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, 20 (17), 6300-7

p12(DOC-1) Is a Novel Cyclin-Dependent Kinase 2-associated Protein

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p12(DOC-1) Is a Novel Cyclin-Dependent Kinase 2-associated Protein

S Shintani et al. Mol Cell Biol.

Abstract

Regulated cyclin-dependent kinase (CDK) levels and activities are critical for the proper progression of the cell division cycle. p12(DOC-1) is a growth suppressor isolated from normal keratinocytes. We report that p12(DOC-1) associates with CDK2. More specifically, p12(DOC-1) associates with the monomeric nonphosphorylated form of CDK2 (p33CDK2). Ectopic expression of p12(DOC-1) resulted in decreased cellular CDK2 and reduced CDK2-associated kinase activities and was accompanied by a shift in the cell cycle positions of p12(DOC-1) transfectants ( upward arrow G(1) and downward arrow S). The p12(DOC-1)-mediated decrease of CDK2 was prevented if the p12(DOC-1) transfectants were grown in the presence of the proteosome inhibitor clasto-lactacystin beta-lactone, suggesting that p12(DOC-1) may target CDK2 for proteolysis. A CDK2 binding mutant was created and was found to revert p12(DOC-1)-mediated, CDK2-associated cell cycle phenotypes. These data support p12(DOC-1) as a specific CDK2-associated protein that negatively regulates CDK2 activities by sequestering the monomeric pool of CDK2 and/or targets CDK2 for proteolysis, reducing the active pool of CDK2.

Figures

FIG. 1
FIG. 1
GST-p12DOC-1 associates with CDK2 in cell lysates. (A) CDK2 immunoblot showing cellular CDK2 from HeLa, 293, and A431 cells associating with the GST-p12DOC-1 fusion protein. Lanes 1, 4, and 7, GST control; lanes 2, 5, and 8, GST-p12DOC-1; lanes 3, 6, and 9, input lysate at 0.1×. (B) CDK2 immunoblot showing that GST-p12DOC-1 associates with the 34-kDa forms of CDK2. Lane 1, GST control; lanes 2 and 3, GST-p12DOC-1 (1 and 2 μg, respectively); lane 4, immunoprecipitation of CDK2. (C) Phosphotyrosine immunoblot showing that the p34CDK2 that associates with GST-p12DOC-1 does not contain phosphotyrosine residues (4G10; Upstate Biotechnology). The samples for panels B and C were run on long SDS-PAGE gels to resolve the 33- and 34-kDa CDK2 bands.
FIG. 2
FIG. 2
Association of p12DOC-1 and CDK2 in cells. (A and B) CDK2 and p12DOC-1 immunoblots showing the coprecipitation of p12DOC-1 with CDK2 in 293 cells cotransfected with pCDK2 and pFLAG-DOC-1-wt or pFLAG-DOC-1-A3. Lanes 1 and 5, lysate (30 μg); lanes 2 and 6, immunoprecipitation using anti-FLAG monoclonal antibody (M5; Sigma Chemicals); lanes 3 and 7, CDK2 immunoprecipitation using anti-CDK2 monoclonal antibody (C18520 Clone 55; Transduction Laboratories); lanes 4 and 8, negative control using nonimmune mouse immunoglobulin G for immunoprecipitation. (C and D) Reprobing of the same membranes shown in panels A and B for cyclins A and E, respectively.
FIG. 3
FIG. 3
Interaction of p12DOC-1 and CDK2 in vivo. (A) Immunoblot to detect p12DOC-1 in normal human tissue lysates. Thirty micrograms of tissue lysates was loaded onto each lane. HUVEC (P3) cells are third passage normal human umbilical endothelial cells. (B and C) Coprecipitation of p12DOC-1 with CDK2 in human lung lysates from two donors. Panel B shows a p12DOC-1 immunoblot using p12DOC-1 Ab3; panel C shows a CDK2 immunoblot using anti-CDK2 antibody (C18520 Clone 55; Transduction Laboratories). Lanes 1 and 5, input lysate (25 μg); lanes 2 and 6, p12DOC-1 immunoprecipitation; lanes 3 and 7, CDK2 immunoprecipitation; lanes 4 and 8, negative control using nonimmune mouse and rabbit immunoglobulin G for panels B and C, respectively. (D and E) Gel filtration chromatograph elution profiles of normal human lung lysate (#2) and HaCaT cells. Top panels show immunoblots for CDK2; bottom panels show immunoblots for p12DOC-1. Thirty micrograms of total proteins was used for the respective input lysate lanes. Approximate molecular sizes (MW) of fractions 21, 22, and 23 are calibrated against known molecular size standards. (F) Phosphotyrosine immunoblot of the same membrane used for panel C to show that the CDK2 coprecipitated with the endogenous p12DOC-1 detected in normal lung lysates was not tyrosine phosphorylated, suggesting that it is the monomeric nonphosphorylated p33CDK2.
FIG. 4
FIG. 4
Amino acids 109 to 111 are necessary for p12DOC-1's association with CDK2. (A) Schematic of the mutagenesis strategy. Mutants were created by the Stratagene QuickChange site-directed mutagenesis system. The gray box in the C terminus (amino acids 62 to 115) is a domain that is homologous to a domain in a C. elegans protein, Y43F4B.7, and a related protein, DOC-1R (15). (B) In vitro association of the A1, A2, and A3 mutants with monomeric CDK2 in 293 cell lysate. One microgram of GST or GST-p12DOC-1 wild-type or mutant protein was mixed with 200 μg of 293 cell lysate. The CDK2 immunoblot was done using anti-CDK2 antibody (C18520 Clone 55; Transduction Laboratories).
FIG. 5
FIG. 5
Ectopic expression of p12DOC-1 and CDK2 kinase activity in 293 cells. (A) Cellular levels of CDK2, FLAG-p12DOC-1-wt, FLAG-p12DOC-1-A3, cyclin A, cyclin E, and actin in control vector and p12DOC-1 transfectants. The samples were run on long SDS-PAGE gels to resolve the 33- and 34-kDa CDK2 bands. (B and C) In vitro phosphorylation using GST-pRBc and histone H1, respectively, as substrates. (D) CDK2, cyclin A, and cyclin E immunoblots to show intracellular levels of these proteins in p12DOC-1-wt (lanes 2, 5, and 8), p12DOC-1-A3 (lanes 3, 6, and 9), and control transfectants (lanes 1, 4, and 7). Lanes 1, 2, and 3, immunoblot for CDK2; lanes 4, 5, and 6, immunoblot for CDK2 and cyclin A; lanes 7, 8, and 9, immunoblot for CDK2 and cyclin E. Signals were quantified by exposing the probed membranes to a quantitative imaging system (Fluor-S MAX MultiImager; Bio-Rad). IP, immunoprecipitation.
FIG. 6
FIG. 6
p12DOC-1 targets CDK2 for proteolysis. 293 cells were transfected with the pFLAG vector or pFLAG-DOC-1 in the presence or absence of the proteosome inhibitor clasto-lactacystin β-lactone solubilized in dimethyl sulfoxide (5 μM) for 24 h. Top panel, immunoblot for β-actin to quantify proteins loaded. Bottom panel, immunoblot for CDK2 and FLAG-p12DOC-1.
FIG. 7
FIG. 7
Effect of ectopic expression of p12DOC-1 on CDK2-mediated phenotypes in 293 cells. (A) Immunodetection of FLAG-p12DOC-1 expression in 293 cells. (B) Effect of p12DOC-1 ectopic expression on cell growth at 12, 24, and 48 h posttransfection. (C) Cell cycle positions at 48 h posttransfection. (D) Tritiated thymidine incorporation at 48 h posttransfection. For panels A and B, 293 cells were transfected with pFLAG vector or pFLAG-DOC-1 for the indicated time points and analyzed. For panels C and D, 293 cells were cotransfected with a neomycin expression vector (pcDNA3) and selected for 2 weeks in the presence of G418 at 400 μg/ml. Data are from three independent experiments. Transfection efficiency of 293 cells with Lipofectamine Plus (Life Technologies/Gibco-BRL, Grand Island, N.Y.) is ∼70%.

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