Journal: Journal of Biological Chemistry

Article Title: Cdk2-dependent Inhibition of p21 Stability via a C-terminal Cyclin-binding Motif

doi: 10.1074/jbc.m407352200

Figure Lengend Snippet: FIG. 1. Cyclin E and Cdk2 stabilize p21. A, Saos-2 cells were transfected with 100 ng of p21 DNA alone or with 3 g each of cyclin E and Cdk2 DNA as indicated. Total DNA in each transfection was brought to 6.1 g by the addition of salmon sperm DNA. 16 h after beginning the transfection, cells were either untreated or exposed to 25 M MG132 for 6 h to inhibit the proteasome. CHX was then added to a final concentration of 25 g/ml, and cell lysates were harvested at the indicated time after CHX addition and immunoblotted for p21. The percentage of p21 remaining at the indicated time points after the CHX addition was quantified by densitometry and is indicated, with the amount of p21 at the zero time point in each case considered as 100%. B, Saos-2 cells were transfected with p21, cyclin E, and Cdk2 DNA as in A. 16 h after transfection, the cells were labeled with 100 Ci of [35S]methionine for 30 min. Cell extracts were then immunoprecipi- tated with anti-p21 polyclonal antibody. Immunoprecipitates were re- solved on a 12% acrylamide gel, and the gel was exposed to film.

Article Snippet: For coimmunoprecipitation of FLAG-tagged p21s with Cdk2 NFG, transfected cell extracts were immunoprecipitated with Cdk2 polyclonal antibody (F7425; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and the immunoprecipitates were examined by immunoblotting with antiFLAG monoclonal antibody (M5; Sigma).

Techniques: Transfection, Concentration Assay, Labeling, Acrylamide Gel Assay

Journal: Journal of Biological Chemistry

Article Title: Cdk2-dependent Inhibition of p21 Stability via a C-terminal Cyclin-binding Motif

doi: 10.1074/jbc.m407352200

Figure Lengend Snippet: FIG. 2. p21 domains required for stabilization by cyclin E and Cdk2. A, schematic of the p21 protein with the CDK-binding site, cy1 and cy2 cyclin-binding sites, and potential CDK phosphorylation sites (Thr-57, Ser-98, and Ser-130) indicated. B and C, cells were transfected with 150 ng of the indicated p21 DNAs and 5 g of Myc-cyclin E and Cdk2 DNA, as indicated. Transfected cell lysates were prepared 24 h after transfection and immunoblotted (IB) for p21, cyclin E, and Cdk2.

Article Snippet: For coimmunoprecipitation of FLAG-tagged p21s with Cdk2 NFG, transfected cell extracts were immunoprecipitated with Cdk2 polyclonal antibody (F7425; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and the immunoprecipitates were examined by immunoblotting with antiFLAG monoclonal antibody (M5; Sigma).

Techniques: Binding Assay, Phospho-proteomics, Transfection

Journal: Journal of Biological Chemistry

Article Title: Cdk2-dependent Inhibition of p21 Stability via a C-terminal Cyclin-binding Motif

doi: 10.1074/jbc.m407352200

Figure Lengend Snippet: FIG. 3. Inactive Cdk2 stabilizes p21 to a greater extent than wild-type Cdk2. A, HeLa cells (left) and Saos-2 cells (right) were transfected with 100 ng of p21 DNA and 7.5 g of DNA encoding wild-type Cdk2 or the catalytically inactive Cdk2 mutant (Cdk2 NFG). p21 and Cdk2 protein levels were monitored by immunoblotting 20 h later. Higher levels of p21 were consistently observed when p21 was expressed with Cdk2 NFG than when expressed with WT Cdk2. B, p21 levels were monitored in Saos-2 cells transfected with 150 ng of p21 DNA and 5 g each of cyclin E and either WT Cdk2 or Cdk2 NFG.

Article Snippet: For coimmunoprecipitation of FLAG-tagged p21s with Cdk2 NFG, transfected cell extracts were immunoprecipitated with Cdk2 polyclonal antibody (F7425; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and the immunoprecipitates were examined by immunoblotting with antiFLAG monoclonal antibody (M5; Sigma).

Techniques: Transfection, Mutagenesis, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Cdk2-dependent Inhibition of p21 Stability via a C-terminal Cyclin-binding Motif

doi: 10.1074/jbc.m407352200

Figure Lengend Snippet: FIG. 4. Effect of Cdk2 on the stability of p21 phosphorylation site mutants. A, Saos-2 cells were transfected with 150 ng of WT p21 or the indicated p21 phosphorylation site mutants either alone or with cotransfection of DNA encoding WT Cdk2 or Cdk2 NFG (5 g each). p21 and Cdk2 protein levels were monitored 20 h later. B, cells were trans- fected with 600 ng of DNA encoding WT p21 or the indicated p21 phosphorylation site mutants. Transfected cells were treated with CHX (25 g/ml) 16 h after transfection, and p21 protein levels were moni- tored at the indicated times after CHX addition. C, p21 levels from at least two separate half-life experiments similar to those in B were quantitated by densitometry and are plotted. IB, immunoblot.

Article Snippet: For coimmunoprecipitation of FLAG-tagged p21s with Cdk2 NFG, transfected cell extracts were immunoprecipitated with Cdk2 polyclonal antibody (F7425; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and the immunoprecipitates were examined by immunoblotting with antiFLAG monoclonal antibody (M5; Sigma).

Techniques: Phospho-proteomics, Transfection, Cotransfection, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Cdk2-dependent Inhibition of p21 Stability via a C-terminal Cyclin-binding Motif

doi: 10.1074/jbc.m407352200

Figure Lengend Snippet: FIG. 5. Cdk2 can bind p21 indirectly through the cyclin-bind- ing sites. Top, HeLa cells were transfected with 1.5 g of DNA encod- ing Cdk2 NFG and the indicated FLAG-tagged forms of p21. 30 h after transfection, cell lysates were immunoprecipitated (IP) with anti-Cdk2 polyclonal antibody and examined by immunoblotting (IB) for the FLAG epitope. Cdk2 NFG could bind p21 CDK. However, deletion of cy1 diminished this binding, and further deletion of cy2 completely abolished this binding. Bottom, levels of Cdk2 and the FLAG-tagged p21 proteins were determined by immunoblotting without prior immunoprecipitation.

Article Snippet: For coimmunoprecipitation of FLAG-tagged p21s with Cdk2 NFG, transfected cell extracts were immunoprecipitated with Cdk2 polyclonal antibody (F7425; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and the immunoprecipitates were examined by immunoblotting with antiFLAG monoclonal antibody (M5; Sigma).

Techniques: Transfection, Immunoprecipitation, Western Blot, FLAG-tag, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Cdk2-dependent Inhibition of p21 Stability via a C-terminal Cyclin-binding Motif

doi: 10.1074/jbc.m407352200

Figure Lengend Snippet: FIG. 6. Effect of Cdk2 on the stability of p21 mutants that lack intact cyclin-binding sites. A, Saos-2 cells were transfected with 150 ng of WT p21 or p21 deletion mutants lacking the cy1 or cy2 cyclin- binding sites either alone or with cotransfection of DNA encoding WT Cdk2 or Cdk2 NFG (5 g each). p21 and Cdk2 protein levels were monitored 20 h later. B, cells were transfected with 600 ng of DNA encoding WT p21 or the indicated cy1 and cy2 deletion mutants. Trans- fected cells were treated with CHX (25 g/ml) 20 h after transfection, and p21 protein levels were monitored at the indicated times after CHX addition. C, p21 levels from at least two separate half-life experiments similar to those in B were quantitated by densitometry and are plotted. D, the half-life of p21 cy2 and p21 cy2, S130A was compared in experiments similar to those in B. IB, immunoblot.

Article Snippet: For coimmunoprecipitation of FLAG-tagged p21s with Cdk2 NFG, transfected cell extracts were immunoprecipitated with Cdk2 polyclonal antibody (F7425; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and the immunoprecipitates were examined by immunoblotting with antiFLAG monoclonal antibody (M5; Sigma).

Techniques: Binding Assay, Transfection, Cotransfection, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Cdk2-dependent Inhibition of p21 Stability via a C-terminal Cyclin-binding Motif

doi: 10.1074/jbc.m407352200

Figure Lengend Snippet: FIG. 7. Cdk2-dependent phosphorylation of p21 occurs at Ser-130 and is dependent in part on cy2. HeLa cells were transfected with DNAs encoding Cdk2 (1.6 g), Myc-tagged cyclin E (1.6 g), and different FLAG-tagged p21 proteins (1.0 g), as indicated. Transfected cell lysates were immunoprecipitated (IP) with an anti-Myc antibody to isolate cyclin E-Cdk2-p21 complexes. A, the immunoprecipitates were incubated under in vitro kinase assay conditions and then resolved on a 12% SDS-polyacrylamide gel. The gel was dried and exposed to film to monitor p21 phosphorylation (p21-P). The resulting autoradiogram is shown. B, the immunoprecipitates were examined by immunoblotting (IB) to detect the level of co-immunoprecipitating FLAG-p21 protein (top). The asterisk indicates the antibody light chain used in the immunoprecipitation. Cell lysates were also examined without prior immunoprecipitation to monitor Myc-cyclin E, Cdk2, and FLAG p21 levels (bottom). The top band in the Myc immunoblot is Myc-tagged cyclin E.

Article Snippet: For coimmunoprecipitation of FLAG-tagged p21s with Cdk2 NFG, transfected cell extracts were immunoprecipitated with Cdk2 polyclonal antibody (F7425; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and the immunoprecipitates were examined by immunoblotting with antiFLAG monoclonal antibody (M5; Sigma).

Techniques: Phospho-proteomics, Transfection, Immunoprecipitation, Incubation, In Vitro, Kinase Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Cdk2-dependent Inhibition of p21 Stability via a C-terminal Cyclin-binding Motif

doi: 10.1074/jbc.m407352200

Figure Lengend Snippet: FIG. 8. A model for Cdk2 regulation of p21 protein stability. Top, cyclins bind cy1 with greater affinity than cy2 and will therefore bind cy1 preferentially when their levels are low. However, excess cyclin levels will bind cy2 and increase the likelihood of indirect binding between Cdk2 and p21 via the cy2 cyclin-binding motif. This indirect interaction is predicted to maintain Cdk2 activity, leading to p21 phos- phorylation and degradation. Bottom, excess p21 levels lead to direct interaction between Cdk2 and p21 and subsequent inhibition of Cdk2 activity. Inactive Cdk2 stabilizes p21 without promoting p21 degradation.

Article Snippet: For coimmunoprecipitation of FLAG-tagged p21s with Cdk2 NFG, transfected cell extracts were immunoprecipitated with Cdk2 polyclonal antibody (F7425; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and the immunoprecipitates were examined by immunoblotting with antiFLAG monoclonal antibody (M5; Sigma).

Techniques: Binding Assay, Activity Assay, Inhibition