Elsevier

Experimental Cell Research

Volume 314, Issue 20, 10 December 2008, Pages 3692-3700
Experimental Cell Research

Research Article
A novel function of CEP135 as a platform protein of C-NAP1 for its centriolar localization

https://doi.org/10.1016/j.yexcr.2008.09.016Get rights and content

Abstract

A proteomic study predicted that about one hundred kinds of proteins constitute a basic structure of the centrosome. Most of the core centrosomal proteins contain extensive coiled-coil domains, suggesting that the protein–protein interaction is a critical force for the core centrosome configuration. In the present study, we investigated a novel interaction between CEP135 and C-NAP1, two core centriolar proteins. Depletion of CEP135 caused a premature centrosome splitting. Reduction of the centrosomal C-NAP1 level was accompanied in a specific manner. Ectopic expression of the CEP135 mutant proteins also caused centrosome splitting in association with the reduction of the centrosomal C-NAP1 levels. Based on these results, we propose that CEP135 acts as a platform protein for C-NAP1 at the centriole.

Introduction

The centrosome comprises a pair of centrioles embedded in an amorphous protein mesh called the pericentriolar material. Since the centrosome is the major microtubule-organizing center at which the minus ends of cellular microtubules are concentrated, it influences a number of microtubule-related processes such as intracellular trafficking and cell morphology and motility. In mitotic cells, centrosomes function as spindle poles for chromosome segregation. Further, the centrosome is involved in a process to release the central microtubules from the midbody for the completion of cell division [1]. The centrosome is also essential for the formation of the primary cilium. The flies without centrioles developed into morphologically normal adults, but had no cilium or flagellum and died shortly after birth [2].

It is believed that a number of proteins are recruited into or removed from the centrosome depending on the physiological status of the cell. Nonetheless, the core structure of the centrosome should be maintained for its integrity. A proteomic analysis identified over 500 proteins from the centrosome-enriched fraction of KE-37 human lymphoblastic cells, of which 114 were considered to be core components of the human centrosome [3]. Many of the core centrosomal proteins include extensive coiled-coil domains, suggesting that the protein–protein interaction is a critical force for the core centrosome configuration.

CEP135 is a core centrosome protein with coiled-coil domains. It is located around the centriolar surface as well as within the proximal lumen of the centrioles [4]. CEP135 overexpression or knockdown was reported to result in abnormal organization of the microtubules in both the interphase and mitotic cells [5]. CEP135 was proposed to play a scaffolding role during centriole biogenesis [4], but exact functions of CEP135 remain to be elucidated.

C-NAP1 is another core centrosome protein with multiple coiled-coil domains. It is located at the proximal end of the centriole [6]. C-NAP1 suppression by a specific antibody injection or siRNA transfection induced premature centrosome splitting [7], [8]. It was proposed that C-NAP1 holds an intercentriolar linker such as rootletin to maintain centrosome cohesion [8]. NEK2 has been considered a key regulator for centrosome separation since its overexpression induced the premature centrosome splitting and since its activity oscillates during the cell cycle [9], [10]. C-NAP1 is the first known substrate of NEK2 for centrosome separation [6]. In addition, NEK2 also phosphorylates rootletin and β-catenin, another intercentrosomal linker protein [8], [11]. However, exact mechanisms remain to be investigated further. For example, it is not clear whether all three proteins should be phosphorylated by NEK2 or not for completion of the centrosome separation.

We have generated a protein–protein interaction network among the core centrosome proteins, and observed a number of novel interactions within them. In the present study, we report a novel interaction of CEP135 with C-NAP1. Our results suggest that CEP135 functions as a platform for C-NAP1 at the centriole.

Section snippets

Preparation of cDNAs

The centrosomal cDNA clones were cloned directly from a HeLa mRNA pool using a PCR-based method; purchased from German Resource Center for Genome Research (www.rzpd.de), Kazusa DNA Research Institute (www.kazusa.or.jp) and Korean UniGene Information (kugi.kribb.re.kr); or kindly gifted from Drs. A. Merdes (PCM-1), Y. Ono (CG-NAP), and J. B. Rattner (C-NAP1). The cDNAs were subcloned into pGADT7 and pGBKT7 (Clontech) for the yeast two-hybrid assays and into pcDNA3.1 (Invitrogen) and pCMV-Tag3

Specific interaction of CEP135 with C-NAP1

We carried out yeast two-hybrid interaction assays with CEP135 as bait and identified 10 centrosomal proteins (data not shown). Among the proteins interacting with CEP135, C-NAP1 revealed the strongest interaction. When C-NAP1 was used for yeast two-hybrid interaction assays among the core centrosome proteins, it exhibited many interactions in addition to known ones such as rootletin and NEK2; thus, it may have additional functions that have not been reported to date (unpublished data).

Discussion

In the present study, we report a novel interaction of CEP135 with C-NAP1. Knockdown of CEP135 caused a premature centrosome splitting. Reduction of the centrosomal C-NAP1 levels was accompanied. Based on these observations, we propose a model in which CEP135 acts as a platform for C-NAP1 at the proximal end of the centriole (Fig. 6). CEP135 suppression allowed release of the centrosomal C-NAP1, resulting in premature centrosome splitting.

We observed that the CEP135 truncated mutant proteins

Acknowledgments

This study was supported by grants from the BioImaging Research Center at GIST, the Basic Research Program (R01-2007-000-20116-0), the Korea Research Promotion Fund (KRF-2006-311-C00471) and the SRC Program (R11-2005-009-03005-0). K. Kim, S. Lee, and J. Chang were supported by the second stage of the Brain Korea 21 Project in 2007.

References (16)

There are more references available in the full text version of this article.

Cited by (52)

  • The Centrosome Linker and Its Role in Cancer and Genetic Disorders

    2020, Trends in Molecular Medicine
    Citation Excerpt :

    Surprisingly little is known on how centriole disengagement and centrosome linker assembly are coordinated [1,28–30]. The ring-like binding of the protein C-Nap1 (encoded by CEP250) to CEP135 at the proximal end of the two centrioles is probably one of the first steps of the centrosome linker assembly process [31]. This priming event then likely triggers the self-assembly of fibers starting from the centriole-associated C-Nap1 ring [30] (Figure 1B).

  • Rootletin prevents Cep68 from VHL-mediated proteasomal degradation to maintain centrosome cohesion

    2017, Biochimica et Biophysica Acta - Molecular Cell Research
    Citation Excerpt :

    Proteins implicated in tethering function include C-Nap1 [12], Rootletin [13], Cep68 [14], CEP215-CDK5RAP2 [8,14], LRCC45 [15], and centlein [16], of which C-Nap1 and Rootletin are the essential components of the intercentrosomal linker [14]. C-Nap1 associated with the proximal ends of the centrioles through CEP135 [17] is supposed to be a centriolar docking protein [10,12], while Rootletin decorates fibers emanating from the proximal ends of parental centrioles [13,14]. At the onset of mitosis, the intercentrosomal linker is disassembled via different mechanisms, coincident with centrosome separation in preparation for mitotic spindle assembly [18].

  • A short CEP135 splice isoform controls centriole duplication

    2015, Current Biology
    Citation Excerpt :

    During G1 phase of the cell cycle, CEP135mini localizes predominantly to the proximal end of centrioles but is slightly spread compared to CEP135full (Figures 1B and S4C–S4F), whereas in G2 it localizes both to centrioles and to the pericentriolar material (PCM) as judged by co-staining with γ-tubulin (Figures 1B, 1C, and S4D–S4F). CEP135full localization to the PCM was not observed in either G1 or G2 (Figures 1B, 1C, and S1I) [16, 20, 22–24]. Immuno-EM localization also places CEP135mini at the proximal end of the centriole co-incident with the triplet microtubules and the PCM (Figures 1D–1F).

View all citing articles on Scopus
1

These authors contributed equally.

View full text