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CCNYL1, but Not CCNY, Cooperates with CDK16 to Regulate Spermatogenesis in Mouse.

Zi Z, Zhang Z, Li Q, An W, Zeng L, Gao D, Yang Y, Zhu X, Zeng R, Shum WW, Wu J - PLoS Genet. (2015)

Bottom Line: These results suggest that Ccnyl1, but not Ccny, is indispensable for male fertility.We identified the phosphorylation sites of CDK16 by mass spectrometry and revealed that several phosphorylation modifications on the N-terminal region of CDK16 were indispensable for the CCNYL1 binding and the modulation of CDK16 kinase activity.Our results therefore reveal a previously unrecognized role of CCNYL1 in regulating spermatogenesis through the interaction and modulation of CDK16.

View Article: PubMed Central - PubMed

Affiliation: Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science & Technology of China, Hefei, Anhui, China.

ABSTRACT
Cyclin Y-like 1 (Ccnyl1) is a newly-identified member of the cyclin family and is highly similar in protein sequences to Cyclin Y (Ccny). However, the function of Ccnyl1 is poorly characterized in any organism. Here we found that Ccnyl1 was most abundantly expressed in the testis of mice and was about seven times higher than the level of Ccny. Male Ccnyl1-/- mice were infertile, whereas both male and female Ccny-/- mice displayed normal fertility. These results suggest that Ccnyl1, but not Ccny, is indispensable for male fertility. Spermatozoa obtained from Ccnyl1-/- mice displayed significantly impaired motility, and represented a thinned annulus region and/or a bent head. We found that the protein, but not the mRNA, level of cyclin-dependent kinase 16 (CDK16) was decreased in the testis of Ccnyl1-/- mice. Further study demonstrated that CCNYL1 interacted with CDK16 and this interaction mutually increased the stability of these two proteins. Moreover, the interaction increased the kinase activity of CDK16. In addition, we observed an alteration of phosphorylation levels of CDK16 in the presence of CCNYL1. We identified the phosphorylation sites of CDK16 by mass spectrometry and revealed that several phosphorylation modifications on the N-terminal region of CDK16 were indispensable for the CCNYL1 binding and the modulation of CDK16 kinase activity. Our results therefore reveal a previously unrecognized role of CCNYL1 in regulating spermatogenesis through the interaction and modulation of CDK16.

No MeSH data available.


Related in: MedlinePlus

The interaction of CCNYL1 and CDK16 and protection of protein stability.(A) CDK16-Flag and CCNYL1-HA were either expressed alone or together in HEK293T cells. Protein expression levels were measured by western blotting. (B) CDK16-Flag and CCNYL1-HA were either expressed alone or together in HEK293T cells for 24 hours followed by incubation with 10 μM cycloheximide for the indicated times. Protein levels were measured by western blotting. (C) CDK16-Flag and CCNYL1-HA were either expressed alone or together in HEK293T cells for 24 h followed by incubation with 10 μM MG132. Then, cells were harvested at 24 h for the detection of CDK16-Flag, or at 6 h for the detection of CCNYL1-HA. Protein levels were measured by western blotting. (D) CDK16-Flag and CCNYL1-HA were either expressed alone or together in HEK293T cells for 24 hours followed by incubation with 10 μM MG132 for 6 h. The CDK16-Flag and CCNYL1-HA protein were immunoprecipitated for the detection of their ubiquitination levels by western blotting.
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pgen.1005485.g006: The interaction of CCNYL1 and CDK16 and protection of protein stability.(A) CDK16-Flag and CCNYL1-HA were either expressed alone or together in HEK293T cells. Protein expression levels were measured by western blotting. (B) CDK16-Flag and CCNYL1-HA were either expressed alone or together in HEK293T cells for 24 hours followed by incubation with 10 μM cycloheximide for the indicated times. Protein levels were measured by western blotting. (C) CDK16-Flag and CCNYL1-HA were either expressed alone or together in HEK293T cells for 24 h followed by incubation with 10 μM MG132. Then, cells were harvested at 24 h for the detection of CDK16-Flag, or at 6 h for the detection of CCNYL1-HA. Protein levels were measured by western blotting. (D) CDK16-Flag and CCNYL1-HA were either expressed alone or together in HEK293T cells for 24 hours followed by incubation with 10 μM MG132 for 6 h. The CDK16-Flag and CCNYL1-HA protein were immunoprecipitated for the detection of their ubiquitination levels by western blotting.

Mentions: Since our data showed that the CDK16 protein level was decreased in the Ccnyl1-/- testis (Fig 4E and 4F), and that CCNYL1 interacted with CDK16 both in vivo and in vitro (Fig 5C and 5D), we proposed that the recruitment of CDK16 by CCNYL1 increases its stability. This hypothesis was supported by the co-expression of CCNYL1 and CDK16 in HEK293T cells resulting in higher levels of both CCNYL1 and CDK16 proteins than single-expression controls (Fig 6A). Protein degradation rates monitored by the cycloheximide chase assay revealed attenuated degradation rates after co-expression (Fig 6B), suggesting that the interaction stabilizes both CCNYL1 and CDK16 proteins. Furthermore, proteasome inhibitor MG132 supplementation led to accumulation of both CCNYL1 and CDK16 proteins, which was more obvious after single expression than co-expression (Fig 6C). Consistent with these results the accumulated ubiquitinated CCNYL1 and CDK16 levels were significantly lower after co-expression than those of after single expression (Fig 6D).


CCNYL1, but Not CCNY, Cooperates with CDK16 to Regulate Spermatogenesis in Mouse.

Zi Z, Zhang Z, Li Q, An W, Zeng L, Gao D, Yang Y, Zhu X, Zeng R, Shum WW, Wu J - PLoS Genet. (2015)

The interaction of CCNYL1 and CDK16 and protection of protein stability.(A) CDK16-Flag and CCNYL1-HA were either expressed alone or together in HEK293T cells. Protein expression levels were measured by western blotting. (B) CDK16-Flag and CCNYL1-HA were either expressed alone or together in HEK293T cells for 24 hours followed by incubation with 10 μM cycloheximide for the indicated times. Protein levels were measured by western blotting. (C) CDK16-Flag and CCNYL1-HA were either expressed alone or together in HEK293T cells for 24 h followed by incubation with 10 μM MG132. Then, cells were harvested at 24 h for the detection of CDK16-Flag, or at 6 h for the detection of CCNYL1-HA. Protein levels were measured by western blotting. (D) CDK16-Flag and CCNYL1-HA were either expressed alone or together in HEK293T cells for 24 hours followed by incubation with 10 μM MG132 for 6 h. The CDK16-Flag and CCNYL1-HA protein were immunoprecipitated for the detection of their ubiquitination levels by western blotting.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4549061&req=5

pgen.1005485.g006: The interaction of CCNYL1 and CDK16 and protection of protein stability.(A) CDK16-Flag and CCNYL1-HA were either expressed alone or together in HEK293T cells. Protein expression levels were measured by western blotting. (B) CDK16-Flag and CCNYL1-HA were either expressed alone or together in HEK293T cells for 24 hours followed by incubation with 10 μM cycloheximide for the indicated times. Protein levels were measured by western blotting. (C) CDK16-Flag and CCNYL1-HA were either expressed alone or together in HEK293T cells for 24 h followed by incubation with 10 μM MG132. Then, cells were harvested at 24 h for the detection of CDK16-Flag, or at 6 h for the detection of CCNYL1-HA. Protein levels were measured by western blotting. (D) CDK16-Flag and CCNYL1-HA were either expressed alone or together in HEK293T cells for 24 hours followed by incubation with 10 μM MG132 for 6 h. The CDK16-Flag and CCNYL1-HA protein were immunoprecipitated for the detection of their ubiquitination levels by western blotting.
Mentions: Since our data showed that the CDK16 protein level was decreased in the Ccnyl1-/- testis (Fig 4E and 4F), and that CCNYL1 interacted with CDK16 both in vivo and in vitro (Fig 5C and 5D), we proposed that the recruitment of CDK16 by CCNYL1 increases its stability. This hypothesis was supported by the co-expression of CCNYL1 and CDK16 in HEK293T cells resulting in higher levels of both CCNYL1 and CDK16 proteins than single-expression controls (Fig 6A). Protein degradation rates monitored by the cycloheximide chase assay revealed attenuated degradation rates after co-expression (Fig 6B), suggesting that the interaction stabilizes both CCNYL1 and CDK16 proteins. Furthermore, proteasome inhibitor MG132 supplementation led to accumulation of both CCNYL1 and CDK16 proteins, which was more obvious after single expression than co-expression (Fig 6C). Consistent with these results the accumulated ubiquitinated CCNYL1 and CDK16 levels were significantly lower after co-expression than those of after single expression (Fig 6D).

Bottom Line: These results suggest that Ccnyl1, but not Ccny, is indispensable for male fertility.We identified the phosphorylation sites of CDK16 by mass spectrometry and revealed that several phosphorylation modifications on the N-terminal region of CDK16 were indispensable for the CCNYL1 binding and the modulation of CDK16 kinase activity.Our results therefore reveal a previously unrecognized role of CCNYL1 in regulating spermatogenesis through the interaction and modulation of CDK16.

View Article: PubMed Central - PubMed

Affiliation: Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science & Technology of China, Hefei, Anhui, China.

ABSTRACT
Cyclin Y-like 1 (Ccnyl1) is a newly-identified member of the cyclin family and is highly similar in protein sequences to Cyclin Y (Ccny). However, the function of Ccnyl1 is poorly characterized in any organism. Here we found that Ccnyl1 was most abundantly expressed in the testis of mice and was about seven times higher than the level of Ccny. Male Ccnyl1-/- mice were infertile, whereas both male and female Ccny-/- mice displayed normal fertility. These results suggest that Ccnyl1, but not Ccny, is indispensable for male fertility. Spermatozoa obtained from Ccnyl1-/- mice displayed significantly impaired motility, and represented a thinned annulus region and/or a bent head. We found that the protein, but not the mRNA, level of cyclin-dependent kinase 16 (CDK16) was decreased in the testis of Ccnyl1-/- mice. Further study demonstrated that CCNYL1 interacted with CDK16 and this interaction mutually increased the stability of these two proteins. Moreover, the interaction increased the kinase activity of CDK16. In addition, we observed an alteration of phosphorylation levels of CDK16 in the presence of CCNYL1. We identified the phosphorylation sites of CDK16 by mass spectrometry and revealed that several phosphorylation modifications on the N-terminal region of CDK16 were indispensable for the CCNYL1 binding and the modulation of CDK16 kinase activity. Our results therefore reveal a previously unrecognized role of CCNYL1 in regulating spermatogenesis through the interaction and modulation of CDK16.

No MeSH data available.


Related in: MedlinePlus