Protein Containing The Ph Domain Of Ckip-1 Subcellular Localization Mechanism

Proper subcellular localization of proteins is essential for execution of proper functions. Abnormality of protein localization might result in terrible or even severe outcomes. CKIP-1 (casein kinase 2 interacting protein-1) is a PH domain-containing protein which is predominantly localized at the plasma membrane. It has been demonstrated that CKIP-1 participates in regulation of multiple cellular processes including muscle cell differentiation, cytoskeleton reorganization, cell apoptosis, tumor cell growth, as well as recruitment of nuclear CK2 and ATM kinases to the plasma membrane. Most recently, our lab established CKIP-1 gene knockout mice model and revealed that CKIP-1 physiologically suppressed osteoblast activity via interaction with and augment of the catalytic activity of E3 ubiquitin ligase Smurfl. The plasma membrane localization of CKIP-1 is critical for these functions; however, the precise mechanism determining its localization remains unclear and was investigated in the present study. Previous studies reported that the PH domain of CKIP-1 could bind to the phospholipids and thus determined its plasma membrane localization. We surprisingly observed that ectopic expressed PH domain of CKIP-1 was exclusively nuclear despite the tag and the cell types. This result implies the complexity of determinant mechanism of CKIP-1 localization than once thought. We further identified a strong and non-classical nuclear localization signal (NLS) R82KSKSRSKK90 within the PH domain, among which R82, K83, K89 and K90 are essential for the NLS function. A series of deletion analysis showed that CKIP-1 C-terminus could interact with the N-terminal PH domain and mask the NLS. This auto-inhibition was involved in the determination of CKIP-1 localization. In addition, overexpression of the CKIP-1 C-terminal truncate C-terml (aa 338-409) could induce a significant nuclear transport of the full-length CKIP-1 in a dose-dependent manner. Taken together, these results suggest a model that the NLS within PH domain and the auto-inhibition between the PH domain and the C-terminus coordinate to determine the plasma membrane localization of CKIP-1 in normal conditions. When cells face stresses or interfering with artificial C-term1, the auto-inhibition could be disrupted, exposing the NLS within the PH domain which recruits CKIP-1 to the nucleus. Our findings provide a novel molecular mechanism determining the CKIP-1 localization and might contribute to full understandings of the regulatory role of PH domains.