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Characterization of Cep85 - a new antagonist of Nek2A that is involved in the regulation of centrosome disjunction.

Chen C, Tian F, Lu L, Wang Y, Xiao Z, Yu C, Yu X - J. Cell. Sci. (2015)

Bottom Line: Opposite to the effects of Nek2A, overexpression of Cep85 in conjunction with inhibition of the motor protein Eg5 (also known as KIF11) leads to the failure of centrosome disjunction.Although the Nek2A-binding domain alone is sufficient to inhibit Nek2A kinase activity in vitro, both domains are indispensable for full suppression of centrosome disjunction in cells.Thus, we propose that Cep85 is a bona fide Nek2A-binding partner that surrounds the proximal ends of centrioles where it cooperates with PP1γ (also known as PPP1CC) to antagonize Nek2A activity in order to maintain the centrosome integrity in interphase in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China chench@xmu.edu.cn yuxw@xmu.edu.cn.

No MeSH data available.


Related in: MedlinePlus

Identification of Cep85 as a binding partner of Nek2A. (A) Flag-tagged kinase-dead (KD) Nek2A was expressed in HEK293T cells, affinity purified with M2 agarose and separated by SDS-PAGE, followed by silver staining to visualize the locations of individual proteins. The protein bands around 85 kDa inside the boxed area were identified by mass spectrometry to contain the Cep85 protein. (B) Myc–Cep85, co-expressed with HA–Nek2A in HEK293T cells, was immunoprecipitated with anti-Myc antibody. HA–Nek2A in the immunoprecipitate (IP) and total cell lysates (TCL) was visualized with anti-HA antibody by western blotting (IB). (C) The endogenous Cep85 in HEK293T cell lysate was immunoprecipitated with anti-Cep85 antibody and anti-Nek2A antibody was used to detect Nek2A in the precipitate. (D) The same experiment as in C was performed using U2OS cell lysates. (E) GST pulldown assays were carried out with GST–Cep85 protein expressed in and purified from E. coli, and HA–Nek2A overexpressed in HEK293T cells. CBB, Coomassie Brilliant Blue. (F) A schematic of Nek2A and its truncated mutants is shown in the upper panel. Myc–Cep85 and HA–Nek2A constructs were co-expressed in HEK293T cells. The binding region in Nek2A to Cep85 defined by co-immunoprecipitation and western blot analysis is shown in the lower panel. Asterisks indicate the positions of Nek2A proteins.
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JCS171637F1: Identification of Cep85 as a binding partner of Nek2A. (A) Flag-tagged kinase-dead (KD) Nek2A was expressed in HEK293T cells, affinity purified with M2 agarose and separated by SDS-PAGE, followed by silver staining to visualize the locations of individual proteins. The protein bands around 85 kDa inside the boxed area were identified by mass spectrometry to contain the Cep85 protein. (B) Myc–Cep85, co-expressed with HA–Nek2A in HEK293T cells, was immunoprecipitated with anti-Myc antibody. HA–Nek2A in the immunoprecipitate (IP) and total cell lysates (TCL) was visualized with anti-HA antibody by western blotting (IB). (C) The endogenous Cep85 in HEK293T cell lysate was immunoprecipitated with anti-Cep85 antibody and anti-Nek2A antibody was used to detect Nek2A in the precipitate. (D) The same experiment as in C was performed using U2OS cell lysates. (E) GST pulldown assays were carried out with GST–Cep85 protein expressed in and purified from E. coli, and HA–Nek2A overexpressed in HEK293T cells. CBB, Coomassie Brilliant Blue. (F) A schematic of Nek2A and its truncated mutants is shown in the upper panel. Myc–Cep85 and HA–Nek2A constructs were co-expressed in HEK293T cells. The binding region in Nek2A to Cep85 defined by co-immunoprecipitation and western blot analysis is shown in the lower panel. Asterisks indicate the positions of Nek2A proteins.

Mentions: Nek2A expression levels remain high in the S to G2 phase, however, its activity at the proximal ends of centrioles needs to be kept low to ensure that the centrosome is not to be separated precociously (Mardin and Schiebel, 2012). In order to identify potential Nek2A-binding partners that might suppress Nek2A activity, we expressed a Flag-tagged kinase-dead mutant Nek2A in human embryonic kidney 293T (HEK293T) cells and used M2 agarose to affinity purify Flag–Nek2A, followed by mass spectrometry analysis to determine the identities of Nek2A-binding proteins (Fig. 1A). Among the identified proteins, centrosomal protein 85 kDa (Cep85), also known as coiled-coil domain-containing protein 21 (CCDC21), was a particular interesting candidate. It was initially identified with other 21 proteins as new centrosomal components by complementary proteomics strategies (Jakobsen et al., 2011). Except for its spindle pole association, to date, no other studies have been performed. Human Cep85 was predicted to contain two coiled-coil domains (supplementary material Fig. S1A). Its orthologs were well conserved in many eukaryotes, including Mus musculus, Xenopus, Danio rerio and Gallus gallus (supplementary material Fig. S1B).Fig. 1.


Characterization of Cep85 - a new antagonist of Nek2A that is involved in the regulation of centrosome disjunction.

Chen C, Tian F, Lu L, Wang Y, Xiao Z, Yu C, Yu X - J. Cell. Sci. (2015)

Identification of Cep85 as a binding partner of Nek2A. (A) Flag-tagged kinase-dead (KD) Nek2A was expressed in HEK293T cells, affinity purified with M2 agarose and separated by SDS-PAGE, followed by silver staining to visualize the locations of individual proteins. The protein bands around 85 kDa inside the boxed area were identified by mass spectrometry to contain the Cep85 protein. (B) Myc–Cep85, co-expressed with HA–Nek2A in HEK293T cells, was immunoprecipitated with anti-Myc antibody. HA–Nek2A in the immunoprecipitate (IP) and total cell lysates (TCL) was visualized with anti-HA antibody by western blotting (IB). (C) The endogenous Cep85 in HEK293T cell lysate was immunoprecipitated with anti-Cep85 antibody and anti-Nek2A antibody was used to detect Nek2A in the precipitate. (D) The same experiment as in C was performed using U2OS cell lysates. (E) GST pulldown assays were carried out with GST–Cep85 protein expressed in and purified from E. coli, and HA–Nek2A overexpressed in HEK293T cells. CBB, Coomassie Brilliant Blue. (F) A schematic of Nek2A and its truncated mutants is shown in the upper panel. Myc–Cep85 and HA–Nek2A constructs were co-expressed in HEK293T cells. The binding region in Nek2A to Cep85 defined by co-immunoprecipitation and western blot analysis is shown in the lower panel. Asterisks indicate the positions of Nek2A proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4582193&req=5

JCS171637F1: Identification of Cep85 as a binding partner of Nek2A. (A) Flag-tagged kinase-dead (KD) Nek2A was expressed in HEK293T cells, affinity purified with M2 agarose and separated by SDS-PAGE, followed by silver staining to visualize the locations of individual proteins. The protein bands around 85 kDa inside the boxed area were identified by mass spectrometry to contain the Cep85 protein. (B) Myc–Cep85, co-expressed with HA–Nek2A in HEK293T cells, was immunoprecipitated with anti-Myc antibody. HA–Nek2A in the immunoprecipitate (IP) and total cell lysates (TCL) was visualized with anti-HA antibody by western blotting (IB). (C) The endogenous Cep85 in HEK293T cell lysate was immunoprecipitated with anti-Cep85 antibody and anti-Nek2A antibody was used to detect Nek2A in the precipitate. (D) The same experiment as in C was performed using U2OS cell lysates. (E) GST pulldown assays were carried out with GST–Cep85 protein expressed in and purified from E. coli, and HA–Nek2A overexpressed in HEK293T cells. CBB, Coomassie Brilliant Blue. (F) A schematic of Nek2A and its truncated mutants is shown in the upper panel. Myc–Cep85 and HA–Nek2A constructs were co-expressed in HEK293T cells. The binding region in Nek2A to Cep85 defined by co-immunoprecipitation and western blot analysis is shown in the lower panel. Asterisks indicate the positions of Nek2A proteins.
Mentions: Nek2A expression levels remain high in the S to G2 phase, however, its activity at the proximal ends of centrioles needs to be kept low to ensure that the centrosome is not to be separated precociously (Mardin and Schiebel, 2012). In order to identify potential Nek2A-binding partners that might suppress Nek2A activity, we expressed a Flag-tagged kinase-dead mutant Nek2A in human embryonic kidney 293T (HEK293T) cells and used M2 agarose to affinity purify Flag–Nek2A, followed by mass spectrometry analysis to determine the identities of Nek2A-binding proteins (Fig. 1A). Among the identified proteins, centrosomal protein 85 kDa (Cep85), also known as coiled-coil domain-containing protein 21 (CCDC21), was a particular interesting candidate. It was initially identified with other 21 proteins as new centrosomal components by complementary proteomics strategies (Jakobsen et al., 2011). Except for its spindle pole association, to date, no other studies have been performed. Human Cep85 was predicted to contain two coiled-coil domains (supplementary material Fig. S1A). Its orthologs were well conserved in many eukaryotes, including Mus musculus, Xenopus, Danio rerio and Gallus gallus (supplementary material Fig. S1B).Fig. 1.

Bottom Line: Opposite to the effects of Nek2A, overexpression of Cep85 in conjunction with inhibition of the motor protein Eg5 (also known as KIF11) leads to the failure of centrosome disjunction.Although the Nek2A-binding domain alone is sufficient to inhibit Nek2A kinase activity in vitro, both domains are indispensable for full suppression of centrosome disjunction in cells.Thus, we propose that Cep85 is a bona fide Nek2A-binding partner that surrounds the proximal ends of centrioles where it cooperates with PP1γ (also known as PPP1CC) to antagonize Nek2A activity in order to maintain the centrosome integrity in interphase in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China chench@xmu.edu.cn yuxw@xmu.edu.cn.

No MeSH data available.


Related in: MedlinePlus