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Curr Biol
2012 May 22;2210:915-21. doi: 10.1016/j.cub.2012.03.048.
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Kendrin is a novel substrate for separase involved in the licensing of centriole duplication.
Matsuo K
,
Ohsumi K
,
Iwabuchi M
,
Kawamata T
,
Ono Y
,
Takahashi M
.
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The centrosome, consisting of a pair of centrioles surrounded by pericentriolar material, directs the formation of bipolar spindles during mitosis. Aberrant centrosome number can promote chromosome instability, which is implicated in tumorigenesis. Thus, centrosome duplication needs to be tightly regulated to occur only once per cell cycle. Separase, a cysteine protease that triggers sister chromatid separation, is involved in centriole disengagement, which licenses centrosomes for the next round of duplication. However, at least two questions remain unsolved: what is the substrate relevant to the disengagement, and how does separase, activated at anaphase onset, act on the disengagement that occurs during late mitosis. Here, we show that kendrin, also named pericentrin, is cleaved by activated separase at a consensus site in vivo and in vitro, and this leads to the delayed release of kendrin from the centrosome later in mitosis. Furthermore, we demonstrate that expression of a noncleavable kendrin mutant suppresses centriole disengagement and subsequent centriole duplication. Based on these results, we propose that kendrin is a novel and crucial substrate for separase at the centrosome, protecting the engaged centrioles from premature disengagement and thereby blocking reduplication until the cell passes through mitosis.
Figure 1. Kendrin Is Specifically Cleaved at a Consensus Site for Separase during Mitosis(A) HeLa cells treated with control or kendrin siRNA (#1 and #2) were immunoblotted with anti-kendrin antibody. The bands labeled by black and white arrowheads indicate full-length kendrin and the fast-migrating band, respectively. The filter was reblotted with anti-p150Glued antibody as a loading control.(B) HeLa cells were arrested at the G1/S boundary by double thymidine treatment and subsequently released in fresh medium for the indicated time periods. Cells lysates were then analyzed by immunoblotting with antibodies against kendrin and the indicated proteins.(C) Schematic representation of kendrin and its mutant. Mutated residues (E2229 and R2231) are indicated by asterisks.(D) HeLa cells expressing HA-kendrinWT-Flag were synchronized at various time points and the cell lysates were analyzed by immunoblotting with the indicated antibodies. Numbers 1–4 correspond to the conditions described above the blots. The full-length and cleaved-kendrin bands are indicated by black and white arrowheads, respectively.(E) Sequence homology around the potential cleavage sites for separase among kendrin orthologs from various species (Homo sapiens: NP_006022.3, Canis lupus familiaris: XP_548735.2, Mus musculus: NP_032813.3, Gallus gallus: XP_421895.2, Danio rerio: XP_699814.3). The alignment was obtained from HomoloGene (NCBI). The position of the consensus sequence is boxed, with an arrow showing the potential cleavage site.(F) HeLa cells expressing HA-kendrinERRE-Flag were synchronized under the same conditions as in (D), and analyzed by immunoblotting.(G) HeLa cells expressing HA-kendrinWT or HA-kendrinERRE were fixed and double-stained with anti-HA and anti-C-Nap1. Centrosomes are indicated by arrows. Scale bar represents 10 μm. Asterisks in (D) and (F) indicate cross-reactive bands.See also Figure S1.
Figure 2. Kendrin Is a Novel Substrate for Separase(A) HeLa cells treated with control or separase siRNA (iSep1-3) were analyzed by immunoblotting with anti-kendrin and anti-separase antibodies. The full-length and cleavage products of kendrin or separase are indicated by arrowheads. The filter (anti-kendrin) was reblotted with anti-p150Glued as a loading control. The anti-cyclin B1 blot revealed that suppression of kendrincleavage was not caused by cell cycle arrest at early mitosis.(B) HeLa cells expressing HA-kendrinWT (WT) or HA-kendrinERRE (ERRE) in combination with Flag-tagged WT or an inactive mutant (CA) of separase were synchronized as in Figure 1D, and the cell lysates were analyzed by immunoblotting. Black and white arrowheads indicate anti-HA- and anti-Flag-specific bands, respectively. The intensity of the fast-migrating bands in the anti-HA blot was quantified as described in Supplemental Experimental Procedures. The cell-cycle progression of these cells was normal, as detected by phosphorylation of MPM2 epitopes. WCL, whole cell lysates. Asterisks indicate cross-reactive bands.(Ca) Flag-tagged separaseSA and separaseCA expressed in 293T cells were immunoprecipitated with anti-Flag (IP), incubated with mitotic extract of Xenopus eggs (M.E.) to degrade securin, and then eluted with 3 × Flag peptide as described in Supplemental Experimental Procedures. The samples were immunoblotted with anti-Flag. The full-length and cleavage products of separase are indicated by arrowheads.(Cb) Purified HA-kendrin-Flag was incubated with purified Flag-separaseSA or Flag-separaseCA for 0 and 90 min, and kendrincleavage was analyzed by immunoblotting with anti-kendrin. The full-length and cleavage products of kendrin are indicated by arrowheads.(D) Time course of kendrincleavage and separase activation during mitosis. HeLa cells synchronized at prometaphase were released into fresh medium for the indicated time periods. Cell lysates were then analyzed by immunoblotting for kendrin and indicated proteins. The anti-separase antibody detected the full-length 220 kDa protein and the 65 kDa autocleavage product, an indicator of separase activation. AS, asynchronous culture.
Figure 3. Kendrin Is Released from Centrosomes upon Limited Cleavage by Separase after a Short Time Lag(A) Subcellular localization of kendrin1-2231. HeLa cells transiently expressing HA-kendrin1-2231 were double-stained with anti-HA and anti-C-Nap1. Centrosomes are indicated by arrows.(B) Changes in kendrin immunofluorescence during the cell cycle. HeLa cells were triple-stained for kendrin (red, upper panel), α-tubulin (green), and DNA (blue). Z series images taken across the cells were projected onto a single view. The centrosomes of the cells at telophase and cytokinesis are indicated by arrows.(C) Interaction between kendrin fragments. Cos7 cells expressing the indicated combinations of the kendrin fragments were immunoprecipitated with anti-Flag or anti-HA antibodies and analyzed by immunoblotting.(D) HeLa cells coexpressing HA-kendrin1-2231 and Flag-kendrinwt were triple-stained with anti-HA, anti-Flag, and DAPI. Centrosomes are indicated by arrows.(E) Relationship between kendrin immunofluorescence intensity and the number of C-Nap1 foci was examined at late mitosis. Arrows indicate disengaged centrioles (2 C-Nap1 foci/centrosome). Scale bars in (A), (B), (D), and (E) represent 10 μm.See also Figure S2.
Figure 4. Expression of a Noncleavable Mutant, KendrinERRE, Suppresses Centriole Disengagement and Subsequent Centriole Duplication(A–D and F–I) U2-OS cells stably expressing centrin2-GFP were nontransfected (A and F) or transfected with γ-tubulin-mCherry (B and G), mCherry-kendrinWT (C and H), or mCherry-kendrinERRE (D and I) expression plasmid. The cells were then synchronized at G1/S phase by double thymidine block (A–D) or released for a further 8 hr to pass through S phase (F–I). Nontransfected cells were immunostained for C-Nap1 and kendrin (A and F), whereas cells expressing mCherry-tagged proteins were stained for C-Nap1 (B–D and G–I). Centrosomes marked by squares are magnified and presented on the right. Dashed circles indicate the positions of the nuclei. C-Nap1 immunofluorescence and mCherry fluorescence are visualized as pseudocolors red and blue, respectively. Scale bar represents 5 μm.(E and J) Quantitative analysis of centriole configurations in the cells shown in (A)–(D) and (F)–(I), respectively. Error bars indicate the SD of triplicate experiments. At least 90 cells were scored per condition. Statistical significance was assessed by analysis of variance and Tukey-Kramer tests. ∗∗p < 0.001 versus normal and versus WT are indicated.(K) U2-OS cells stably expressing centrin2-EGFP were immunostained for C-Nap1 (blue) and kendrin (red). Localization of kendrin before (a) and after (b) centriole duplication was examined by taking z series sections. Images of a typical single z section are shown. Arrowheads indicate the location of centrin2 detected outside of the kendrin cylinder. Scale bar represents 1 μm.See also Figure S3.
