Pericentrin contains five NESs and an NLS essential for its nucleocytoplasmic trafficking during the cell cycle

Abstract

Pericentrin, a conserved centrosomal component, provides the structural scaffold to anchor numerous centrosomal proteins, and thus plays an essential role in the organization and function of the centrosome and the mitotic spindle. Although pericentrin was shown to localize in the cytoplasm and reported to be sensitive to leptomycin B (LMB), a specific inhibitor of Crm1, the regions within pericentrin that serve as signals for transporting in and out of the nucleus have not yet been identified. In this study, we identified five novel nuclear export signals (NESs) in pericentrin with diverse export activities. All of the five NESs could bind to Crm1 in a LMB-sensitive way when mediating the nuclear export of pericentrin. We also demonstrated that the region of amino acids 8-42 in pericentrin contains a tripartite nuclear localization signal (NLS) consisting of three clusters of basic amino acids. The NLS of pericentrin binds to importin β directly or via the adaptor importin α to form the import complex, which could be disrupted by RanQ69L, a dominant-negative Ran GTPase possessing high affinity for importin β. Furthermore, we found that mutation of the NESs in full-length pericentrin results in both nuclear and cytoplasmic localization, and mutation of the NLS abolishes the nuclear import of pericentrin. On the basis of these results, we suggest that the NESs and NLS of pericentrin are essential for its subcellular localization and nucleocytoplasmic trafficking during the cell cycle.

Calumenin-15 facilitates filopodia formation by promoting TGF-β superfamily cytokine GDF-15 transcription

“…32, 33 Besides, immunoprecipitation showed that Calu-15 also interacted with Ran GTPase (Figure 2d), which has been reported to disassociate the importin–cargo complex and be required for the nuclear import process. 33, 34 Moreover, the cotransfection with RanQ69L, which is a mutant defective in GTPase activity, 35 largely inhibited the nuclear localization of Calu-15 (Figure 2e). Therefore, the nuclear import process of Calu-15 is mediated by importin- α and regulated by Ran GTPase.…”

Nuclear roles for cilia-associated proteins

“…As examples, both the ciliary axoneme-associated radial spoke protein 3 (RSPH3–OMIM: 615876) [100] and the pericentriolar and ciliary basal body-associated protein pericentrin (PCNT: OMIM: 605925) [51] have been found to contain functional nuclear localization (NLS) and nuclear exclusion (NES) sequences. Similarly parafusin, a signaling scaffolding protein, has been found localized to the base of primary cilia in a variety of mammalian cell types and within the nuclei of fibroblasts [77].…”

Chromatin-bound NLS proteins recruit membrane vesicles and nucleoporins for nuclear envelope assembly via importin-α/β

“…The small GTPase Ran regulates NE and NPC assembly, as well as nucleocytoplasmic transport and mitotic spindle formation [10,[14][15][16][17][18][19][20][21]. Chromatin-independent NE assembly can be induced using artificial beads coated with Ran in the extracts from Xenopus eggs or somatic…”