MCF-7, T-47-D, BT-474, PC-3 and 293 cell lines were obtained from American Type Culture Collection (Manassas, VA, USA) and maintained in RPMI media obtained from Thermo Fisher Scientific (Waltham, MA, USA, Cat no. SH-30027) containing 10% fetal bovine serum (FBS) and 100 U/ml penicillin-streptomycin antibiotic solution (from Life Technologies, Grand Island, NY, USA, Cat no. 15140122). HMECs were from Lonza (Allendale, NJ, USA, Cat no. CC-2551) and HMEC 184A1 cells (a carcinogen immortalized clone of HMEC) was a gift from Dr Steven Wiley (Pacific Northwest Labs, Richland, WA, USA) were maintained in mammary epithelial growth medium (MEGM, from Lonza, Cat no. CC-3150) which contains human epidermal growth factor, insulin, and hydrocortisone. All cells were maintained at 5% CO₂ and 37°C. β-clasto-lactacystin was obtained from Boston Biochem (Cambridge, MA, USA, Cat no. I-100), MG-132 was from SelleckChem (Houston, TX, USA, Cat no. S-2619), epoximicin was from Apex Bio, (Houston, TX, USA, Cat no. A2606), and cycloheximide was from Sigma-Aldrich (St. Louis, MO, USA, Cat no. C7698).

Epitope-tagged wild-type human Cks1 expression plasmids (pIIB-CMV-HA-Cks1, pIIB-CMV-FLAG-Cks1 and pIIB-CMV-FLAG-Cks2) were constructed using a vector containing the strong CMV promoter-enhancer previously described by us (7). An additional HA-Cks1 expression plasmid was constructed by ligating a PCR generated HA-Cks1 fragment with BamHI and NotI overhangs into the pcDNA3 vector. The QuikChange™ kit from Agilent Technologies (Santa Clara, CA, USA, Cat no. 210518) was employed to generate all the Y-F, Y-E and K-R mutants of Cks1. pIIB-CMV-HA-Cks1 plasmid was used as the template for these site directed mutagenesis reactions. Briefly, mutagenic primers were employed to introduce single nucleotide changes within the Y or K codons present in the Cks1 coding region. All constructs were sequenced and verified to confirm the location and identity of the substitution.

In general, cells were plated in 6-cm UltraCruz™ tissue culture dishes (from Santa Cruz, Dallas, TX, USA, Cat no. 351863) at density of 0.6–1 million cells to achieve a confluence of 30–50% on the day of transfection. Cells were transfected using Lipofectamine™ (Life Technologies, Cat no. 18324-012) according to the manufacturer’s instructions. Briefly, 3–4 μg of DNA was complexed with 10–12 μl of Lipofectamine in serum-free OptiMEM™ medium (Life Technologies, Cat no. 31985-070). DNA-Lipofectamine complexes were added to the cells and incubated in serum-free medium for 16 h. The medium was then replaced with RPMI medium containing 10% FBS for 20 h. Epoximicin was added to the cells as indicated in the text. To ensure equal input for each transfection DNA concentration of each plasmid preparation was measured and normalized to a concentration of 1 μg/ml. Transfection efficiency was assessed by measuring exogenous Cks1 gene expression by a custom TaqMan™ probe (see Real-time quantitative PCR) designed to detect only the exogenous HA-tagged Cks1 mRNA. Exogenous Cks1 protein was assessed by immunoblotting with anti-HA antibody.

To assess the turnover of endogenous Cks1, de novo protein synthesis was blocked by adding Chx to cells and then assessing the turnover of the Cks1 protein in the presence or absence of proteasomal inhibitors. MCF-7 cells were pretreated with epoximicin for 30 min. Following this the cells were briefly washed with 1X phosphate buffered saline and were exposed to Chx in presence or absence of epoximicin. Cells were then harvested at regular intervals and protein lysates from these cells were subjected to immunoblotting with anti-Cks1 antibody. To compare the turnover of WT and mutant Cks1 protein, pIIBCMV-HA-Cks1 WT plasmid or the plasmids bearing Y-F, Y-E or K-R transfection were transfected into MCF-7 cells as described above (see transfection of WT and mutant Cks1). Chx chase experiments were carried out as described above. Immunoblotting was done with an anti-HA antibody to compare the turnover of mutant and WT Cks1 proteins over time.

A HA-ubiquitin plasmid (obtained from Dr Harish Ramanathan, NIH, Bethesda, MD, USA) was transfected into MCF-7 as described above. Cells were then treated with proteasomal inhibitors for 8 h and harvested. Protein lysate (100–200 μg) from the harvested cells was subjected to immunoprecipitation (IP) with 1–2 μg of Cks1 antibody using the Exacta Cruz™ F kit (from Santa Cruz, Cat no. 45043) according to the manufacturer’s instructions. Normal rabbit IgG serum (Santa Cruz, Cat no. 2027) was used as an isotype control for non specific binding during IP experiments. The IPs were then transferred to nitrocellulose membranes by overnight wet transfer and subjected to immunoblotting as described below to assess changes in ubiquitinated Cks1.

Total RNA was isolated from cells using the TRI reagent obtained from Molecular Research Center (Cincinnati, OH, USA, Cat no. TR118) according to the manufacturer’s protocol. The samples were then treated with amplification grade DNAse (from Life Technologies, Cat no. 18068-015) to remove genomic DNA contamination. DNAse was inactivated by heating at 65°C for 20 min in the presence of 1 mM EDTA. First-strand cDNA was synthesized using the iScript™ kit (from Bio-Rad, Hercules, CA, USA, Cat no. 170-8891). TaqMan™ gene expression assays from Life Technologies were employed to measure endogenous Cks1 (Cat no. 4331182, gene ID: Hs02518862_g1) and GAPDH (Cat no. 4331182, gene ID: Hs99999905_m1) transcript levels. The reactions were carried out in Chromo4™ Continuous Fluorescence Detector (from Bio-Rad) using the following cycling conditions: 40 cycles of 20 sec each at 95°C and 1 min at 60°C. The comparative CT method was used to represent the relative expression level of Cks1. A custom TaqMan probe designed to detect only the HA-tagged Cks1 mRNA was employed to determine transfection efficiency. The relative expression level is expressed as a unitless number and calculated as 2^−ΔΔCT as previously described (31).

To assess the rate of translation for WT and mutant Cks1, a kit for in vitro expression of Cks1 from (Thermo Fisher Scientific Cat no. 88881) was employed. WT and mutant Cks1 constructs (described above) were excised from the pIIB-CMV-HA-Cks1 backbone and ligated under a T7 promoter into the pT7CFE vector. This system allows robust in vitro expression of the WT or the mutant Cks1 protein in HeLa cell lysates. Following priming of the lysates, with the T7 expressing Cks1 construct, an energy source, and a translation-transcription mix containing accessory proteins and cofactors to drive in vitro synthesis of the proteins, aliquots were taken at regular intervals. The aliquots were then electrophoresed on SDS-PAGE gels and immunoblotted with anti-HA antibodies to assess the rate of synthesis of WT and the mutant proteins.

Cells harvested following each experiment were lysed with 1X lysis buffer from Cell Signaling (Danvers, MA, USA, Cat no. 9803). Lysates (25–40 μg) were run on Mini-PROTEAN™ or Criterion™ gels (Bio-Rad) and transferred on to nitrocellulose membranes by overnight wet transfer. The membranes were blocked for 1 h with 5% milk in 0.05% TBST and probed with the following antibodies: anti-Cks1 (Life Technologies, Cat no. 366-800, 1:500–1:2,000), anti-HA Clone-6E2 (Cell Signaling Technology, Cat no. 2367, 1:2,000–1:3,000), anti-FLAG Clone-M2 (Sigma, St. Louis, MO, USA, F1804, 1:500–1:1,000), anti-His (Thermo Fischer, Cat no. MA1-21315) and anti-GAPDH Clone-14C10 (Cell Signaling Technology, Cat no. 2118, 1:20,000–1:50,000). The membranes were then washed with 1X TBST three times, 5 min each, and probed with appropriate HRP-tagged secondary antibodies for 1 h. Supersignal™ West Pico enhanced chemiluminescence kit was used (from Thermo Fisher, Cat no. 34080) for imaging. Image acquisition was done using the LAS Image Quant™ 4010 system (from GE Healthcare, Waukesha, WI, USA).

We have previously shown that mammary tumors excised from transgenic models of mammary tumorigenesis and carcinogen initiated rat mammary tumors have very high levels of Cks1 protein in comparison to normal mammary glands, in which the Cks1 protein was nearly undetectable (7). This analysis was extended by comparing relative Cks1 protein levels in primary HMECs, HMEC 184A1 and the breast carcinoma cell lines MCF-7, T47D and BT-474 (Fig. 1A). Immunoblot analysis revealed that primary HMECs, similarly to normal mammary glands, did not have detectable Cks1 protein (Fig. 1A). Interestingly HMEC 184A1, a carcinogen immortalized HMEC line did exhibit a modest level of Cks1 protein, although even in this line the Cks1 protein was substantially less as compared to that in the breast cancer cell lines (Fig. 1A). In the breast cancer lines, Cks1 levels were comparable in the MCF-7 and BT-474 lines while the levels in T-47D were somewhat higher (Fig. 1A).

We next compared Cks1 mRNA levels in the HMEC lines versus the three breast cancer lines. Interestingly, despite displaying no detectable protein there was significant Cks1 mRNA in HMEC which was comparable to that in the breast cancer lines MCF-7 and BT-474 (Fig. 1B). Indeed, the immortalized HMEC line 184A1, which also had very low protein levels, expressed even more Cks1 mRNA than the MCF-7 line (Fig. 1B). These results indicate that, in general, Cks1 mRNA expression does not correlate with protein levels suggesting that a major component of its regulation could be at the translational or post-translational level. These data are also in agreement with similar findings from analysis of datasets from the Oncomine™ database wherein transcript levels of Cks1 were found to be largely similar between normal breast tissues and ductal breast carcinomas in a number of studies (not shown). Immunoblot analysis also revealed that Cks1 protein was not detectable in a panel of lysates from several normal human tissues as well as three separate lysates from normal breast (Fig. 1C).

Cks1 protein turnover is known to be regulated by the UPS pathway in the 293 and HeLa cell lines (16,17). We surmised therefore that the marked differences in Cks1 protein observed between HMECs and MCF-7 may be due to differences in its rate of proteasomal degradation in these cell types. To test this we first compared Cks1 protein levels in parallel time course experiments where both HMECs and MCF-7 cells were treated with the proteasomal inhibitor epoximicin (0.5 μM) (Fig. 2A and B). Our results indicate a time-dependent stabilization of Cks1 protein levels in HMECs and in MCF-7 when treated with epoximicin. Because of the high basal levels of Cks1 in MCF-7 cells stabilization was only noticeable when the western blots were exposed to film for short periods (Fig. 2B). We also observed that Cks1 mRNA was not altered following epoximicin treatment, confirming that the Cks1 increases exhibited by both cell types was indeed due to blockade of proteasomal degradation and not due to alterations in its message (Fig. 2B). As in the case of MCF-7, Cks1 protein stabilization after epoximicin treatment was also observed in T47D breast carcinoma cells and in the PC-3 prostate carcinoma cell line, indicating that Cks1 is regulated through the proteasomal degradation pathway in multiple cancer cell types (Fig. 2C). However, given high basal Cks1 levels in cancer cells in general, further stabilization by proteasomal blocakade is relatively modest (Fig. 2C).

We next assessed the effects of proteasomal inhibition on the constitutive turnover kinetics of Cks1 in MCF-7 cells after blocking translation with cycloheximide (Chx). We observed that Cks1 was markedly depleted in MCF-7 but only after ~8 h of Chx treatment (Fig. 2D). Epoximicin significantly blocked Cks1 depletion in the presence of Chx (Fig. 2D). Of note, Cks1 protein was still present 16 h after Chx treatment in the absence of epoximicin, suggesting that Cks1 is considerably stable in carcinoma cells. Nonetheless, the fact that it is stabilized further by epoximicin suggests that concomitant degradation by the proteasomal pathway, even in cancer cells, is a predominant regulator of steady-state Cks1 levels (Fig. 2D).

Ubiquitin ligation to either the ɛ-amino group of lysines or the amino terminus of a protein can serve as a tag to direct proteins to degradation by the 26S proteasome. To test whether Cks1 in MCF-7 cells is ubiquitinated we transfected these cells with an expression plasmid for HA-tagged ubiquitin and assessed the ubiquitination status of immunoprecipitated Cks1 (Fig. 3). We find that Cks1-specific immunoprecipitates when probed with an anti-HA antibody reveal a large smear of polyubiquitinated Cks1 when compared to that from a control isotype-matched IgG (Fig. 3A). The proteasomal inhibitors lactacystin and epoximicin caused a marked increase in the intensity of the polyubiquitinated Cks1 smears indicating a reduced degradation of the polyubiquitinitated forms of the protein (Fig. 3B).

To begin a systematic analysis of the residues involved in Cks1 turnover we first examined the degradation kinetics of a transiently transfected WT Cks1 in MCF-7 cells. We constructed two separate Cks1 expression plasmids driven by the strong CMV promoter-enhancer. As shown in Fig. 4A, pIIB-CMV-HA-Cks1, when transiently transfected, resulted in detectable Cks1 protein only in cells exposed to epoximicin for 24 h. To confirm that this was not artifactual we examined another cDNA (HA-Cdc2) in the same vector. Our results demonstrate that unlike Cks1, HA-Cdc2 protein was amply produced even without proteasomal blockade. Data in Fig. 4A also show that HA-Cks1 mRNA expression was similar in the presence or absence of epoximicin. Taken together these results indicate that transiently expressed Cks1 is extremely unstable in MCF-7 cells. We further confirmed this finding using another CMV driven vector (pcDNA3-HA-Cks1) and again found that detectable HA-Cks1 was only found in the presence of epoximicin (Fig. 4B). Furthermore, identical results were obtained when MCF-7 cells were transfected with the pIIB-CMV-FLAG-Cks1 vector which like the HA tagged Cks1 vector expressed detectable Cks1 protein only in the presence of epoximicin (Fig. 4C). This indicates that the marked instability of Cks1 was not related to a specific tag fused to its open reading frame.

To further dissect the stability of transiently expressed Cks1, we transfected PC-3 and 293 cells with the pIIB-CMV-HA-Cks1 vector (Fig. 4D). Like the MCF-7 cells, PC-3 cells express detectable Cks1 protein only in the presence of epoximicin (Fig. 4C). Similarly, the 293 cell line, which is considered to be an efficient model for transient transfection and mammalian protein expression, also expressed only a modest level of Cks1 protein that exhibited marked stabilization following epoxomicin treatment.

We also observed that rapid proteasomal kinetics of transiently expressed Cks1 was recapitulated by the Cks2 protein, a paralog of Cks1 which bears >81% sequence identity to the Cks1 protein (Fig. 4D). Transfection of MCF-7 cells with the FLAG-Cks2 vector revealed that similar to the transiently transfected Cks1 protein, the expression of Cks2 protein was detectable only upon epoxomicin treatment (Fig. 4D).

Collectively our data suggest that although sufficient Cks1 mRNA expression can be achieved from transiently transfected CMV-driven Cks1 expression plasmids, its protein (and also that of Cks2) exhibits rapid proteasomal turnover in MCF-7 carcinoma cells.

To identify structural determinants that are critical for Cks1 protein stability we introduced specific Y-F substitutions into its primary sequence and assessed their effects on the kinetics of degradation as compared to WT Cks1 (Fig. 5). We transiently transfected WT and Y-F Cks1 mutants into MCF-7 cells, followed by epoximicin treatment for 24 h. The cells were then washed and protein turnover was followed after addition of Chx to inhibit further translation. As shown in Fig. 5, substitution of Y residues 8, 12 and 19 with F significantly increased the turnover of exogenous Cks1 protein (Fig. 5). On the other hand the turnover kinetics of Y57F and Y7F were nearly identical to that of the WT Cks1 (Fig. 5). We further observed that proteasomal degradation was even more accelerated in the case of the combination with mutants Y-7-8-F, Y-7-8-12-F and Y-7-8-19-F (Fig. 6). All of these mutations markedly increased the rate of degradation, depleting the protein within 8–10 h following Chx treatment (Fig. 6). In sum, our results indicate that Y residues 8, 12 and 19 in Cks1 are critical for its proteasomal turnover.

Y residues within a protein can, in principle, regulate stability through multiple mechanisms. For instance phosphorylation of Y residues can create phosphodegrons allowing the recognition of the substrate protein by a specific ubiquitin ligase which in turn can append ubiquitin molecules to the protein and target it for proteasomal degradation (32). Our results with specific phospho-defective Y-F Cks1 substitutions prompted us to investigate the effects of potential phospho-mimicking ones at the same residues. We introduced Y-E substitutions on residues 8, 12 and 19 both individually and in combination. Like the WT and Y-F Cks1 protein, Y-E mutant proteins were detectable only upon epoximicin treatment. However, in contrast to the Y-F mutations, Y-E mutations at residues 8, 12 and 19 substantially slowed down the turnover of Cks1 following Chx addition (Fig. 7 and data not shown). Furthermore, the rate of degradation of Cks1 protein bearing the triple Y-E substitution on residues 8, 12 and 19 was markedly slower than that of Cks1 protein bearing single Y-E substitutions on these residues (Fig. 7B).

The role of specific K residues, the ɛ-amino group of which serves as the most common ubiquitin attachment site, has not been elucidated in the context of Cks1. Cks1 has nine K residues each of which could potentially serve as a ubiquitin attachment site. We individually mutated each one of these K residues of Cks1 to an arginine (R). We also simultaneously mutated all four terminal K residues of Cks1 to R. K-R substitution retains the positive charge of the amino acid but abrogates ubiquitination. Like the Y-F and Y-E mutants, all K-R mutants of Cks1 expressed detectable amount of Cks1 protein only upon epoxomicin treatment (Fig. 8). We compared the degradation kinetics of K-R mutants of Cks1 with that of WT Cks1. We found that K-R substitutions at residues 4 and 34, and to a larger extent in the more conserved lysine residues 26 and 30, greatly stabilized Cks1. On the other hand K-R substitutions at residues 11, 75, 76 did not markedly affect Cks1 stability (Fig. 8). Strikingly, replacing the terminal K79 residue with R greatly increased the turnover of Cks1 (Fig. 8). This was further corroborated with the K-R combination mutant where Rs were introduced in place of all four K residues in the C-terminus (Fig. 8).

To demonstrate that impaired transcription of mutant Cks1 cDNAs, or differences in transfection efficiency, could not account for the differences in the turnover exhibited by these proteins as compared to WT Cks1, we measured their mRNA levels in the transfected MCF-7 cells, both in the presence and absence of epoximicin (Tables I and II). We employed a custom TaqMan probe targeted to detect only the transfected HA-Cks1 transcript. The TaqMan probe was designed to detect the WT and mutant HA-Cks1 transcripts while excluding hybridization to any region of Cks1 cDNA sequence that contains Y-F or K-R substitutions. No amplification was detected in the untransfected samples confirming the specificity of the probe (Tables I and II). Furthermore, no significant differences in C(t) values were found between WT and any of the mutant Cks1 cDNA samples obtained from total mRNA harvested from the transfected cells (Tables I and II). Similarly to the WT Cks1 described in Fig. 4, expression was not altered after epoximicin treatment in the transfected Cks1 mutants (Tables I and II). These results allow us to conclude that the differences in turnover observed for mutants with point mutations introduced into their sequence were not the result of defects in their transcription.

To assess if the inherent instability of the transfected Y-F mutant HA-Cks1 molecules is due to defects in their translation, we employed an in vitro system for mammalian protein expression. This system allows robust in vitro translation of protein derived from message under control of a T7 promoter in HeLa cell lysates. Aliquots were taken at regular intervals after start of reaction and electrophoresed and subjected to immunoblotting (Fig. 9). We found that abundant levels of WT and the three mutant Cks1 molecules Y8F, Y19F and Y57F were expressed within the first 10 min of the in vitro reaction (Fig. 9). Cks1 protein translation in vitro was time-dependent and was independent of mutation within the Cks1. We found similar rates of translation with the other mutants as well (data not shown). The synthesis of WT Cks1 and all the mutants achieved almost full saturation after first hour of the reaction indicating that they were not impaired in translation (Fig. 9C and D and data not shown).