Differential requirements for the EF-hand domains of human centrin 2 in primary ciliogenesis and nucleotide excision repair

Abstract

Centrin 2 is a small conserved calcium-binding protein that localizes to the centriolar distal lumen in human cells. It is required for efficient primary ciliogenesis and nucleotide excision repair (NER). Centrin 2 forms part of the xeroderma pigmentosum group C protein complex. To explore how centrin 2 contributes to these distinct processes, we mutated the four calcium-binding EF-hand domains of human centrin 2. Centrin 2 in which all four EF-hands had been mutated to ablate calcium binding (4DA mutant) was capable of supporting in vitro NER and was as effective as the wild-type protein in rescuing the UV sensitivity of centrin 2-null cells. However, we found that mutation of any of the EF-hand domains impaired primary ciliogenesis in human TERT-RPE1 cells to the same extent as deletion of centrin 2. Phenotypic analysis of the 4DA mutant revealed defects in centrosome localization, centriole satellite assembly, ciliary assembly and function and in interactions with POC5 and SFI1. These observations indicate that centrin 2 requires calcium-binding capacity for its primary ciliogenesis functions, but not for NER, and suggest that these functions require centrin 2 to be capable of forming complexes with partner proteins. This article has an associated First Person interview with the first author of the paper.

Systematic Discovery of Short Linear Motifs Decodes Calcineurin Phosphatase Signaling

“…However, our as-yet-unsuccessful attempts to directly detect CN localization at centrosomes (data not shown) suggest that CN-centrosome interactions are limited, transient, and/or temporally regulated. Investigations of Ca 2+ signaling at centrosomes, which contain multiple Ca 2+ -and/or calmodulin-binding proteins (Galletta et al, 2014;Khouj et al, 2019;Spektor et al, 2007;Tsang et al, 2006), and cilia, which constitute a distinct Ca 2+ compartment (Delling et al, 2013(Delling et al, , 2016, are ongoing. Our findings point to CN as a likely downstream effector of Ca 2+ signals at these organelles.…”

CCDC15 localizes to the centriole inner scaffold and regulates centriole integrity and ciliogenesis

“…As shown in Fig. S1, Centrin-2 and POC5 proximity interactomes were enriched for GO categories that are relevant for their published functions during centrosomal and/or nuclear biological processes and related cellular compartments (Azimzadeh et al, 2009; Dantas et al, 2013; Heydeck et al, 2020; Khouj et al, 2019; Resendes et al, 2008; Salisbury et al, 2002; Steib et al, 2020; Yang et al, 2010; Ying et al, 2019). For example, the highly enriched GO categories for biological processes include cell division, protein folding, mRNA splicing, and chromatin remodeling for Centrin-2 and centriole replication, protein localization to centrosome and cilium assembly for POC5 (Fig.…”

Calcineurin associates with centrosomes and regulates cilia length maintenance

“…Ca 2+ signals have been observed at centrosomes (Helassa et al, 2019), where they activate downstream effectors like CaM (Plotnikova et al, 2012) and centrin. In fact, centrin requires Ca 2+ to localize to centrosomes and to interact with the structural protein POC5 (Khouj et al, 2019). CN binds directly to POC5 through a PxIxIT motif and alters POC5 distribution within the centriole, suggesting that it may directly regulate this protein despite our inability to detect CN-dependent changes in POC5 phosphorylation in vivo .…”