Figure 7.
Figure 7. CDKN3 modulates progression through mitosis via regulation of CDC2 phosphorylation at Thr-161. (A) The CDKN3 interface is conserved in human CDK2 (Protein Database [PDB] accession no. 1B39) and CDC2 (PDB accession no. 3LFQ) kinases (GDSEID/DYK motifs, blue). Activation loops (magenta) include putative CDKN3 target residue (pThr160/161; yellow). CDC25 target sites are green. Regions of full conservation are red; other regions are gray. (B) Endogenous CDKN3 colocalizes with endogenous CDC2 on centrosomes during mitosis. (C) Endogenous CDC2pThr161 localizes to centrosomes and the mitotic spindle during cell division. Dephosphorylation of CDC2pThr161 occurs in anaphase. HeLa cells were stained with antibody recognizing CDC2pThr-161, anti–α-tubulin antibody, and Hoechst 33342. (D) CDCpThr161 localizes to kinetochores in early mitosis. CDC2pThr161 (red) colocalizes with the kinetochore marker GFP-CENPA (green) in prometaphase but not in anaphase. (E) CDC2pThr161 is dephosphorylated at exit from mitosis. Cells were arrested in G2 through 24 h of exposure to RO3306 and washed to trigger mitotic entry. Decreasing cyclin B1 levels indicate cell cycle progression toward the mitotic exit. (F) Hyperphosphorylation of CDC2pThr161 in HeLa cells transfected with CDKN3 siRNA. Total CDC2 and CDC2pTyr15 levels are unaffected by CDKN3 siRNA. (G) Recombinant CDKN3 inactivates recombinant CDC2/cyclin B in an in vitro kinase assay in a dose-dependent manner. Increasing amounts of recombinant CDKN3 (0.5–5 µg) were incubated for 30 min in kinase buffer with a constant amount of active CDC2–cyclin B kinase complex, CDC2 substrate (histone H1), and radioactive [P32]γ-ATP. CDC2-dependent H1 phosphorylation was detected by autoradiography. 150 mM olomoucine (a CDK kinase inhibitor) was used as a control. (H) Cells transfected with CDKN3 siRNA fail to dephosphorylate CDC2pThr161 in early anaphase. Note the normal CDC2pThr-161 metaphase signal in control and CDKN3 siRNA cells. The CDC2pThr-161 signal persists in CDKN3 siRNA cells during anaphase.

CDKN3 modulates progression through mitosis via regulation of CDC2 phosphorylation at Thr-161. (A) The CDKN3 interface is conserved in human CDK2 (Protein Database [PDB] accession no. 1B39) and CDC2 (PDB accession no. 3LFQ) kinases (GDSEID/DYK motifs, blue). Activation loops (magenta) include putative CDKN3 target residue (pThr160/161; yellow). CDC25 target sites are green. Regions of full conservation are red; other regions are gray. (B) Endogenous CDKN3 colocalizes with endogenous CDC2 on centrosomes during mitosis. (C) Endogenous CDC2pThr161 localizes to centrosomes and the mitotic spindle during cell division. Dephosphorylation of CDC2pThr161 occurs in anaphase. HeLa cells were stained with antibody recognizing CDC2pThr-161, anti–α-tubulin antibody, and Hoechst 33342. (D) CDCpThr161 localizes to kinetochores in early mitosis. CDC2pThr161 (red) colocalizes with the kinetochore marker GFP-CENPA (green) in prometaphase but not in anaphase. (E) CDC2pThr161 is dephosphorylated at exit from mitosis. Cells were arrested in G2 through 24 h of exposure to RO3306 and washed to trigger mitotic entry. Decreasing cyclin B1 levels indicate cell cycle progression toward the mitotic exit. (F) Hyperphosphorylation of CDC2pThr161 in HeLa cells transfected with CDKN3 siRNA. Total CDC2 and CDC2pTyr15 levels are unaffected by CDKN3 siRNA. (G) Recombinant CDKN3 inactivates recombinant CDC2/cyclin B in an in vitro kinase assay in a dose-dependent manner. Increasing amounts of recombinant CDKN3 (0.5–5 µg) were incubated for 30 min in kinase buffer with a constant amount of active CDC2–cyclin B kinase complex, CDC2 substrate (histone H1), and radioactive [P32]γ-ATP. CDC2-dependent H1 phosphorylation was detected by autoradiography. 150 mM olomoucine (a CDK kinase inhibitor) was used as a control. (H) Cells transfected with CDKN3 siRNA fail to dephosphorylate CDC2pThr161 in early anaphase. Note the normal CDC2pThr-161 metaphase signal in control and CDKN3 siRNA cells. The CDC2pThr-161 signal persists in CDKN3 siRNA cells during anaphase.

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