Abstract
Centrosomes, the principal microtubule-organising centres in animal cells, contain centrins, small, conserved calcium-binding proteins unique to eukaryotes. Centrin2 binds to xeroderma pigmentosum group C protein (XPC), stabilising it, and its presence slightly increases nucleotide excision repair (NER) activity in vitro. In previous work, we deleted all three centrin isoforms present in chicken DT40 cells and observed delayed repair of UV-induced DNA lesions, but no centrosome abnormalities. Here, we explore how centrin2 controls NER. In the centrin null cells, we expressed centrin2 mutants that cannot bind calcium or that lack sites for phosphorylation by regulatory kinases. Expression of any of these mutants restored the UV sensitivity of centrin null cells to normal as effectively as expression of wild-type centrin. However, calcium-binding-deficient and T118A mutants showed greatly compromised localisation to centrosomes. XPC recruitment to laser-induced UV-like lesions was only slightly slower in centrin-deficient cells than in controls, and levels of XPC and its partner HRAD23B were unaffected by centrin deficiency. Interestingly, we found that overexpression of the centrin interactor POC5 leads to the assembly of linear, centrin-dependent structures that recruit other centrosomal proteins such as PCM-1 and NEDD1. Together, these observations suggest that assembly of centrins into complex structures requires calcium binding capacity, but that such assembly is not required for centrin activity in NER.
Highlights
As the principal microtubule organising centre in animal somatic cells, centrosomes play important roles in controlling cell shape and polarity, as well as directing the formation of the mitotic spindle through establishing its poles
We used site-directed mutagenesis to mutate each of these 3 residues to alanine (A) in a myc-centrin2 construct which expresses a protein that reproduces the subcellular localisation of endogenous centrin2 and rescues the nucleotide excision repair (NER) defect of centrindeficient cells [25]
Our results suggest that centrosomal localisation of centrin2 is regulated by calcium binding and that, even though this capacity is not required for NER, it appears to be critical for centrin assembly into larger complexes
Summary
As the principal microtubule organising centre in animal somatic cells, centrosomes play important roles in controlling cell shape and polarity, as well as directing the formation of the mitotic spindle through establishing its poles. Evolutionarily conserved, calcium-binding proteins that are crucial for basal body assembly and/or function in lower eukaryotes [4,5,6]. They localise to centrosomes and basal bodies in mammalian cells [7,8,9,10,11,12], the bulk of cellular centrin is not centrosomal [12]. CETN1 is restricted to certain cell types in its expression pattern and CETN4 is a pseudogene in human cells, while CETN2 is expressed ubiquitously in humans [17,18]
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