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Cdc6 Protein Activates p27KIP1-bound Cdk2 Protein Only after the Bound p27 Protein Undergoes C-terminal Phosphorylation

Uranbileg, Baasanjav, Yamamoto, Hanako, Park, Jung-ha, Mohanty, Atish R., Arakawa-Takeuchi, Shiho, Jinno, Shigeki, Okayama, Hiroto·2012·Journal of Biological Chemistry
DOI 10.1074/jbc.m111.318295
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FIGURE 1 Enforced expression of Cdk6 and cyclin D3 activates Cdk2 in the absence of anchorage. A , Rb remains phosphorylated at Ser 807/811 , a Cdk2specific phosphorylation site in anchorage-deprived REF-K6D3 cells. Rapidly proliferating REF and REF-K6D3 were cultured in MC for 48 h and analyzed for the indicated factors by immunoblotting as described (14). B , Cdk2 activation is prolonged in anchorage-deprived REF-K6D3. Cdk2 and Cdk6 in A were assayed for their amounts and activities as described (14). C , E2F-dependent genes driving S phase onset are up-regulated in anchorage-deprived REF-K6D3. RNA was prepared from the cells harvested in A , and cyclin A, E2F1 , and Cdc6 mRNAs were quantified by reverse transcription-coupled real time PCR as described (14). The data shown are averaged values with S.D obtained from three independently isolated samples.
FIGURE 1 Enforced expression of Cdk6 and cyclin D3 activates Cdk2 in the absence of anchorage. A , Rb remains phosphorylated at Ser 807/811 , a Cdk2specific phosphorylation site in anchorage-deprived REF-K6D3 cells. Rapidly proliferating REF and REF-K6D3 were cultured in MC for 48 h and analyzed for the indicated factors by immunoblotting as described (14). B , Cdk2 activation is prolonged in anchorage-deprived REF-K6D3. Cdk2 and Cdk6 in A were assayed for their amounts and activities as described (14). C , E2F-dependent genes driving S phase onset are up-regulated in anchorage-deprived REF-K6D3. RNA was prepared from the cells harvested in A , and cyclin A, E2F1 , and Cdc6 mRNAs were quantified by reverse transcription-coupled real time PCR as described (14). The data shown are averaged values with S.D obtained from three independently isolated samples.CC-BY · From the PDF
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FIGURE 2 C-terminal phosphorylation of p27 is essential for the continuedactivation of Cdk2 in anchorage-deprived REF-K6D3. A , REF and REFK6D3 were incubated in MC for 48 h and analyzed for the levels of Cdk2 phosphorylation at Thr 160 and p27 phosphorylation at Ser 10 , Thr 187 , and Thr 197 and the indicated factors by immunoblotting. In parallel, Cdk2 was
FIGURE 2 C-terminal phosphorylation of p27 is essential for the continuedactivation of Cdk2 in anchorage-deprived REF-K6D3. A , REF and REFK6D3 were incubated in MC for 48 h and analyzed for the levels of Cdk2 phosphorylation at Thr 160 and p27 phosphorylation at Ser 10 , Thr 187 , and Thr 197 and the indicated factors by immunoblotting. In parallel, Cdk2 wasCC-BY · From the PDF
FIGURE3 ROCK1phosphorylatesC-terminalThr 197 of p27. A , ROCK is active in anchorage-deprived REF-K6D3. The same experiment as in Fig. 1 was carried out with immunoblot analysis of the indicated factors. B , treatment with Y29762, a specific ROCK inhibitor, diminishes p27 Thr 197 phosphorylation. REF-K6D3 was incubated in MC for 12 h and then with no inhibitor, 20 𝛍 M GW5074, 20 𝛍 M BI-D1870, or 30 𝛍 M Y27632 for an additional 36 h. The cells were lysed and analyzed for p27 Thr 197 phosphorylation. C , in REF expressing an active ROCK1, Thr 197 phosphorylation of p27 persists during anchorage deprivation. REF-RK and REF-aRK were cultured in MC and analyzed for the indicated factors as in Fig. 2 A . The arrow indicates the constitutively active truncated ROCK1. D , E. coli -expressed active ROCK1 phosphorylates E. coli -expressed p27 Thr 197 in vitro . N-terminal histidine oligomer-tagged, C-terminal-truncated aRK was incubated with similarly tagged p27 or its E. coli empty vector control lysate ( EV ) in the reaction mixture for 30 min with or without the addition of 50 𝛍 M Y29762 ( Y2 ) as described (17) and analyzed for p27 Thr 197 phosphorylation by immune-blotting.
FIGURE3 ROCK1phosphorylatesC-terminalThr 197 of p27. A , ROCK is active in anchorage-deprived REF-K6D3. The same experiment as in Fig. 1 was carried out with immunoblot analysis of the indicated factors. B , treatment with Y29762, a specific ROCK inhibitor, diminishes p27 Thr 197 phosphorylation. REF-K6D3 was incubated in MC for 12 h and then with no inhibitor, 20 𝛍 M GW5074, 20 𝛍 M BI-D1870, or 30 𝛍 M Y27632 for an additional 36 h. The cells were lysed and analyzed for p27 Thr 197 phosphorylation. C , in REF expressing an active ROCK1, Thr 197 phosphorylation of p27 persists during anchorage deprivation. REF-RK and REF-aRK were cultured in MC and analyzed for the indicated factors as in Fig. 2 A . The arrow indicates the constitutively active truncated ROCK1. D , E. coli -expressed active ROCK1 phosphorylates E. coli -expressed p27 Thr 197 in vitro . N-terminal histidine oligomer-tagged, C-terminal-truncated aRK was incubated with similarly tagged p27 or its E. coli empty vector control lysate ( EV ) in the reaction mixture for 30 min with or without the addition of 50 𝛍 M Y29762 ( Y2 ) as described (17) and analyzed for p27 Thr 197 phosphorylation by immune-blotting.CC-BY · From the PDF
FIGURE4 Cdc6isrequiredfortheCdk2activationinanchorage-deprived REF-K6D3 cells. A , proliferating REF-K6D3 was transfected with the Cdc6specific siRNA or a control RNA. Two days later the transfected cells and rapidly proliferating REF-K6D3-Cdc6 cells were cultured in MC and determined for Cdk2 activity and the indicated factors as in Fig. 2 A . IP , immunoprecipitate. B , both the ATPase domain and the Cy motif are essential for Cdc6 to activate Cdk2. Rapidly proliferating REF-K6D3-iCdc6, REF-K6D3-iCdc6 Cy , and REFK6D3-iCdc6 WB cells were induced for the corresponding Cdc6 proteins by withdrawal of doxycycline, then cultured in MC and determined for Cdk2 activity and the levels of the indicated factors as in Fig. 2 B .
FIGURE4 Cdc6isrequiredfortheCdk2activationinanchorage-deprived REF-K6D3 cells. A , proliferating REF-K6D3 was transfected with the Cdc6specific siRNA or a control RNA. Two days later the transfected cells and rapidly proliferating REF-K6D3-Cdc6 cells were cultured in MC and determined for Cdk2 activity and the indicated factors as in Fig. 2 A . IP , immunoprecipitate. B , both the ATPase domain and the Cy motif are essential for Cdc6 to activate Cdk2. Rapidly proliferating REF-K6D3-iCdc6, REF-K6D3-iCdc6 Cy , and REFK6D3-iCdc6 WB cells were induced for the corresponding Cdc6 proteins by withdrawal of doxycycline, then cultured in MC and determined for Cdk2 activity and the levels of the indicated factors as in Fig. 2 B .CC-BY · From the PDF
FIGURE5 ActiveROCK1phosphorylatestheCterminusofCdk2-boundp27andCdc6activatestheCdk2initsATPase-andCy-dependentmanneronly after boundp27isC-terminal-phosphorylated. A , agarose bead-bound inactive Cdk2 immunoprecipitated ( IP ) from 12-h anchorage-deprived REF cells was incubated first with either E. coli -expressed aRK or its negative control preparation ( C1 ) and then with either reticulocyte-synthesized Cdc6 or its negative control preparation ( C2 ). After incubation, a half of the reaction mixture was denatured and analyzed for the amounts of Cdk2, Cdc6, p27, and its Thr 197 phosphorylated form. From the other half, bead-bound Cdk2 was collected and analyzed for its activity and coprecipitated p27-, Cdc6-, and Thr 197 -phosphorylated p27. B , the immunoprecipitated Cdk2 as above was incubated first with aRK and then with in vitro synthesized recombinant Cdc6, Cdc6 WB , or Cdc6 Cy and analyzed as in A . C , baculovirus-produced and highly purified Cdk2-cyclin A complexes were incubated with E. coli -expressed p27 S10D or an empty vector counterpart ( C1 ) and immunoprecipitated with anti-Cdk2 antibody-conjugated beads. The bead-bound Cdk2 was then incubated with aRK or C1 and finally with baculovirus-produced double affinity-purified Cdc6 or its negative control preparation ( C3 ) and analyzed as in A .
FIGURE5 ActiveROCK1phosphorylatestheCterminusofCdk2-boundp27andCdc6activatestheCdk2initsATPase-andCy-dependentmanneronly after boundp27isC-terminal-phosphorylated. A , agarose bead-bound inactive Cdk2 immunoprecipitated ( IP ) from 12-h anchorage-deprived REF cells was incubated first with either E. coli -expressed aRK or its negative control preparation ( C1 ) and then with either reticulocyte-synthesized Cdc6 or its negative control preparation ( C2 ). After incubation, a half of the reaction mixture was denatured and analyzed for the amounts of Cdk2, Cdc6, p27, and its Thr 197 phosphorylated form. From the other half, bead-bound Cdk2 was collected and analyzed for its activity and coprecipitated p27-, Cdc6-, and Thr 197 -phosphorylated p27. B , the immunoprecipitated Cdk2 as above was incubated first with aRK and then with in vitro synthesized recombinant Cdc6, Cdc6 WB , or Cdc6 Cy and analyzed as in A . C , baculovirus-produced and highly purified Cdk2-cyclin A complexes were incubated with E. coli -expressed p27 S10D or an empty vector counterpart ( C1 ) and immunoprecipitated with anti-Cdk2 antibody-conjugated beads. The bead-bound Cdk2 was then incubated with aRK or C1 and finally with baculovirus-produced double affinity-purified Cdc6 or its negative control preparation ( C3 ) and analyzed as in A .CC-BY · From the PDF
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Metadata

Authors
Uranbileg, B., Yamamoto, H., Park, J.-H., Mohanty, A. R., Arakawa-Takeuchi, S., Jinno, S., & Okayama, H.
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2012-02
Volume
287
Issue
9
Pages
6275-6283
ISSN
0021-9258
DOI
10.1074/jbc.m111.318295
Cited by
19
References
1
Language
en
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CoChat. (2026). Citation check: Cdc6 Protein Activates p27KIP1-bound Cdk2 Protein Only after the Bound p27 Protein Undergoes C-terminal Phosphorylation. https://app.cochat.ai/papers/10.1074/jbc.m111.318295
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Uranbileg, B., Yamamoto, H., Park, J.-H., Mohanty, A. R., Arakawa-Takeuchi, S., Jinno, S., & Okayama, H. (2012). Cdc6 Protein Activates p27KIP1-bound Cdk2 Protein Only after the Bound p27 Protein Undergoes C-terminal Phosphorylation. Journal of Biological Chemistry. https://doi.org/10.1074/jbc.m111.318295