Chemical reagents, including Tris, HCl, and SDS for molecular biology and buffer preparation, were purchased from Sigma-Aldrich (St. Louis, MO). The checkmate mammalian two-hybrid system was from Promega Co. (Madison, WI). Cell culture media and supplements were obtained from Life Technologies (Rockville, MD). Cdk3, Cdk2, and JNK1 active kinases were from Upstate Biotechnology, Inc. (Lake Placid, NY). Antibodies against His, Cdk3, phospho-c-Jun (Ser63/73), Cyc C, and JNKs were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against phospho-c-Jun (Ser63), phospho-c-Jun (Ser73), c-Jun (mouse), β-actin, GST and phospho-JNKs were from Cell Signal Technology, Inc. (Beverly, MA). The QuikChange II Site-Directed Mutagenesis Kit was obtained from Stratagene, Inc. (La Jolla, CA) and Taq DNA polymerase from Qiagen, Inc. (Valencia, CA). JetPEI was purchased from Qbiogen, Inc. (Montreal, Quebec, Canada). G418 was from Biomol International, L.P. (Plymouth Meeting, PA) and EGF was purchased from BD Bioscience (Bedford, MA).
Cell Culture and Transfections
HEK293 (10% FBS-DMEM), SaoS-2 (15% FBS-McCoy’s 5A), and JB6 C141 mouse skin epidermal cells (5% FBS-MEM) were cultured with growth medium supplemented with antibiotics at 37°C and 5% CO2. NIH3T3 cells were cultured with DMEM with 10% calf bovine serum (CBS) and antibiotics at 37°C and 5% CO2. Cells were maintained by splitting at 80–90% confluence and media changed every 3 days. For transfection experiments, cells were split and the expression vector induced when cells were 50 to 60% confluent using jetPEI (Qbiogen, Inc.), following the manufacturer’s suggested protocol. For stable transfection, JB6 cells (5.0×105) in 5% FBS-MEM were seeded in 100-mm culture dishes. After culturing at 37°C in 5% CO2 for 16 h, the cells were transfected with 2 μg of pRcCMV-HA-Cdk3, pCMV-HA-Cdk3DN, or pcDNA3.1 (“mock”) using jetPEI. Cdk3, Cdk3-DN, and mock stably-transfected cells were obtained by selection for G418 resistance (400 μg/ml) and further confirmed by assessing Cdk3 activity and expression.
Construction of Expression Vectors
pCMV-HA-Cdk2 (pHA-Cdk2, Cdk2 in pCMV-Bam-neo), pCMV-HA-Cdk2-DN (pHA-Cdk2-DN, dominant-negative Cdk2 in pCMV-Bam-neo), pRcCMV-HA-Cdk3 (pHA-Cdk3, Cdk3 in pRcCMV), and pcDNA3-Cyc C were gifts from Dr. Barrett J. Rollins (Department of Medical Oncology, Harvard Medical School, Boston, Massachusetts) (19
). The pHis6-tagged c-Jun was kindly provided by Dr. Dirk Bohmann (European Molecular Biology Laboratory, Heidelberg, Germany) (21
). The c-Jun fragment was generated by PCR and subcloned into the pGEX-5X-1 vector (Amersham Biosciences Corp., Piscataway, NJ) at the Bam
I site to generate a glutathione S-transferase (GST)-c-Jun plasmid (pGST-c-Jun) (22
). The mutant GST-c-Jun plasmid was generated by the QuikChange II Site-Directed Mutagenesis Kit and c-Jun mutant primers (for Ser63 mutation, sense: 5′-CGACCTTCTATGACGATGCC-3′; antisense: 5′-GGGCATCGTCATAGAAGGTCG-3′; and for Ser73, sense: 5′-AGCGGACCTTATGGCTACAGT-3′; antisense: 5′-ACTGTAGCCATAAGGTCCGCT-3′). Mutant plasmids were confirmed by DNA sequencing.
Reporter Gene Assays
The AP-1 luciferase reporter plasmid construct contains the −73 to +63 collagenase promoter sequence (23
). AP-1 transactivation activity was analyzed by transfection of the AP-1-luc reporter plasmid with various expression vector combinations of Cdk3, Cdk2, c-Jun, c-Jun M63/73, Cdk3-DN, and/or Cyc C. Cells were disrupted with lysis buffer (Dual Luciferase Reporter Assay System, Promega) at room temperature for 30 min by gentle shaking and analyzed for firefly luciferase activity. The AP-1-luc luciferase activity was normalized against Renilla
luciferase activity (phRL-SV40).
To estimate cell proliferation, SaoS-2 cells harboring pU6pro-si-mock (“mock”) or pU6pro-si-Cdk3 (“si-Cdk3”) were seeded (4 × 103) into 96-well plates in 100 μl of 15% FBS/McCoy’s 5A medium and incubated at 37°C, 5% CO2. After culturing for 24 h, 20 μl of the CellTiter 96® Aqueous One Solution (Promega, Madison, WI, USA) were added to each well and cells were then incubated for 1 h in a 37°C, 5% CO2 incubator. To stop the reaction, 25 μl of 10% SDS were added and absorbance was measured at 492 and 690 nm.
Construction of si-RNA vectors
The pU6pro vector (provided by David L. Turner, University of Michigan, Ann Arbor, MI) was used to construct pU6pro-si-mock (“si-mock”) and Pu6pro-si-Cdk3 (“si-Cdk3) following the recommended protocol at (http://sitemaker.umich.edu/dlturner.vectors
). For the si-mock and si-Cdk3, we synthesized primers for the si-mock (General scramble: sense, 5′-TTTGACTACCGTTGTTATAGGTGTTCAAGAGACACCTATAACAACGGTAGTTTTTT-3′ and antisense, 5′-CTAGAAAAAACTACCGTTGTTATAGGTGTCTCTTGAACACCTATAACAACGGTAGT-3′) and for si-Cdk3 (sense, 5′-TTTGTGAGTTGGGTGCCATCAAGTTCAAGAGACTTGATGGCACCCAACTCATTTTT-3′ and antisense, 5′-CTAGAAAAATGAGTTGGGTGCCATCAAGTCTCTTGAACTTGATGGCACCCAACTCA-3′). All constructs were confirmed by restriction enzyme mapping and DNA sequencing.
Anchorage-Independent Cell Transformation Assay
To determine Cdk3 function in cell transformation induced by growth factors, JB6 cells were stably transfected with a mock vector or Cdk3. Cells (8 × 103
/ml) were exposed to EGF (20 ng/ml) in 1 ml of 0.3% basal medium Eagle’s agar/10% FBS. Cultures were maintained in a 37°C, 5% CO2
incubator for 10 days, and colonies were scored using a microscope and the Image-Pro PLUS computer software program (v.4, Media Cybermetics, Silver Spring, MD) as described (24
Foci Formation Assay
Transformation of NIH3T3 cells was performed following standard protocols (25
). Cells were transiently transfected with combinations of pcDNA3-H-RasG12V
(100 ng), Cdk3 (2.5 μg), c-Jun (2.5 μg), or c-Jun M63/73 (2.5 μg) and pcDNA3-mock (as compensation to achieve equal amount of DNA) DNA and then cultured in 5% calf serum-DMEM for 2 weeks. The media were changed every 3 days. Foci were fixed, stained with 0.5% crystal violet, counted with a microscope and the Image-Pro PLUS (v.4) software program.
Mammalian Two-Hybrid Assay
HEK293 cells (2.0×104) were seeded into 48-well plates and incubated for 18 h before transfection. The DNAs, pACT-c-Jun, pBIND-Cdk3, and pG5-Luciferase (pG5-luc), were combined in the same molar ratio and the total amount of DNA was not more than 100 ng per well. The transfection was performed using jetPEI following the manufacturer’s recommended protocol. The cells were disrupted by addition of lysis buffer [25 mM Tris-HCl (pH 7.5), 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2, 1 mM aprotinin and 1 mM PMSF] directly into each well of the 48-well plate and aliquots of 20 μl were added to each well of a 96-well luminescence plate. The luminescence activity was measured automatically by computer program (MTX Lab, Inc., Vienna, VA). Equal transfection efficiency was normalized with Renilla luciferase activity and the relative firefly luciferase activity was calculated and normalized based on the pG5-luciferase basal control.
IP/Kinase Assay of Cdk3
To study the effect of EGF on the induction of Cdk3 activity, Cdk3 or Cdk3-DN stably-transfected cells (1.0×106) were cultured for 12–24 h in 100-mm dishes. At 70–80% confluence, cells were stimulated with EGF at various doses (0, 10, 20, 40 ng/ml) or 20 ng EGF for different times (0.25, 0.5, 1, 3, 6 h), washed once with ice-cold PBS, harvested and disrupted in 250 μl of lysis buffer [25 mM Tris-HCl (pH 7.5), 5mM β-glycerophosphate, 0.1 mM Na3 VO4, 10 mM MgCl2, 1 mM aprotinin, and 1 mM PMSF]. The clarified supernatant fractions containing equal amounts of protein were subjected to immunoprecipitation using a Cdk3 antibody. The Cdk3 kinase assay was carried out as described by Upstate (Upstate Biotechnology, Inc., Lake Placid, NY). Briefly, the immune complex was added to 2.5 μl of 10x kinase buffer [250 mM Tris-HCl (pH 7.5), 50 mM β-glycerophosphate, 20 mM DTT, 1 mM Na3 VO4, 100 mM MgCl2], 2.5 μl (2.5 μg) of a GST-c-Jun fusion protein, 10 μl diluted ATP/cocktail (Upstate Biotechnology, Inc.), 10 μCi of [γ-32P] ATP and H2O to a final volume of 25 μl. The reaction was incubated at 30°C for 30 min and then resolved by 12% SDS-PAGE. Phosphorylated-GST-c-Jun was visualized by autoradiography.
Cdk3 and mock stably-transfected cells (1.0 × 103) were seeded in eight-chamber slides and incubated 24 h at 37°C, 5% CO2. The cells were washed at each time point, fixed in 4% formalin and permeabilized with 0.5% Triton X-100/1X PBS for 10 min. The cells were hybridized with a c-Jun mouse monoclonal antibody (1:200) and Cdk3 rabbit antibody (1:100) together at room temperature for 4 h. The cells were washed and hybridized at room temperature for 1 h with an anti-mouse goat antibody conjugated with Texas Red for detection of c-Jun and an anti-rabbit goat antibody conjugated with FITC for detection of Cdk3. Cells were washed again and observed under a fluorescence microscope (X200). To analyze co-localization of endogenous Cdk3 and phosphorylated c-Jun, SaoS-2 cells (3 × 104) were seeded into 2-chamber slides and cultured overnight. The cells were fixed with 4% formalin, permeabilized with 0.5% Triton X-100 and then hybridized with a Cdk3 rabbit antibody and a phospho-c-Jun (Ser63/73) mouse antibody at 37 °C for 2 h. The slides were washed and incubated with anti-mouse goat antibody conjugated with Texas Red and anti-rabbit goat antibody conjugated with FITC at 37 °C for 1 h. The slides were observed under a confocal microscope.