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Genetic characterization of p27(kip1) and stathmin in controlling cell proliferation in vivo.

Berton S, Pellizzari I, Fabris L, D'Andrea S, Segatto I, Canzonieri V, Marconi D, Schiappacassi M, Benevol S, Gattei V, Colombatti A, Belletti B, Baldassarre G - Cell Cycle (2014)

Bottom Line: We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27(kip1) binding partner.In vivo analyses showed a reduced proliferation rate in DKO compared to p27(kip1) mice, linked, at molecular level, to decreased kinase activity of CDK4/6, rather than of CDK1 and CDK2.Taken together, our results demonstrate that stathmin cooperates with p27(kip1) to control the early phase of G1 to S phase transition and that this function may be of particular relevance in the context of tumor progression.

View Article: PubMed Central - PubMed

Affiliation: a Department of Translational Research; Division of Experimental Oncology 2; CRO of Aviano, National Cancer Institute ; Aviano , Italy.

ABSTRACT
The CDK inhibitor p27(kip1) is a critical regulator of cell cycle progression, but the mechanisms by which p27(kip1) controls cell proliferation in vivo are still not fully elucidated. We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27(kip1) binding partner. To get more insights into the in vivo significance of this interaction, we generated p27(kip1) and stathmin double knock-out (DKO) mice. Interestingly, thorough characterization of DKO mice demonstrated that most of the phenotypes of p27(kip1) mice linked to the hyper-proliferative behavior, such as the increased body and organ weight, the outgrowth of the retina basal layer and the development of pituitary adenomas, were reverted by co-ablation of stathmin. In vivo analyses showed a reduced proliferation rate in DKO compared to p27(kip1) mice, linked, at molecular level, to decreased kinase activity of CDK4/6, rather than of CDK1 and CDK2. Gene expression profiling of mouse thymuses confirmed the phenotypes observed in vivo, showing that DKO clustered with WT more than with p27 knock-out tissue. Taken together, our results demonstrate that stathmin cooperates with p27(kip1) to control the early phase of G1 to S phase transition and that this function may be of particular relevance in the context of tumor progression.

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Related in: MedlinePlus

CDK4/6 activity is decreased in thymus from DKO mice. (A-B) Kinase activity (using 32P-HH1 or 32P-RB as substrates, as indicated) associated with the indicated immunoprecipitated (IP) proteins, from thymuses of WT, p27KO, DKO and StmKO FVB (A) and C57BL/6 (B) mice. Numbers at the bottom of the panels indicate the kinase activity normalized for the associated IP protein. (C) Kinase activity associated with the indicated immunoprecipitated (IP) proteins from lysates of exponentially growing SCC9 head and neck carcinoma cells, using histone H1 as substrate (32P-HH1). Lane 1 is the IP (C), lane 2 Is the IP with imidazole (B), lanes from 3 to 6 are IPs incubated with increasing doses of recombinant stathmin (0.25-0.5-1-2 μg), prior to immunoprecipitation. Values in the graphs represent the mean of 3 independent experiments +/− SD. Differences in kinase activity in the presence of stathmin are not statistically significant. On top of the graph, the kinase activity observed in a typical experiment is shown. (D) Kinase activity associated with recombinant CDK2/CycA2 complex, using histone H1 (32P-HH1) as substrate, in the presence of recombinant p27 (0-0.5-2 μg) without or with increasing doses of stathmin (0-0.65-1.3-2.6 μg). On top of the graph, the kinase activity is shown.
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f0006: CDK4/6 activity is decreased in thymus from DKO mice. (A-B) Kinase activity (using 32P-HH1 or 32P-RB as substrates, as indicated) associated with the indicated immunoprecipitated (IP) proteins, from thymuses of WT, p27KO, DKO and StmKO FVB (A) and C57BL/6 (B) mice. Numbers at the bottom of the panels indicate the kinase activity normalized for the associated IP protein. (C) Kinase activity associated with the indicated immunoprecipitated (IP) proteins from lysates of exponentially growing SCC9 head and neck carcinoma cells, using histone H1 as substrate (32P-HH1). Lane 1 is the IP (C), lane 2 Is the IP with imidazole (B), lanes from 3 to 6 are IPs incubated with increasing doses of recombinant stathmin (0.25-0.5-1-2 μg), prior to immunoprecipitation. Values in the graphs represent the mean of 3 independent experiments +/− SD. Differences in kinase activity in the presence of stathmin are not statistically significant. On top of the graph, the kinase activity observed in a typical experiment is shown. (D) Kinase activity associated with recombinant CDK2/CycA2 complex, using histone H1 (32P-HH1) as substrate, in the presence of recombinant p27 (0-0.5-2 μg) without or with increasing doses of stathmin (0-0.65-1.3-2.6 μg). On top of the graph, the kinase activity is shown.

Mentions: The above data supported that p27 controls cell proliferation in vitro and in vivo, at least partially, via stathmin. Previous works suggested that increased proliferation rate observed in p27 animals was linked to increased CDK2 and, to lesser extent, CDK1 activity.8-12 We thus wondered whether stathmin could impact on the activities of CDK2 and/or CDK1. As expected, CDK2- and CDK1-associated kinase activities were higher in p27KO respect to WT but, unexpectedly, not different between p27KO and DKO thymuses, both in the FVB (Fig. 6A) and the C57BL/6 (Fig. 6B) strains, in all the experiments performed. Since stathmin is a substrate of both CDK2 and CDK1,23,26,27 we tested whether its expression could directly or indirectly influence their kinase activity. To test this possibility, increasing doses of recombinant stathmin protein were added to CDK2-containing complexes and their kinase activity was evaluated. The presence of stathmin did not significantly change the activity of CDK2 (Fig. 6C). Moreover, recombinant stathmin did not alter the kinase activity of cyclin A2/CDK2 recombinant complexes, neither it significantly overcame the block imposed by p27 on cyclin A2/CDK2 activity (Fig. 6D). Overall, these data confirmed that p27 is a master regulator of CDK2 activity, both in vitro and in vivo and further highlighted that the higher CDK2 activity observed in p27 cells and organs is not sufficient to explain their increased proliferation rate, as previously reported also by other groups.11,12Figure 6.


Genetic characterization of p27(kip1) and stathmin in controlling cell proliferation in vivo.

Berton S, Pellizzari I, Fabris L, D'Andrea S, Segatto I, Canzonieri V, Marconi D, Schiappacassi M, Benevol S, Gattei V, Colombatti A, Belletti B, Baldassarre G - Cell Cycle (2014)

CDK4/6 activity is decreased in thymus from DKO mice. (A-B) Kinase activity (using 32P-HH1 or 32P-RB as substrates, as indicated) associated with the indicated immunoprecipitated (IP) proteins, from thymuses of WT, p27KO, DKO and StmKO FVB (A) and C57BL/6 (B) mice. Numbers at the bottom of the panels indicate the kinase activity normalized for the associated IP protein. (C) Kinase activity associated with the indicated immunoprecipitated (IP) proteins from lysates of exponentially growing SCC9 head and neck carcinoma cells, using histone H1 as substrate (32P-HH1). Lane 1 is the IP (C), lane 2 Is the IP with imidazole (B), lanes from 3 to 6 are IPs incubated with increasing doses of recombinant stathmin (0.25-0.5-1-2 μg), prior to immunoprecipitation. Values in the graphs represent the mean of 3 independent experiments +/− SD. Differences in kinase activity in the presence of stathmin are not statistically significant. On top of the graph, the kinase activity observed in a typical experiment is shown. (D) Kinase activity associated with recombinant CDK2/CycA2 complex, using histone H1 (32P-HH1) as substrate, in the presence of recombinant p27 (0-0.5-2 μg) without or with increasing doses of stathmin (0-0.65-1.3-2.6 μg). On top of the graph, the kinase activity is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f0006: CDK4/6 activity is decreased in thymus from DKO mice. (A-B) Kinase activity (using 32P-HH1 or 32P-RB as substrates, as indicated) associated with the indicated immunoprecipitated (IP) proteins, from thymuses of WT, p27KO, DKO and StmKO FVB (A) and C57BL/6 (B) mice. Numbers at the bottom of the panels indicate the kinase activity normalized for the associated IP protein. (C) Kinase activity associated with the indicated immunoprecipitated (IP) proteins from lysates of exponentially growing SCC9 head and neck carcinoma cells, using histone H1 as substrate (32P-HH1). Lane 1 is the IP (C), lane 2 Is the IP with imidazole (B), lanes from 3 to 6 are IPs incubated with increasing doses of recombinant stathmin (0.25-0.5-1-2 μg), prior to immunoprecipitation. Values in the graphs represent the mean of 3 independent experiments +/− SD. Differences in kinase activity in the presence of stathmin are not statistically significant. On top of the graph, the kinase activity observed in a typical experiment is shown. (D) Kinase activity associated with recombinant CDK2/CycA2 complex, using histone H1 (32P-HH1) as substrate, in the presence of recombinant p27 (0-0.5-2 μg) without or with increasing doses of stathmin (0-0.65-1.3-2.6 μg). On top of the graph, the kinase activity is shown.
Mentions: The above data supported that p27 controls cell proliferation in vitro and in vivo, at least partially, via stathmin. Previous works suggested that increased proliferation rate observed in p27 animals was linked to increased CDK2 and, to lesser extent, CDK1 activity.8-12 We thus wondered whether stathmin could impact on the activities of CDK2 and/or CDK1. As expected, CDK2- and CDK1-associated kinase activities were higher in p27KO respect to WT but, unexpectedly, not different between p27KO and DKO thymuses, both in the FVB (Fig. 6A) and the C57BL/6 (Fig. 6B) strains, in all the experiments performed. Since stathmin is a substrate of both CDK2 and CDK1,23,26,27 we tested whether its expression could directly or indirectly influence their kinase activity. To test this possibility, increasing doses of recombinant stathmin protein were added to CDK2-containing complexes and their kinase activity was evaluated. The presence of stathmin did not significantly change the activity of CDK2 (Fig. 6C). Moreover, recombinant stathmin did not alter the kinase activity of cyclin A2/CDK2 recombinant complexes, neither it significantly overcame the block imposed by p27 on cyclin A2/CDK2 activity (Fig. 6D). Overall, these data confirmed that p27 is a master regulator of CDK2 activity, both in vitro and in vivo and further highlighted that the higher CDK2 activity observed in p27 cells and organs is not sufficient to explain their increased proliferation rate, as previously reported also by other groups.11,12Figure 6.

Bottom Line: We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27(kip1) binding partner.In vivo analyses showed a reduced proliferation rate in DKO compared to p27(kip1) mice, linked, at molecular level, to decreased kinase activity of CDK4/6, rather than of CDK1 and CDK2.Taken together, our results demonstrate that stathmin cooperates with p27(kip1) to control the early phase of G1 to S phase transition and that this function may be of particular relevance in the context of tumor progression.

View Article: PubMed Central - PubMed

Affiliation: a Department of Translational Research; Division of Experimental Oncology 2; CRO of Aviano, National Cancer Institute ; Aviano , Italy.

ABSTRACT
The CDK inhibitor p27(kip1) is a critical regulator of cell cycle progression, but the mechanisms by which p27(kip1) controls cell proliferation in vivo are still not fully elucidated. We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27(kip1) binding partner. To get more insights into the in vivo significance of this interaction, we generated p27(kip1) and stathmin double knock-out (DKO) mice. Interestingly, thorough characterization of DKO mice demonstrated that most of the phenotypes of p27(kip1) mice linked to the hyper-proliferative behavior, such as the increased body and organ weight, the outgrowth of the retina basal layer and the development of pituitary adenomas, were reverted by co-ablation of stathmin. In vivo analyses showed a reduced proliferation rate in DKO compared to p27(kip1) mice, linked, at molecular level, to decreased kinase activity of CDK4/6, rather than of CDK1 and CDK2. Gene expression profiling of mouse thymuses confirmed the phenotypes observed in vivo, showing that DKO clustered with WT more than with p27 knock-out tissue. Taken together, our results demonstrate that stathmin cooperates with p27(kip1) to control the early phase of G1 to S phase transition and that this function may be of particular relevance in the context of tumor progression.

Show MeSH
Related in: MedlinePlus