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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: 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.

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

Identification and functional study of phosphorylation modifications of CDK16.(A) Testicular lysates from two pairs of WT and Ccnyl1-/- mice were incubated with or without calf intestinal phosphatase (CIP) at 37°C for 30 min and analyzed by western blotting. (B) CDK16-Flag was either expressed alone or together with CCNYL1-HA in HEK293T cells. The cell lysates were incubated with or without CIP, and analyzed by western blotting. (C) Phosphorylation sites on CDK16 identified by mass spectrometry. Sites that changed significantly in phosphorylation levels between single- (CDK16-Flag) and double- (CDK16-Flag and CCNYL1-HA) expressed groups are labeled red with asterisks. (D) Various CDK16-Flag mutants were coexpressed with CCNYL1-HA in HEK293T cells. The interactions were analyzed by CoIP experiments followed by western blot. (E) Varies CDK16-Flag mutants were either expressed alone or coexpressed with CCNYL1-HA in HEK293T cells. The CDK16-Flag protein was immunoprecipitated, and incubated with MBP as substrate in the kinase buffer. Reaction products were separated by SDS-PAGE and followed by autoradiography.
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pgen.1005485.g007: Identification and functional study of phosphorylation modifications of CDK16.(A) Testicular lysates from two pairs of WT and Ccnyl1-/- mice were incubated with or without calf intestinal phosphatase (CIP) at 37°C for 30 min and analyzed by western blotting. (B) CDK16-Flag was either expressed alone or together with CCNYL1-HA in HEK293T cells. The cell lysates were incubated with or without CIP, and analyzed by western blotting. (C) Phosphorylation sites on CDK16 identified by mass spectrometry. Sites that changed significantly in phosphorylation levels between single- (CDK16-Flag) and double- (CDK16-Flag and CCNYL1-HA) expressed groups are labeled red with asterisks. (D) Various CDK16-Flag mutants were coexpressed with CCNYL1-HA in HEK293T cells. The interactions were analyzed by CoIP experiments followed by western blot. (E) Varies CDK16-Flag mutants were either expressed alone or coexpressed with CCNYL1-HA in HEK293T cells. The CDK16-Flag protein was immunoprecipitated, and incubated with MBP as substrate in the kinase buffer. Reaction products were separated by SDS-PAGE and followed by autoradiography.

Mentions: We found that CDK16 was not only decreased in the Ccnyl1-/- testis, but also shifted to a slightly lower mass than that of WT controls by SDS-PAGE (Figs 5B and 7A). This band-shift was eliminated by incubating the total testicular cell lysates with phosphatase (Fig 7A), an observation also found in HEK293T cells (Fig 7B), implying that the presence of CCNYL1 is related to the phosphorylation status of CDK16. To check this implication, we co-expressed CCNYL1 with kinase dead CDK16 (K194R). While the interaction between these two proteins was not affected by the kinase dead mutation of CDK16, the slight band-shift of CDK16 was absent (S7A Fig), indicating that the phosphorylation modifications of CDK16 in the presence of CCNYL1 might also depend on the kinase activity of CDK16. Analysis of the phosphorylation sites on CDK16 by mass spectrometry showed that the N-terminal region of CDK16 was highly phosphorylated (Fig 7C). In total, 22 phosphorylation sites were identified, and 19 of them determined with high confidence (S2 Table and S1 Dataset). Of those 19 sites, nine have not been reported. They were S36, S64, S65, S89, S146, T175, T380, S391, and S478. Our results showed that the phosphorylation levels of CDK16 were increased at four sites (S36, S146, T175, and S480) and decreased at one site (S78), when co-expressed with CCNYL1 (S2 Table), consistent with the band-shift of CDK16 reflecting altered phosphorylation levels. Phosphorylation of CDKs has been reported to be involved in modifying their interaction with the cyclins and activation of their own Ser/Thr protein kinase activity [5, 15, 24]. To determine if the altered phosphorylation of CDK16 can affect its binding activity, we generated mutations of these phosphorylation sites for use in CoIP assays. We found that the S36A, S36D, S480A, and S480D mutations had no effect on CCNYL1 binding; but the S78A and T175D mutations increased and the T175A mutation decreased binding activity (Fig 7D). Most significant was the S146 site, with both S146A and S146D mutation almost entirely abolishing the binding to CCNYL1, confirming the importance of this site for the binding activity. In addition, we found S110, S119 and S153 were also highly phosphorylated, of these sites S119 and S153 are reported to be critical for the binding of the CCNY [5]. We mutated the S110, S119 and S153 sites, and found that the S110A, S110D, S119A and S153D mutations decreased the binding towards CCNYL1 to different degrees, whereas the S153A mutation increased the binding to CCNYL1 (Fig 7D and S7B Fig).


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)

Identification and functional study of phosphorylation modifications of CDK16.(A) Testicular lysates from two pairs of WT and Ccnyl1-/- mice were incubated with or without calf intestinal phosphatase (CIP) at 37°C for 30 min and analyzed by western blotting. (B) CDK16-Flag was either expressed alone or together with CCNYL1-HA in HEK293T cells. The cell lysates were incubated with or without CIP, and analyzed by western blotting. (C) Phosphorylation sites on CDK16 identified by mass spectrometry. Sites that changed significantly in phosphorylation levels between single- (CDK16-Flag) and double- (CDK16-Flag and CCNYL1-HA) expressed groups are labeled red with asterisks. (D) Various CDK16-Flag mutants were coexpressed with CCNYL1-HA in HEK293T cells. The interactions were analyzed by CoIP experiments followed by western blot. (E) Varies CDK16-Flag mutants were either expressed alone or coexpressed with CCNYL1-HA in HEK293T cells. The CDK16-Flag protein was immunoprecipitated, and incubated with MBP as substrate in the kinase buffer. Reaction products were separated by SDS-PAGE and followed by autoradiography.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005485.g007: Identification and functional study of phosphorylation modifications of CDK16.(A) Testicular lysates from two pairs of WT and Ccnyl1-/- mice were incubated with or without calf intestinal phosphatase (CIP) at 37°C for 30 min and analyzed by western blotting. (B) CDK16-Flag was either expressed alone or together with CCNYL1-HA in HEK293T cells. The cell lysates were incubated with or without CIP, and analyzed by western blotting. (C) Phosphorylation sites on CDK16 identified by mass spectrometry. Sites that changed significantly in phosphorylation levels between single- (CDK16-Flag) and double- (CDK16-Flag and CCNYL1-HA) expressed groups are labeled red with asterisks. (D) Various CDK16-Flag mutants were coexpressed with CCNYL1-HA in HEK293T cells. The interactions were analyzed by CoIP experiments followed by western blot. (E) Varies CDK16-Flag mutants were either expressed alone or coexpressed with CCNYL1-HA in HEK293T cells. The CDK16-Flag protein was immunoprecipitated, and incubated with MBP as substrate in the kinase buffer. Reaction products were separated by SDS-PAGE and followed by autoradiography.
Mentions: We found that CDK16 was not only decreased in the Ccnyl1-/- testis, but also shifted to a slightly lower mass than that of WT controls by SDS-PAGE (Figs 5B and 7A). This band-shift was eliminated by incubating the total testicular cell lysates with phosphatase (Fig 7A), an observation also found in HEK293T cells (Fig 7B), implying that the presence of CCNYL1 is related to the phosphorylation status of CDK16. To check this implication, we co-expressed CCNYL1 with kinase dead CDK16 (K194R). While the interaction between these two proteins was not affected by the kinase dead mutation of CDK16, the slight band-shift of CDK16 was absent (S7A Fig), indicating that the phosphorylation modifications of CDK16 in the presence of CCNYL1 might also depend on the kinase activity of CDK16. Analysis of the phosphorylation sites on CDK16 by mass spectrometry showed that the N-terminal region of CDK16 was highly phosphorylated (Fig 7C). In total, 22 phosphorylation sites were identified, and 19 of them determined with high confidence (S2 Table and S1 Dataset). Of those 19 sites, nine have not been reported. They were S36, S64, S65, S89, S146, T175, T380, S391, and S478. Our results showed that the phosphorylation levels of CDK16 were increased at four sites (S36, S146, T175, and S480) and decreased at one site (S78), when co-expressed with CCNYL1 (S2 Table), consistent with the band-shift of CDK16 reflecting altered phosphorylation levels. Phosphorylation of CDKs has been reported to be involved in modifying their interaction with the cyclins and activation of their own Ser/Thr protein kinase activity [5, 15, 24]. To determine if the altered phosphorylation of CDK16 can affect its binding activity, we generated mutations of these phosphorylation sites for use in CoIP assays. We found that the S36A, S36D, S480A, and S480D mutations had no effect on CCNYL1 binding; but the S78A and T175D mutations increased and the T175A mutation decreased binding activity (Fig 7D). Most significant was the S146 site, with both S146A and S146D mutation almost entirely abolishing the binding to CCNYL1, confirming the importance of this site for the binding activity. In addition, we found S110, S119 and S153 were also highly phosphorylated, of these sites S119 and S153 are reported to be critical for the binding of the CCNY [5]. We mutated the S110, S119 and S153 sites, and found that the S110A, S110D, S119A and S153D mutations decreased the binding towards CCNYL1 to different degrees, whereas the S153A mutation increased the binding to CCNYL1 (Fig 7D and S7B Fig).

Bottom Line: 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.

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