Expression of NPAT, a novel substrate of cyclin E-CDK2, promotes S-phase entry

Abstract

To understand the mechanisms by which CDKs regulate cell cycle progression, it is necessary to identify and characterize the physiological substrates of these kinases. We have developed a screening method to identify novel CDK substrates. One of the cDNAs identified in the screen is identical to the recently isolated NPAT gene. Here we show that NPAT associates with cyclin E-CDK2 in vivo and can be phosphorylated by this CDK. The protein level of NPAT peaks at the G1/S boundary. Overexpression of NPAT accelerates S-phase entry, and this effect is enhanced by coexpression of cyclin E-CDK2. These results suggest that NPAT is a substrate of cyclin E-CDK2 and plays a role in S-phase entry.

Figure 1

Binding screen for cyclin E–CDK2-interacting proteins. (Top) Outline of the binding screen (for details, see Materials and Methods). (Bottom) Autoradiographs showing a positive clone identified in primary (left) and tertiary (right) screens, respectively. In the primary screen, the λgt11 phage library was plated at a density of 1.8 × 10⁴ PFU/15-cm plate. In the tertiary screen, 50–100 PFU was plated on a 10-cm plate. The arrow indicates the positive clone in the primary screen.

Figure 1

Binding screen for cyclin E–CDK2-interacting proteins. (Top) Outline of the binding screen (for details, see Materials and Methods). (Bottom) Autoradiographs showing a positive clone identified in primary (left) and tertiary (right) screens, respectively. In the primary screen, the λgt11 phage library was plated at a density of 1.8 × 10⁴ PFU/15-cm plate. In the tertiary screen, 50–100 PFU was plated on a 10-cm plate. The arrow indicates the positive clone in the primary screen.

Figure 2

In vivo association of NPAT with cyclin E–CDK2. (A) Lysates (1 mg of total protein) from U2OS cells were immunoprecipitated with an anti-NPAT monoclonal antibody (DH3), anti-cyclin E antibody (HE67) or a control monoclonal antibody (anti-SV40-Tg antibody, PAB419), respectively. The immunoprecipitates were analyzed by Western blotting with a mouse anti-NPAT polyclonal antibody. The first lane was loaded with total protein (25 μg) from U2OS cells transfected with pCMV–NPAT, and the second lane was loaded with 50 μg of total protein from U2OS cells. (B) Same as described in A except that the lysates (0.25 mg of total protein) were prepared from U2OS cells transfected with NPAT (10 μg), cyclin E (2.5 μg), and CDK2 (2.5 μg) expression plasmids. (C, D) Lysates (1 mg of total protein) from U2OS cells were immunoprecipitated with indicated antibodies. The immunoprecipitates were analyzed by Western blotting with anti-cyclin E monoclonal antibody (HE12), (C) or anti-CDK2 antibody (D).

Figure 2

In vivo association of NPAT with cyclin E–CDK2. (A) Lysates (1 mg of total protein) from U2OS cells were immunoprecipitated with an anti-NPAT monoclonal antibody (DH3), anti-cyclin E antibody (HE67) or a control monoclonal antibody (anti-SV40-Tg antibody, PAB419), respectively. The immunoprecipitates were analyzed by Western blotting with a mouse anti-NPAT polyclonal antibody. The first lane was loaded with total protein (25 μg) from U2OS cells transfected with pCMV–NPAT, and the second lane was loaded with 50 μg of total protein from U2OS cells. (B) Same as described in A except that the lysates (0.25 mg of total protein) were prepared from U2OS cells transfected with NPAT (10 μg), cyclin E (2.5 μg), and CDK2 (2.5 μg) expression plasmids. (C, D) Lysates (1 mg of total protein) from U2OS cells were immunoprecipitated with indicated antibodies. The immunoprecipitates were analyzed by Western blotting with anti-cyclin E monoclonal antibody (HE12), (C) or anti-CDK2 antibody (D).

Figure 3

The level of NPAT protein peaks at the G₁/S boundary. (A) DNA synthesis in U2OS cells after removal of nocodazole was monitored by [³H]thymidine incorporation. (B) Lysates (50 μg of total protein) prepared from cells at the indicated times (hr) after release from nocodazole block were analyzed by Western blotting with a rabbit anti-NPAT polyclonal antibody (top). As a control, the lysates were also subjected to Western blotting analysis with anti-CDK2 antibody (bottom).

Figure 3

The level of NPAT protein peaks at the G₁/S boundary. (A) DNA synthesis in U2OS cells after removal of nocodazole was monitored by [³H]thymidine incorporation. (B) Lysates (50 μg of total protein) prepared from cells at the indicated times (hr) after release from nocodazole block were analyzed by Western blotting with a rabbit anti-NPAT polyclonal antibody (top). As a control, the lysates were also subjected to Western blotting analysis with anti-CDK2 antibody (bottom).

Figure 4

Effects of overexpression of NPAT on U2OS cell cycle distribution. (A) cells were transfected with control pCMV vector alone or with indicated amounts of pCMV–NPAT, and were analyzed by FACS. The results represent the means from triplicate samples. (B) Cells were transfected with 20 μg of vector or 20 μg of pCMV–NPAT and analyzed as in A. The absolute percentage differences between NPAT-transfected cells and vector-transfected cells (% NPAT-transfected cells-% vector-transfected cells) were calculated. The mean results from duplicate samples in four independent experiments (1–4) are depicted. (C) Cells were transfected with 20 μg of vector or pCMV–NPAT in the presence (vector + NOC; NPAT + NOC) or absence of nocodazole and were analyzed as in A. Shown are the mean results from duplicate samples; similar results have also been obtained in four other independent experiments. (D) Cells were transfected with the indicated expression plasmids (10 μg of NPAT; 2.5 μg of cyclin E; and 2.5 μg of CDK2) and analyzed as in A. The mean results from duplicate samples are presented. (░⃞) G₁ phase; (▪) S phase; (□) G₂/M phase.

Figure 4

Effects of overexpression of NPAT on U2OS cell cycle distribution. (A) cells were transfected with control pCMV vector alone or with indicated amounts of pCMV–NPAT, and were analyzed by FACS. The results represent the means from triplicate samples. (B) Cells were transfected with 20 μg of vector or 20 μg of pCMV–NPAT and analyzed as in A. The absolute percentage differences between NPAT-transfected cells and vector-transfected cells (% NPAT-transfected cells-% vector-transfected cells) were calculated. The mean results from duplicate samples in four independent experiments (1–4) are depicted. (C) Cells were transfected with 20 μg of vector or pCMV–NPAT in the presence (vector + NOC; NPAT + NOC) or absence of nocodazole and were analyzed as in A. Shown are the mean results from duplicate samples; similar results have also been obtained in four other independent experiments. (D) Cells were transfected with the indicated expression plasmids (10 μg of NPAT; 2.5 μg of cyclin E; and 2.5 μg of CDK2) and analyzed as in A. The mean results from duplicate samples are presented. (░⃞) G₁ phase; (▪) S phase; (□) G₂/M phase.

Figure 4

Effects of overexpression of NPAT on U2OS cell cycle distribution. (A) cells were transfected with control pCMV vector alone or with indicated amounts of pCMV–NPAT, and were analyzed by FACS. The results represent the means from triplicate samples. (B) Cells were transfected with 20 μg of vector or 20 μg of pCMV–NPAT and analyzed as in A. The absolute percentage differences between NPAT-transfected cells and vector-transfected cells (% NPAT-transfected cells-% vector-transfected cells) were calculated. The mean results from duplicate samples in four independent experiments (1–4) are depicted. (C) Cells were transfected with 20 μg of vector or pCMV–NPAT in the presence (vector + NOC; NPAT + NOC) or absence of nocodazole and were analyzed as in A. Shown are the mean results from duplicate samples; similar results have also been obtained in four other independent experiments. (D) Cells were transfected with the indicated expression plasmids (10 μg of NPAT; 2.5 μg of cyclin E; and 2.5 μg of CDK2) and analyzed as in A. The mean results from duplicate samples are presented. (░⃞) G₁ phase; (▪) S phase; (□) G₂/M phase.

Figure 4

Effects of overexpression of NPAT on U2OS cell cycle distribution. (A) cells were transfected with control pCMV vector alone or with indicated amounts of pCMV–NPAT, and were analyzed by FACS. The results represent the means from triplicate samples. (B) Cells were transfected with 20 μg of vector or 20 μg of pCMV–NPAT and analyzed as in A. The absolute percentage differences between NPAT-transfected cells and vector-transfected cells (% NPAT-transfected cells-% vector-transfected cells) were calculated. The mean results from duplicate samples in four independent experiments (1–4) are depicted. (C) Cells were transfected with 20 μg of vector or pCMV–NPAT in the presence (vector + NOC; NPAT + NOC) or absence of nocodazole and were analyzed as in A. Shown are the mean results from duplicate samples; similar results have also been obtained in four other independent experiments. (D) Cells were transfected with the indicated expression plasmids (10 μg of NPAT; 2.5 μg of cyclin E; and 2.5 μg of CDK2) and analyzed as in A. The mean results from duplicate samples are presented. (░⃞) G₁ phase; (▪) S phase; (□) G₂/M phase.

Figure 5

Phosphorylation of NPAT by cyclin E–CDK2. (A) Lysates from U2OS cells (1 mg of total protein) or from U2OS cells (0.25 mg of total protein) transfected with plasmids expressing indicated proteins were precipitated with anti-cyclin E (HE 67) or a control antibody (pAb 419) as indicated. The immunoprecipitates were incubated with [γ-³²p]ATP in the absence (−) or presence (+) of purified CDK inhibitor p21 protein (20 ng) as indicated. The phosphorylation of NPAT was determined by reimmunoprecipitation of the phosphorylated protein with anti-NPAT antibody (DH3). The amounts of expression plasmids used in the transfection experiments were 15 μg of NPAT, 4 μg of cyclin E, and 6 μg of CDK2 and CDK2dn, respectively. (B) U2OS cells were transfected with indicated expression plasmids, and the cellular phosphoproteins were labeled with [³²P]orthophosphate. Lysates were immunoprecipitated with anti-NPAT antibody (DH3) or a control antibody as indicated. The immunoprecipitates were denatured and reimmunoprecipitated with anti-HA antibody (12CA5). Fifteen micrograms of HA-tagged NPAT, 4 μg of cyclin E, 6 μg of CDK2/CDK2dn, 4 μg of cyclin D1, and 6 μg of CDK4 expression plasmids were used in the transfection experiments.