Nuclear Localization Signals - metabolism; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Nuclear Transport; Phosphatase; Phosphoprotein Phosphatases - genetics; Phosphoprotein Phosphatases - metabolism; PP2C; Protein Structure, Tertiary; Ribosomal Protein S6 Kinases, 90-kDa - biosynthesis; Ribosomal Protein S6 Kinases, 90-kDa - genetics; RSK2; Signal Peptidase; Signal Transduction - physiology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.432195

Background: As a phosphatase belonging to the PP2C family, ILKAP plays key roles in the regulation of cell survival and apoptosis. Results: ILKAP interacts with importin α proteins; nuclear ILKAP interacts with RSK2 and induces apoptosis by inhibiting RSK2 activity. Conclusion: ILKAP contains a functional NLS and induces apoptosis by regulating RSK2 signaling. Significance: ILKAP may regulate cell survival and apoptosis through the activation of nuclear pathways. Integrin-linked kinase-associated phosphatase (ILKAP) is a serine/threonine (S/T) phosphatase that belongs to the protein phosphatase 2C (PP2C) family. Many previous studies have demonstrated that ILKAP plays key roles in the regulation of cell survival and apoptosis. Researchers have thus far considered ILKAP a cytoplasmic protein that negatively regulates integrin signaling by interacting with and phosphorylating integrin-linked kinase 1 (ILK1). In this study, we found that both endogenous and tagged ILKAP mainly localize to the nucleus and that the nuclear transport of ILKAP is nuclear localization signal (NLS) importin-mediated. The ILKAP protein interacts directly with importin α1, α3, and α5. The NLS in ILKAP is located in the N-terminal region between amino acids 71 and 86, and the NLS-deleted ILKAP protein was distributed in the cytoplasm. In addition, we show that Lys-78 and Arg-79 are critical for the binding of ILKAP to importin α. We also found that nuclear ILKAP interacts with ribosomal protein S6 kinase-2 (RSK2) and induces apoptosis by inhibiting RSK2 activity and down-regulating the expression level of the RSK2 downstream substrate cyclin D1. These results indicate that ILKAP is a nuclear protein that regulates cell survival and apoptosis through the regulation of RSK2 signaling.