A Role for Centrin 3 in Centrosome Reproduction

Abstract

Centrosome reproduction by duplication is essential for the bipolarity of cell division, but the molecular basis of this process is still unknown. Mutations in Saccharomyces cerevisiae CDC31 gene prevent the duplication of the spindle pole body (SPB). The product of this gene belongs to the calmodulin super-family and is concentrated at the half bridge of the SPB. We present a functional analysis of HsCEN3, a human centrin gene closely related to the CDC31 gene. Tran- sient overexpression of wild-type or mutant forms of HsCen3p in human cells demonstrates that centriole localization depends on a functional fourth EF-hand, but does not produce mitotic phenotype. However, injection of recombinant HsCen3p or of RNA encoding HsCen3p in one blastomere of two-cell stage Xenopus laevis embryos resulted in undercleavage and inhibition of centrosome duplication. Furthermore, HsCEN3 does not complement mutations or deletion of CDC31 in S. cerevisiae, but specifically blocks SPB duplication, indicating that the human protein acts as a dominant negative mutant of CDC31. Several lines of evidence indicate that HsCen3p acts by titrating Cdc31p-binding protein(s).Our results demonstrate that, in spite of the large differences in centrosome structure among widely divergent species, the centrosome pathway of reproduction is conserved.

Centrobin-mediated Regulation of the Centrosomal Protein 4.1-associated Protein (CPAP) Level Limits Centriole Length during Elongation Stage

“…1A, left panel). As shown previously by others (54,55), tubulin and centrin staining along the length of these structures confirms that these are in fact microtubule-based, elongated centrioles.…”

Differential Expression and Interaction with the Visual G-protein Transducin of Centrin Isoforms in Mammalian Photoreceptor Cells

“…Although a recent study showed Ca 2ϩ -independent binding of centrin to a target protein (50), in most cases the activation of centrins by Ca 2ϩ is necessary for the interaction with their binding partners (16,18). In the yeast Saccharomyces cerevisiae, Ca 2ϩ facilitates binding of Cdc31p, the yeast centrin homologue, and of the human centrin isoforms 1 and 2 to Kar1p, which is necessary for division of the yeast cell (48,51,52). In our previous centrin blot overlay assays, the binding of recombinant MmCen1 to all detected interacting proteins was Ca 2ϩ -dependent (15,16).…”

Centrin/Cdc31 Is a Novel Regulator of Protein Degradation

“…The three centrin proteins that are expressed in human cells (30,31) display differential tissue-specific levels (27,36,49). However, all three isoforms share the key structural features that are present in EF-hand proteins (32).…”

“…The SPB is a proteinaceous structure that is embedded in the nuclear envelope, and its duplication marks exit from the G 1 phase and the onset of chromosome duplication. The composition of the SPB has been defined, and the steps that lead to its duplication have been studied extensively using biochemical, genetic, and microscopic methods (3,5,6,18,24,28,30,43,44). An important component of the SPB is Cdc31 (3, 13), which in yeast is encoded by the essential CDC31 gene.…”

“…However, all three isoforms share the key structural features that are present in EF-hand proteins (32). Human CEN3 is closely related to Cdc31 and can interfere with SPB duplication when overexpressed in yeast (30). An N-terminal regulatory domain is proposed to regulate the activity of the C-terminal domain, possibly through Ca 2ϩ -mediated signaling (42).…”