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Table S1. Co-occurrence analysis of PPP1R1B and IGF-1R transcripts Table S2. Hazard ratios associated with PPP1R1B and IGF-1R expression Table S3. Multivariate analysis of PPP1R1B and IGF-1R with HER2 for OS

Figure S1. Protein levels of PPP1R1B and IGF-1R. Figure S2. Comparison of respiration rates in SK-BR-3 parental and derived cell lines. Figure S3. siRNA knockdown of IGF-1R and IR. Figure S4. The effect of recombinant t-Darpp on oxygen consumption rate (OCR) of SK-BR-3 cells. Figure S5. Western analysis of IGF-1R and Akt phosphorylation in L6 myotubes by treatment with recombinant t-Darpp. Figure S6. Relative endogenous receptor levels in SK-BR-3 and derived cell lines.

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Purpose: Increased glycolysis and glucose dependence is a hallmark of malignancy that enables tumors to maximize cell proliferation. In HER2+ cancers, an increase in glycolytic capacity is associated with trastuzumab resistance. IGF-1R activation and t-Darpp overexpression both confer trastuzumab resistance in breast cancer. We therefore investigated a role for IGF-1R and t-Darpp in regulating glycolytic capacity in HER2+ breast cancers.Experimental Design: We examined the relationship between t-Darpp and IGF-1R expression in breast tumors and their respective relationships with patient survival. To assess t-Darpp's metabolic effects, we used the Seahorse flux analyzer to measure glucose metabolism in trastuzumab-resistant SK-BR-3 cells (SK.HerR) that have high endogenous t-Darpp levels and SK.tDrp cells that stably overexpress exogenous t-Darpp. To investigate t-Darpp's mechanism of action, we evaluated t-Darpp:IGF-1R complexes by coimmunoprecipitation and proximity ligation assays. We used pathway-specific inhibitors to study the dependence of t-Darpp effects on IGF-1R signaling. We used siRNA knockdown to determine whether glucose reliance in SK.HerR cells was mediated by t-Darpp.Results: In breast tumors, PPP1R1B mRNA levels were inversely correlated with IGF-1R mRNA levels and directly associated with shorter overall survival. t-Darpp overexpression was sufficient to increase glucose metabolism in SK.tDrp cells and essential for the glycolytic phenotype of SK.HerR cells. Recombinant t-Darpp stimulated glucose uptake, glycolysis, and IGF-1R–Akt signaling in SK-BR-3 cells. Finally, t-Darpp stimulated IGF-1R heterodimerization with ErbB receptors and required IGF-1R signaling to confer its metabolic effects.Conclusions: t-Darpp activates IGF-1R signaling through heterodimerization with EGFR and HER2 to stimulate glycolysis and confer trastuzumab resistance. Clin Cancer Res; 24(5); 1216–26. ©2017 AACR.

We set out to uncover transcriptome and chromatin landscape changes that occur in HER2 + breast cancer (BC) cells upon acquiring resistance to trastuzumab. RNA-seq analysis was applied to two independently-derived BC cell lines with acquired resistance to trastuzumab (SKBr3.HerR and BT-474HerR) and their parental drug-sensitive cell lines (SKBr3 and BT-474). Chromatin landscape analysis indicated that the most significant increase in accessibility in resistant cells occurs in PPP1R1B within a segment spanning introns 1b through intron 3. Footprint analysis of this segment revealed that FoxJ3 (within intron 2) and Pou5A1/Sox2 (within inton 3) transcription factor motifs are protected in resistant cells. Overall, 344 shared genes were upregulated in both resistant cell lines relative to their parental counterparts and 453 shared genes were downregulated in both resistant cell lines relative to their parental counterparts. In resistant cells, genes associated with autophagy and mitochondria organization are upregulated and genes associated with ribosome assembly and cell cycle are downregulated relative to parental cells. The five top upregulated genes in drug-resistant breast cancer cells are APOD, AZGP1, ETV5, ALPP, and PPP1R1B. This is the first report of increased chromatin accessibility within PPP1R1B associated with its t-Darpp transcript increase, and points to a possible mechanism for its activation in trastuzumab-resistant cells.

Docking on the p53-binding site of murine double minute 2 (MDM2) by small molecules restores p53's tumor-suppressor function. We previously assessed 3244 FDA-approved drugs via “computational conformer selection” for inhibiting MDM2 and p53 interaction. Here, we developed a surface plasmon resonance method to experimentally confirm the inhibitory effects of the known MDM2 inhibitor, nutlin-3a, and two drug candidates predicted by our computational method. This p53/MDM2 interaction displayed a dosage-dependent weakening when MDM2 is pre-mixed with drug candidates. The inhibition efficiency order is nutlin-3a (IC50 = 97 nM) > bepridil (206 nM) > azelastine (307 nM). Furthermore, we verified their anti-proliferation effects on SJSA-1 (wild-type p53 and overexpressed MDM2), SW480 (mutated p53), and SaOs-2 (deleted p53) cancer cell lines. The inhibitory order towards SJSA-1 cell line is nutlin-3a (IC50 = 0.8 μM) > bepridil (23 μM) > azelastine (25 μM). Our experimental results are in line with the computational prediction, and the higher IC50 values from the cell-based assays are due to the requirement of higher drug concentrations to penetrate cell membranes. The anti-proliferation effects of bepridil and azelastine on the cell lines with mutated and deleted p53 implied some p53-independent anti-proliferation effects.

The PPP1R1B gene is located on chromosome 17q12 (39,626,208–39,636,626[GRCh38/hg38]), which codes for multiple transcripts and two experimentally-documented proteins Darpp-32 and t-Darpp. Darpp-32 (Dopamine and cAMP Regulated Phosphoprotein), discovered in the early 1980s, is a protein whose phosphorylation is upregulated in response to cAMP in dopamine-responsive tissues in the brain. It’s phosphorylation profile modulates its ability to bind and inhibit Protein Phosphatase 1 activity, which, in turn, controls the activity of hundreds of phosphorylated proteins. PPP1R1B knockout mice exhibit subtle learning defects. In 2002, the second protein product of PPP1R1B was discovered in gastric cancers: t-Darpp (truncated Darpp-32). The start codon of t-Darpp is amino acid residue 37 of Darpp-32 and it lacks the domain responsible for modulating Protein Phosphatase 1. Aside from gastric cancers, t-Darpp and/or Darpp-32 is overexpressed in tumor cells from breast, colon, esophagus, lung and prostate tissues. More than one research team has demonstrated that these proteins, through mechanisms that to date remain cloudy, activate AKT, a protein whose phosphorylation leads to cell survival and blocks apoptosis. Furthermore, in Her2 positive breast cancers (an aggressive form of breast cancer), t-Darpp/Darpp-32 overexpression causes resistance to the frequently-administered anti-Her2 drug, trastuzumab (Herceptin), likely through AKT activation. Here we briefly describe how Darpp-32 and t-Darpp were discovered and report on the current state of knowledge of their involvement in cancers. We present a case for the development of an anti-t-Darpp therapeutic agent and outline the unique challenges this endeavor will likely encounter.

t-Darpp is a protein encoded by the PPP1R1B gene and is expressed in breast, colon, esophageal, gastric, and prostate cancers, as well as in normal adult brain striatal cells. Overexpression of t-Darpp in cultured cells leads to increased protein kinase A activity and increased phosphorylation of AKT (protein kinase B). In HER2+ breast cancer cells t-Darpp confers resistance to the chemotherapeutic agent trastuzumab. To shed light on t-Darpp function, we studied its secondary structure, oligomerization status, metal-binding properties, and phosphorylation by cyclin dependent kinases 1 and 5. t-Darpp exhibits 12% alpha helix, 29% beta strand, 24% beta turn and 35% random coil structures. t-Darpp binds to calcium, but not to other metals commonly found in biological systems. The T39 site, critical for t-Darpp activation of the AKT signaling pathway, is a substrate for phosphorylation by cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 5 (CDK5). Gel filtration chromatography, sedimentation equilibrium analysis, blue native gel electrophoresis, and glutaraldehyde-mediated crosslinking experiments demonstrate that the majority of t-Darpp exists as a monomer, but forms low levels (< 3%) of hetero-oligomers with its longer isoform Darpp-32. t-Darpp has a large Stokes radius of 4.4 nm relative to its mass of 19 kDa, indicating that it has an elongated structure.

Purpose: Increased glycolysis and glucose dependence is a hallmark of malignancy that enables tumors to maximize cell proliferation. In HER2+ cancers, an increase in glycolytic capacity is associated with trastuzumab resistance. IGF-1R activation and t-Darpp overexpression both confer trastuzumab resistance in breast cancer. We therefore investigated a role for IGF-1R and t-Darpp in regulating glycolytic capacity in HER2+ breast cancers. Experimental Design: We examined the relationship between t-Darpp and IGF-1R expression in breast tumors and their respective relationships with patient survival. To assess t-Darpp's metabolic effects, we used the Seahorse flux analyzer to measure glucose metabolism in trastuzumab-resistant SK-BR-3 cells (SK.HerR) that have high endogenous t-Darpp levels and SK.tDrp cells that stably overexpress exogenous t-Darpp. To investigate t-Darpp's mechanism of action, we evaluated t-Darpp:IGF-1R complexes by coimmunoprecipitation and proximity ligation assays. We used pathway-specific inhibitors to study the dependence of t-Darpp effects on IGF-1R signaling. We used siRNA knockdown to determine whether glucose reliance in SK.HerR cells was mediated by t-Darpp. Results: In breast tumors, PPP1R1B mRNA levels were inversely correlated with IGF-1R mRNA levels and directly associated with shorter overall survival. t-Darpp overexpression was sufficient to increase glucose metabolism in SK.tDrp cells and essential for the glycolytic phenotype of SK.HerR cells. Recombinant t-Darpp stimulated glucose uptake, glycolysis, and IGF-1R–Akt signaling in SK-BR-3 cells. Finally, t-Darpp stimulated IGF-1R heterodimerization with ErbB receptors and required IGF-1R signaling to confer its metabolic effects. Conclusions: t-Darpp activates IGF-1R signaling through heterodimerization with EGFR and HER2 to stimulate glycolysis and confer trastuzumab resistance. Clin Cancer Res; 24(5); 1216–26. ©2017 AACR.

t-Darpp is the truncated form of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (Darpp-32) and has been demonstrated to confer resistance to trastuzumab, a Her2-targeted anticancer agent, via sustained signaling through the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt pathway and activation of protein kinase A (PKA). The mechanism of t-Darpp-mediated PKA activation is poorly understood. In the PKA holoenzyme, when the catalytic subunits are bound to regulatory subunits RI or RII, kinase activity is inhibited. We investigated PKA activity and holoenzyme composition in cell lines overexpressing t-Darpp (SK.tDp) or a T39A phosphorylation mutant (SK.tDpT39A), as well as an empty vector control cell line (SK.empty). We also evaluated protein-protein interactions between t-Darpp and PKA catalytic (PKAc) or regulatory subunits RI and RII in those cell lines. SK.tDp cells had elevated PKA activity and showed diminished association of RI with PKAc, whereas SK.tDpT39A cells did not have these properties. Moreover, wild type t-Darpp associates with RI. Concurrent expression of Darpp-32 reversed t-Darrp’s effects on PKA holoenzyme state, consistent with earlier observations that Darpp-32 reverses t-Darpp’s activation of PKA. Together, t-Darpp phosphorylation at T39 seems to be crucial for t-Darpp—mediated PKA activation and this activation appears to occur through an association with RI and sequestering of RI away from PKAc. The t-Darpp-RI interaction could be a druggable target to reduce PKA activity in drug-resistant cancer.

HER2 is a member of the ErbB/HER family of receptor tyrosine kinases that promotes the proliferation of a subset of human breast cancers. HER2 is over-expressed in 20–25% of breast cancers and high expression is associated with poor clinical outcomes if not treated with an HER2-targeted drug. Although HER2 does not have a known extracellular ligand, HER2 is the preferred heterodimerization partner for the other HER-family receptors and HER2-containing heterodimers have increased affinity for ligands that bind to those heterodimer partners. The downstream effect is strong signal transduction and tumor cell proliferation. Even in the absence of a ligand, HER2 over-expression by itself can drive tumorigenesis and tumor growth. Given its elevated expression and oncogenic activity, its preferred status as a HER-family signaling partner, and the enhanced activity of HER2-containing heterodimers, HER2 is a valuable pharmacological target for the treatment of HER2+ breast cancer. HER2-targeted agents fall into three general categories: therapeutic antibodies, antibody-drug conjugates, and tyrosine kinase inhibitors. The resistance mechanisms associated with each of these classes of drugs will be reviewed here.

The integrity of the p53 tumor suppressor pathway is compromised in the majority of cancers. In 7% of cancers p53 is inactivated by abnormally high levels of MDM2--an E3 ubiquitin ligase that polyubiquitinates p53, marking it for degradation. MDM2 engages p53 through its hydrophobic cleft, and blockage of that cleft by small molecules can re-establish p53 activity. Small molecule MDM2 inhibitors have been developed, but there is likely to be a high cost and long time period before effective drugs reach the market. An alternative is to repurpose FDA-approved drugs. This report describes a new approach, called Computational Conformer Selection, to screen for compounds that potentially inhibit MDM2. This screen was used to computationally generate up to 600 conformers of 3244 FDA-approved drugs. Drug conformer similarities to 41 computationally-generated conformers of MDM2 inhibitor nutlin 3a were ranked by shape and charge distribution. Quantification of similarities by Tanimoto combo scoring resulted in scores that ranged from 0.142 to 0.802. In silico docking of drugs to MDM2 was used to calculate binding energies and to visualize contacts between the top-ranking drugs and the MDM2 hydrophobic cleft. We present 15 FDA-approved drugs predicted to inhibit p53/MDM2 interaction.

MDM2 and MDM4 are proto-oncoproteins that bind to and inhibit members of the p53 protein family, p53, p73 and possibly p63. p53 is a mammalian tumor suppressor and p63 and p73 are critical for development. With the sequencing of genomes from multiple organisms there is mounting evidence for a consensus scenario of p53 gene family evolution. A single p53/p63/p73 gene is in invertebrates and required for maintenance of germline DNA. Gene duplication occurred in an ancestor in common with cartilaginous fishes, giving rise to a separate p53 gene and at least one ancestral p63/p73 gene. In bony vertebrates, all three p53 gene family paralogs, p53, p63, and p73 are distinct genes. This raises the question of how MDM2 and MDM4 genes evolved. We show evidence that MDM2 and MDM4 arose from a gene duplication event prior to the emergence of bony vertebrates more than 440 millionyears ago. Comparative genome studies indicate that invertebrate organisms have only one MDM homolog. In jawed vertebrates, the p53-binding domains of MDM2 and MDM4 proteins evolved at a high rate, approaching the evolution rate of the MDM2-binding domain of p53. However, the MDM2-binding domain of p73 exhibits markedly stronger conservation suggesting novel p53-independent functions. The most conserved domain within all MDM2 family members is the RING domain of the MDM2 ortholog which is responsible for ubiquitination of p53 and heterodimerization with MDM4. We suggest a model where oligomerization is an ancient function of MDM and ubiquitination activity was acquired later near the MDM gene duplication event coinciding with the time of the emergence of p53 as a distinct gene.

Trastuzumab (Herceptin) resistance is a persistent challenge in the treatment of metastatic Her2+ breast cancer, with an average relapse time of 1 year after treatment. Human breast cancer cell-lines (SKBr3 and BT474) selected for trastuzumab resistance have a PPP1R1B gene (chr17:39,626,924-39,636,626) that is transcriptionally activated compared to their drug-sensitive counterparts. PPP1R1B codes for two related protein products, Darpp-32 (long) and t-Darpp (short), that are overexpressed in 50% of clinical breast cancer samples in comparison to normal breast tissue; reintroduction of t-Darpp into drug-sensitive cells confers drug resistance. The mechanism controlling overexpression of these transcripts is unknown. To gain insight into this mechanism we used ATAC-seq to map open chromatin areas in trastuzumab-resistant breast cancer cell lines and compared their open chromatin areas to those in the parental trastuzumab-sensitive counterparts. The PPP1R1B gene consists of 7 canonical exons. In each trastuzumab-resistant line there is two significantly increased open chromatin peaks, one within exon 2 and one within exon 3. We are performing deep sequencing (>200 million reads) to identify transcription factor footprints in these peaks. Furthermore, we performed RNA-seq analysis and identified genes that are similarly regulated in the resistant lines in comparison to their drug-sensitive counterparts. In summary, these results shed light on an area of high clinical importance and point to potential drug targets to be developed to overcome trastuzumab resistance in Her2+ breast cancer. Citation Format: Momand J, Murad R, Ma X, Geng S, Mortazavi A. Mechanism of activation of PPP1R1B drug-resistance gene in trastuzumab-resistant Her2+ breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-03-15.

Protein cysteine thiols can be divided into four groups based on their reactivities: those that form permanent structural disulfide bonds, those that coordinate with metals, those that remain in the reduced state, and those that are susceptible to reversible oxidation. Physicochemical parameters of oxidation-susceptible protein thiols were organized into a database named the Balanced Oxidation Susceptible Cysteine Thiol Database (BALOSCTdb). BALOSCTdb contains 161 cysteine thiols that undergo reversible oxidation and 161 cysteine thiols that are not susceptible to oxidation. Each cysteine was represented by a set of 12 parameters, one of which was a label (1/0) to indicate whether its thiol moiety is susceptible to oxidation. A computer program (the C4.5 decision tree classifier re-implemented as the J48 classifier) segregated cysteines into oxidation-susceptible and oxidation-non-susceptible classes. The classifier selected three parameters critical for prediction of thiol oxidation susceptibility: (1) distance to the nearest cysteine sulfur atom, (2) solvent accessibility, and (3) pKa. The classifier was optimized to correctly predict 136 of the 161 cysteine thiols susceptible to oxidation. Leave-one-out cross-validation analysis showed that the percent of correctly classified cysteines was 80.1% and that 16.1% of the oxidation-susceptible cysteine thiols were incorrectly classified. The algorithm developed from these parameters, named the Cysteine Oxidation Prediction Algorithm (COPA), is presented here. COPA prediction of oxidation-susceptible sites can be utilized to locate protein cysteines susceptible to redox-mediated regulation and identify possible enzyme catalytic sites with reactive cysteine thiols.

Reactive oxygen species, generated either by cellular respiration or upon exposure to environmental agents such as ionizing radiation (IR), attack DNA to form a variety of oxidized base and sugar modifications. Accumulation of oxidative DNA damage has been associated with age-related disease as well as the aging process. Single-strand breaks harboring oxidative 3' obstructive termini, e.g. 3' phosphates and 3' phosphoglycolates, must be removed prior to DNA repair synthesis or ligation. In addition, 3' tyrosyl-linked protein damage, resulting from therapeutic agents such as camptothecin (CPT), must be processed to initiate repair. Several nucleases participate in DNA repair and the excision of 3' obstructive ends. As the protein defective in the segmental progeroid Werner syndrome (WRN) possesses 3' to 5' exonuclease activity, and Werner syndrome cells are hypersensitive to IR and CPT, we examined for WRN exonuclease activity on 3' blocking lesions. Moreover, we compared side-by-side the activity of four prominent human 3' to 5' exonucleases (WRN, APE1, TREX1, and p53) on substrates containing 3' phosphates, phosphoglycolates, and tyrosyl residues. Our studies reveal that while WRN degrades 3' hydroxyl containing substrates in a nonprocessive manner, it does not excise 3' phosphate, phosphoglycolate, or tyrosyl groups. In addition, we found that APE1 was most active at excising 3' blocking termini in comparison to the disease-related exonucleases TREX1, WRN, and p53 under identical physiological reaction conditions, and that TREX1 was the most powerful 3' to 5' exonuclease on undamaged oligonucleotide substrates.

We describe the presentation, treatment, clinical outcome, and targeted genome analysis of a metastatic salivary acinic cell carcinoma (AciCC). A 71-year-old male presented with a 3 cm right tail of a parotid lesion, first detected as a nodule by the patient seven months earlier. He had a right total parotidectomy with cranial nerve VII resection, right facial nerve resection and grafting, resection of the right conchal cartilage, and right modified radical neck dissection. The primary tumor revealed AciCC with two distinct areas: a well-differentiated component with glandular architecture and a dedifferentiated component with infiltrative growth pattern associated with prominent stromal response, necrosis, perineural invasion, and cellular pleomorphism. Tumor staging was pT4 N0 MX. Immunohistochemistry staining showed pankeratin (+), CD56 (−), and a Ki67 proliferation index of 15%. Upon microscopic inspection, 49 local lymph nodes resected during parotidectomy were negative for cancer cells. Targeted sequencing of the primary tumor revealed deletions of CDKN2A and CDKN2B, a nonsense mutation in ARID2, and single missense mutations of unknown significance in nine other genes. Despite postoperative localized radiation treatment, follow-up whole body PET/CT scan showed lung, soft tissue, bone, and liver metastases. The patient expired 9 months after resection of the primary tumor.

t-Darpp is the truncated form of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (Darpp-32) and has been demonstrated to confer resistance to trastuzumab, a Her2-targeted anticancer agent, via sustained signaling through the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt pathway. t-Darpp over-expression results in enhanced activity of protein kinase A (PKA), but the mechanism of t-Darpp-mediated PKA activation is poorly understood. In the PKA holoenzyme, when the catalytic subunits are bound to regulatory subunits RI or RII, kinase activity is inhibited. We investigated PKA activity and holoenzyme composition in cell lines that over-express t-Darpp (SK.tDrp) or a T75A phosphorylation mutant (SK.tDrp.T75A), as well as an empty vector control cell line (SK.empty). We also evaluated protein-protein interactions between t-Darpp and PKA catalytic (PKAc) or regulatory subunits RI and RII in those cell lines. Whereas SK.tDrp cells had elevated PKA activity, SK.tDrp.T75A cells did not. In agreement with the effects on activity, a proximity ligation assay showed that SK.tDrp cells, but not SK.tDrp.T75A cells, had diminished association of RI with PKAc. Moreover, t-Darpp was seen to bind directly to RI in co-immunoprecipitation studies. Cells that express both Darpp-32 and t-Darpp had PKA activity levels and RI-PKAc association that were similar to control SK.empty cells. This is consistent with our published findings that Darpp-32 reverses t-Darpp’s effects on trastuzumab resistance and CREB DNA binding activity (downstream of PKA). Taken together, our data suggest that T75 phosphorylation is crucial for t-Darpp-mediated PKA activation and this activation appears to occur through direct binding to RI and release of RI from PKAc. The t-Darpp-RI interaction could be a druggable target to reduce PKA activity in drug-resistant cancer. Citation Format: Dirk Theile, Shuhui Geng, Erin Denny, Jamil Momand, Susan E. Kane. t-Darpp stimulates protein kinase A activity by interacting with its RI regulatory subunit [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4093. doi:10.1158/1538-7445.AM2017-4093

t-Darpp, one of two protein isoforms encoded by the PPP1R1B gene, is overexpressed in gastric, esophageal, colon, prostate, and breast cancers; and confers resistance to trastuzumab in Her2+ breast cancer cells. To gain insight into the trastuzumab-resistance mechanism of t-Darpp, we studied its structure, oligomerization status, metal-binding properties, and sites of phosphorylation. Circular dichroism spectroscopy analysis showed that recombinant t-Darpp exhibits 12% alpha helix, 29% beta strand, 24% beta turn and 35% random coil character at 25°C. Upon mild heat treatment (50°C for five min) the secondary structure does not appreciably change. Metal ion-binding analyses through inductively coupled plasma-mass spectrometry and graphite furnace-atomic absorption indicated that t-Darpp co-purifies with calcium, but not other metals commonly found in biological systems. The T75 site, critical for t-Darpp activation of the Akt signaling pathway, is a substrate for phosphorylation by cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 5 (CDK5). Gel filtration chromatography, sedimentation equilibrium analysis, native blue gel electrophoresis, and glutaraldehyde-mediated crosslinking experiments showed that the majority of t-Darpp (calculated mass = 19 kDa) exists as a monomer, but forms low levels (< 3%) of hetero-oligomers with its longer isoform Darpp-32. t-Darpp has a relatively large Stokes radius of 4.4 nm, suggesting an elongated structure. In summary, this study indicates that t-Darpp is an elongated, mild-heat-stable, and monomeric calcium-binding protein that is capable of being phosphorylated at T75 by CDK1 and CDK5. Blockage of t-Darpp calcium binding capacity or T75 phosphorylation may therefore help sustain Her2+ breast cancer sensitivity to trastuzumab therapy. Citation Format: Patrycja Magdziarz, You Feng, Dianlu Jiang, Elizabeth Parga, Arianna Celis, Erin Denny, Xiaoying Wang, Martin Phillips, Feimeng Zhou, Susan E. Kane, Jamil Momand. t-Darpp chemoresistence protein: insights into its structure, oligomerization status, calcium-binding properties and phosphorylation sites [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5866. doi:10.1158/1538-7445.AM2017-5866

A novel method was developed to determine the oxidation status of proteins in cultured cells. Methoxy-polyethylene glycol-maleimide MW 2000 (MAL-PEG) was used to covalently tag p53 protein that was oxidized at cysteine residues in cultured cells. Treatment of MCF7 breast cancer cells with pyrrolidine dithiocarbamate (PDTC), a metal chelator, resulted in a minimum of 25% oxidation of p53. The oxidized p53 had an average of one cysteine residue oxidized per p53 protein molecule. The effect of PDTC treatment on downstream components of the p53 signal-transduction pathway was tested. PDTC treatment prevented actinomycin D-mediated up-regulation of two p53 effector gene products, murine double minute clone 2 oncoprotein and p21WAF1/CIP1 (where WAF1 corresponds to wild-type p53-activated fragment 1 and CIP1 corresponds to cyclin-dependent kinase-interacting protein 1). Actinomycin D treatment led to accumulation of p53 protein in the nucleus. However, when cells were simultaneously treated with PDTC and actinomycin D, p53 accumulated in both the nucleus and the cytoplasm. The data indicate that an average of one cysteine residue per p53 protein molecule is highly sensitive to oxidation and that p53 can be efficiently oxidized by PDTC in cultured cells. PDTC-mediated oxidation of p53 correlates with altered p53 subcellular localization and reduced activation of p53 downstream effector genes. The novel method for detecting protein oxidation detailed in the present study may be used to determine the oxidation status of specific proteins in cells.

MDM2 is an oncogene that mainly functions to modulate p53 tumor suppressor activity. In normal cells the MDM2 protein binds to the p53 protein and maintains p53 at low levels by increasing its susceptibility to proteolysis by the 26S proteosome. Immediately after the application of cellular stress, the ability of MDM2 to bind to p53 is blocked or altered in a fashion that prevents MDM2-mediated degradation. As a result, p53 levels rise, causing cell cycle arrest or apoptosis. In this review, we present evidence for the existence of three highly conserved regions (CRs) shared by MDM2 proteins and MDMX proteins of different species. These highly conserved regions encompass residues 42-94 (CR1), 301-329 (CR2), and 444-483 (CR3) on human MDM2. These three domains are respectively important for binding p53, for binding the retinoblastoma protein, and for transferring ubiquitin to p53. This review discusses the major milestones uncovered in MDM2 research during the past 12 years and potential uses of this knowledge in the fight against cancer.

Pyrrolidine dithiocarbamate (PDTC) is a thiol compound widely used to study the activation of redox-sensitive transcription factors. Although normally used as an antioxidant, PDTC has been shown to exert pro-oxidant activity on proteins both in vitro and in vivo. Because p53 redox status has been shown to alter its DNA binding capability, we decided to test the effect of PDTC on p53 activation. In this communication, we report that PDTC inhibits the activation of temperature-sensitive murine p53(Val-135) (TSp53) in the transformed rat embryo fibroblast line, A1-5, as well as wild-type human p53 in the normal diploid fibroblast line, WS1neo. In A1-5 cells, PDTC abrogated UV- and temperature shift-induced TSp53 nuclear translocation and p53-mediated transactivation of MDM2. PDTC also blocked UV-induced accumulation of wild-type p53 in WS1neo cells. Continual presence of PDTC was required for its effect as both UV-induced nuclear translocation and accumulation resumed after PDTC removal. We next investigated whether PDTC treatment altered the p53 redox state. We found that PDTC increased p53 cysteine residue oxidation in vivo. This represents the first direct evidence showing that the p53 redox state can be altered in vivo and that increased oxidation correlates with its inability to perform its downstream functions.

The tumor suppressor protein p53 has a transcriptional activation activity thought to mediate its biologic function including G1 arrest and perhaps apoptosis. To learn more about p53's transactivator function in vivo, we performed genomic footprinting experiments examining p53-DNA interactions in the regulatory regions of the p53-regulated genes p21, GADD45, and MDM2. Using ionizing radiation to induce DNA damage in human ML-1 myeloblastic leukemia cells, the promoter and intronic regions of these genes containing p53-consensus binding sites were examined for in vivo footprints. There was a uniform and sustained expression of p53 protein as well as a strong induction of p21, GADD45, and MDM2 mRNA following irradiation. At the two p53 consensus binding sites in the p21 promoter, reduced DNaseI cleavage was observed in irradiated cells beginning 1 to 2 h after irradiation, being most pronounced after 2 h and diminishing after 8 h. A partial in vivo footprint was also observed in the third intron of the GADD45 gene beginning 2 h after irradiation. No in vivo footprints were seen at the two p53 binding sites in the MDM2 gene. Our study provides direct evidence that the DNA damage-induced activity of p53 is mediated by its consensus DNA binding sites in the p21 and GADD45 genes. We suggest that the transient nature and relative instability of p53-DNA interactions in vivo may make the p53 protein more accessible to a rapid turnover pathway which might be impaired under conditions when the protein is stably bound to DNA.

MDM2 is an oncoprotein that blocks p53 tumor suppressor-mediated transcriptional transactivation, escorts p53 from the cell nucleus to the cytoplasm, and polyubiquitylates p53. Polyubiquitylated p53 is rapidly degraded in the cytoplasm by the 26S proteasome. MDM2 is abnormally upregulated in several types of cancers, especially those of mesenchymal origin. MDM4 is a homolog of MDM2 that also inhibits p53 by blocking p53-mediated transactivation. MDM4 is required for MDM2-mediated polyubiquitylated of p53 and is abnormally upregulated in several cancer types. MDM2 and MDM4 genes have been detected in all vertebrates to date and only a single gene homolog, named MDM, has been detected in some invertebrates. MDM2, MDM4, and MDM have similar gene structures, suggesting that MDM2 and MDM4 arose through a duplication event more than 440 million years ago. All members of this small MDM2 gene family contain a single really interesting new gene (RING) domain (with the possible exception of lancelet MDM) which places them in the RING-domain superfamily. Similar to MDM2, the vast majority of proteins with RING domains are E3 ubiquitin ligases. Other RING domain E3 ubiquitin ligases that target p53 are COP1, Pirh2, and MSL2. In this report, we present evidence that COP1, Pirh2, and MSL2 evolved independently of MDM2 and MDM4. We also show, through structure homology models of invertebrate MDM RING domains, that MDM2 is more evolutionarily conserved than MDM4.

Cellular DNA damage results in the increased expression and accumulation of the p53 tumor suppressor protein within the nucleus which leads to cell cycle arrest or apoptosis. In some cases, however, wild-type p53 and some mutant forms of p53 reside in the cytoplasm of cancer cells. To understand the mechanism responsible for its cytoplasmic retention, studies were undertaken to determine if unique proteins form a complex with mutant p53 within the cytoplasm of transformed cells. One protein, with an apparent molecular mass of 92 kDa (p92), was observed to form a complex with a temperature-sensitive mutant p53 (TSp53Val-135) in the cytoplasm of transformed rat embryo fibroblasts at the non-permissive temperature. p92 copurified with TSp53Val-135 on a p53-specific immunoaffinity column and a gel filtration column. The protein was purified to homogeneity and identified as hsp84 by partial amino acid sequence analysis. hsp84 is a member of the hsp90 class of proteins. At the non-permissive temperature, TSp53Val-135 and hsp84 colocalized in the cytoplasm near the nuclear envelope. At the permissive temperature, TSp53Val-135 resides in the nucleus and expresses a “wild-type like” conformation. Under these conditions hsp84 continued to reside in the cytoplasm and little or no hsp84 formed a complex with p53. The results suggest that hsp84 binds mutant p53 in a spatial and/or conformation dependent manner.

p53 activates transcription of genes with a p53 response element, and it can repress genes lacking the element. Here we demonstrate that wild-type but not mutant p53 inhibits transcription in a HeLa nuclear extract from minimal promoters. Wild-type but not mutant p53 binds to human TATA-binding protein (TBP). p53 does not bind to yeast TBP, and it cannot inhibit transcription in a HeLa extract where yeast TBP substitutes for human TBP. These results suggest a model in which p53 binds to TBP and interferes with transcriptional initiation.

A cellular phosphoprotein with an apparent molecular mass of 90 kd (p90) that forms a complex with both mutant and wild-type p53 protein has been characterized, purified, and identified. The protein was identified as a product of the murine double minute 2 gene (mdm-2). The mdm-2 gene enhances the tumorigenic potential of cells when it is overexpressed and encodes a putative transcription factor. To determine if mdm-2 could modulate p53 transactivation, a p53-responsive element from the muscle creatine kinase gene was employed. A wild-type p53-expressing plasmid enhanced the expression of the p53-responsive element when cotransfected into cells that contain no endogenous p53. When a cosmid expressing mdm-2 was transfected with this p53-expressing plasmid, the transactivation of the p53-responsive element was inhibited. Thus, a product of the mdm-2 oncogene forms a tight complex with the p53 protein, and the mdm-2 oncogene can inhibit p53-mediated transactivation.

The cell cycle is composed of a series of steps which can be negatively or positively regulated by various factors. Chief among the negative regulators is the p53 protein. Alteration or inactivation of p53 by mutation, or by its interactions with oncogene products of DNA tumour viruses, can lead to cancer. These mutations seem to be the most common genetic change in human cancers.

p53, a tumor suppressor protein and a transcription factor, is capable of inhibiting the growth of tumor cells by eliciting either cell-cycle arrest or apoptosis through a cascade of events. p53 binds sites within the promoters of several genes that conform to a sequence commonly defined as the consensus site. In more than 50% of cancer cases, the p53 gene has been found to be mutated and the p53 protein loses its ability to bind the consensus DNA. In this work, double-stranded (ds-) oligonucleotides (ODNs) containing the consensus site are immobilized onto gold electrodes to capture wild-type p53. The cysteine residues on the exterior of the p53 molecule were derivatized for the attachment of gold nanoparticle/streptavidin conjugates capped with multiple ferrocene (Fc) groups. Well-defined voltammetric peaks of high signal intensity were obtained, and p53 concentration as low as 2.2 pM was measured. The peak heights were found to be dependent on the surface density of the consensus ds-ODN, the sequence of the immobilized ODNs, and the p53 concentration. With base pair(s) in the full consensus binding sequence altered, the level of p53 binding was found to decrease sharply, and no p53 binding occurred at electrodes covered with nonconsensus ds-ODNs. The amenability of this method to the analyses of p53 from normal and cancer cell lysates was also demonstrated. Owing to the p53 mutation in the cancer cells, the concentration of the wild-type p53 was found to decrease significantly (by about 50-182 times). The sensitivity and amenability for real sample analysis of the method compared well with enzyme-linked immunosorbant assay (ELISA), and complements ELISA in that wild-type p53, instead of total p53 (wild-type and mutant p53) concentration, is measured. The method described herein is simple and selective and does not require the use of p53 antibodies.

We have tested the hypothesis that isoaspartic acid residues in proteins can arise via errors that occur during protein synthesis. One such error involves a mischarging step in which the aspartic acid side-chain beta-carboxyl group is linked to the tRNA(Asp) instead of the main chain alpha-carboxyl group. If this altered Asp-tRNA(Asp) is a substrate for the ribosomal elongation reactions, a polypeptide will be made with an isoaspartic acid, or beta-linkage, in which the peptide chain is branched at the side chain of the aspartic acid residue. Using an ammonium sulfate fraction of aspartyl-tRNA(Asp) synthetase from Escherichia coli and [3H]aspartic acid, we have prepared [3H]aspartyl-tRNA(Asp) complexes and directly analyzed the linkage of the [3H]aspartate to the tRNA by identifying the products of ammonolysis. Normal attachment of the alpha-carboxyl group of aspartate to the tRNA produces [3H]isoasparagine, while the mischarging reaction leads to [3H]asparagine formation after ammonolysis. We have separated [3H]isoasparagine from [3H]asparagine and found an upper limit of 1 asparagine per 10,000 isoasparagines. These results show that the bacterial aminoacyl-tRNA synthetase can very accurately distinguish between the alpha- and beta-carboxyl groups of aspartic acid and suggest that only a very small fraction of the isoaspartic acid residues found to occur in cellular proteins may be the result of mischarging steps.

MDM2 possesses three activities that, together, effectively inhibit the p53 tumor suppressor. First, it binds to p53 and sterically blocks p53 interaction with TATA box protein accessory factors thereby shutting down its transcriptional transactivation function. Second, MDM2 shuttles p53 from its site of action within the nucleus into the cytoplasm. Third, MDM2 is an E3 ligase that transfers ubiquitin onto lysine residues of p53. Ubiquitinated p53 is rapidly degraded by the 26S proteosome. Because the MDM2 oncoprotein mediates three progressive stages of inhibition, it is the principal regulator of p53 activity. TheMDM2gene is located on chromosome 12q14.3-q15 and is amplified in several types of neoplasms, most of which are of mesenchymal tissue origin. MDM2 binding to p53 can be inhibited by phosphorylation of either MDM2 or p53. The kinases responsible for this phosphorylation are activated by cell stressors in general (hypoxia, nitric oxide, hydrogen peroxide) and DNA damaging agents in particular (ionizing radiation, UV-light). MDM2 can be inhibited by RAS or MYC oncoproteins. RAS and MYC activate the tumor suppressor protein p19Arf which sequesters MDM2 into the nucleolus and, in doing so, allows p53 levels to rise. The MDM2gene is activated by p53, which means that, in effect, p53 inhibits itself through MDM2. The cell requires a fine balance of MDM2 and p53 to maintain cell growth and a rapid response to stressors. MDMX is a paralog of MDM2 that has retained the ability to inhibit p53 binding to TATA box protein accessory factors. MDMX does not possess other p53 inhibitory activities. There has been recent progress in the development of small molecules that block MDM2 from binding to p53. Although these molecules are in the early stages of development, it is hoped that they will contribute to the war on cancer. This chapter summarizes the key studies that have increased our understanding of the interplay between p53, MDM2, and MDMX.

A summer program was created for undergraduates and graduate students that teaches bioinformatics concepts, offers skills in professional development, and provides research opportunities in academic and industrial institutions. We estimate that 34 of 38 graduates (89%) are in a career trajectory that will use bioinformatics. Evidence from open-ended research mentor and student survey responses, student exit interview responses, and research mentor exit interview/survey responses identified skills and knowledge from the fields of computer science, biology, and mathematics that are critical for students considering bioinformatics research. Programming knowledge and general computer skills were essential to success on bioinformatics research projects. General mathematics skills obtained through current undergraduate natural sciences programs were adequate for the research projects, although knowledge of probability and statistics should be strengthened. Biology knowledge obtained through the didactic phase of the program and prior undergraduate education was adequate, but advanced or specific knowledge could help students progress on research projects. The curriculum and assessment instruments developed for this program are available for adoption by other bioinformatics programs at http://www.calstatela.edu/SoCalBSI .

Oligonucleotide (ODN)-capped gold nanoparticles (Au-NPs) were used in a sandwich assay of ODN or polynucleotide by a flow injection surface plasmon resonance (SPR). A carboxylated dextran film was immobilized onto the SPR sensor surface to eliminate nonspecific adsorption of ODN-capped Au-NPs. The tandem use of signal amplification via the adlayer of the ODN-capped Au-NPs and the differential signal detection by the bicell detector on the SPR resulted in a remarkable DNA detection level. A 39-mer target at a quantity as low as 2.1 x 10(-20)mol, corresponding to 1.38 fM in a 15 microl solution, can be measured. To our knowledge, both the concentration and quantity detection levels are the lowest among all the gene analyses conducted with SPR to this point. The method is shown to be reproducible (relative standard deviation values

We describe a method for purifying recombinant p53 from baculovirus infected cells in one step by anion exchange chromatography. The p53 is full-length with no flanking sequences and its expression is driven by the baculovirus polyhedron promoter. We also describe how to concentrate the p53 up to 0.9 mg/mL. By gel filtration analysis, we demonstrate that 20% of the p53 forms a tetramer, and 80% forms a monomer. In a DNA binding assay known as the electromobility shift assay, the purified p53/DNA complex forms a single band the gel. This simple procedure should be useful for investigations into the biochemistry of the p53 protein.

The p53 tumor suppressor protein is susceptible to oxidation, which prevents it from binding to its DNA response element. The goal of the current research was to determine the nature of the cysteine residue thiol oxidation that prevents p53 from binding its DNA target and its effect on p53 structure. Recombinant p53, purified in the presence of the reducing agent dithiothreitol (DTT), contains five free thiol groups on the surface of the protein. In the absence of DTT, p53 contains only four thiol groups, indicating that an average of one surface thiol group is readily susceptible to oxidation. Sulfite-mediated disulfide bond cleavage followed by reaction with 2-nitro-5-thiosulfobenzoate showed that oxidized p53 contains a single disulfide bond per monomer. By atomic force microscopy, we determined that reduced p53 binds to a double-stranded DNA containing the p53 promoter element of the MDM2 gene. The DNA-bound reduced p53 has an average cross-sectional diameter of 8.61 nm and a height of 4.12 nm. The amount of oxidized p53 that bound to the promoter element was ninefold lower, and it has an 18% larger average cross-sectional diameter. Electromobility shift assays showed that binding of oxidized p53 to DNA was enhanced upon addition of DTT, indicating that oxidation is reversible. The possibility that oxidized p53 contained significant amounts of sulfenic (-SOH), sulfinic (-SO2H), or sulfonic acid (-SO3H) was ruled out. Gel filtration chromatography indicated that oxidation increases the percentage of p53 monomers and high-molecular-weight oligomers (1,000 kDa) relative to tetrameric p53. Protein modeling studies suggest that a mixed disulfide glutathione adduct on Cys182 could account for the observed stoichiometry of oxidized thiols and structural changes. The glutathione adduct may prevent proper helix-helix interaction within the DNA binding domain and contribute to tetramer dissociation.

We describe a method for purifying recombinant p53 from baculovirus infected cells in one step by anion exchange chromatography. The p53 is full-length with no flanking sequences and its expression is driven by the baculovirus polyhedron promoter. We also describe how to concentrate the p53 up to 0.9 mg/mL. By gel filtration analysis, we demonstrate that 20% of the p53 forms a tetramer, and 80% forms a monomer. In a DNA binding assay known as the electromobility shift assay, the purified p53/DNA complex forms a single band the gel. This simple procedure should be useful for investigations into the biochemistry of the p53 protein.

The p53 tumour-suppressor protein is a transcription factor that activates the expression of genes involved in cell cycle arrest, apoptosis and DNA repair. The p53 protein is vulnerable to oxidation at cysteine thiol groups. The metal-chelating dithiocarbamates, pyrrolidine dithiocarbamate (PDTC), diethyldithiocarbamate, ethylene(bis)dithiocarbamate and H(2)O(2) were tested for their oxidative effects on p53 in cultured human breast cancer cells. Only PDTC oxidized p53, although all oxidants tested increased the p53 level. Inductively coupled plasma MS analysis indicated that the addition of 60 microM PDTC increased the cellular copper concentration by 4-fold, which was the highest level of copper accumulated amongst all the oxidants tested. Bathocuproinedisulphonic acid, a membrane-impermeable Cu(I) chelator inhibited the PDTC-mediated copper accumulation. Bathocuproinedisulphonic acid as well as the hydroxyl radical scavenger d-mannitol inhibited the PDTC-dependent increase in p53 protein and oxidation. Our results show that a low level of copper accumulation in the range of 25-40 microg/g of cellular protein increases the steady-state levels of p53. At copper accumulation levels higher than 60 microg/g of cellular protein, p53 is oxidized. These results suggest that p53 is vulnerable to free radical-mediated oxidation at cysteine residues.

Protein sulfhydryl groups can undergo reversible oxidation reactions in response to reactive oxygen and reactive nitrogen species. Sensitive detection of sulfhydryl group oxidation in specific proteins is required to further our understanding of protein redox changes in biological systems. In general, to detect reversible oxidation reactions the oxidized sulfur atom is reduced to a sulfhydryl group followed by a reaction with a quantifiable agent. Our aim was to develop a sensitive method to detect reversibly oxidized protein sulfhydryl groups in a Western blot format. Conjugation of methoxypolyethylene glycol-maleimide (MAL-PEG) to protein sulfhydryl groups was optimized. Once MAL-PEG forms a covalent bond with the protein, the MAL-PEG-protein conjugate can be detected as a band shift by western analysis. The efficiency of MAL-PEG conjugation to protein was determined with creatine kinase. MAL-PEG conjugated to approximately 100% of the available sulfhydryl groups on creatine kinase within 30 min. Band shift detection sensitivity was measured using the redox-regulated protein p53. MAL-PEG conjugation coupled to western analysis detected a minimum of 0.23 pmol of oxidized p53. The MAL-PEG conjugation method described in this communication can be used to assess the reversible sulfhydryl oxidation status of proteins for which antibodies suitable for western analysis are available.

The p53 tumor suppressor gene is inactivated in human tumors by several distinct mechanisms. The best characterized inactivation mechanisms are: (i) gene mutation; (ii) p53 protein association with viral proteins; (iii) p53 protein association with the MDM2 cellular oncoprotein. The MDM2 gene has been shown to be abnormally up-regulated in human tumors and tumor cell lines by gene amplification, increased transcript levels and enhanced translation. This communication presents a brief review of the spectrum of MDM2 abnormalities in human tumors and compares the tissue distribution of MDM2 amplification and p53 mutation frequencies. In this study, 3889 samples from tumors or xenografts from 28 tumor types were examined for MDM2 amplification from previously published sources. The overall frequency of MDM2 amplification in these human tumors was 7%. Gene amplification was observed in 19 tumor types, with the highest frequency observed in soft tissue tumors (20%), osteosarcomas (16%) and esophageal carcinomas (13%). Tumors which showed a higher incidence of MDM2 amplification than p53 mutation were soft tissue tumors, testicular germ cell cancers and neuro-blastomas. Data from studies where both MDM2 amplification and p53 mutations were analyzed within the same samples showed that mutations in these two genes do not generally occur within the same tumor. In these studies, 29 out of a total of 33 MDM2 amplification-positive tumors had wild-type p53. We hypothesize that heretofore uncharacterized carcinogens favor MDM2 amplification over p53 mutations in certain tumor types. A database listing the MDM2 gene amplifications is available on the World Wide Web at http://www. infosci.coh.org/mdm2 . Charts of MDM2 amplification frequencies and comparisons with p53 genetic alterations are also available at this Web site.

p53 is a tumor suppressor protein that acts in the nucleus to effect cell cycle arrest and apoptosis. In some cells p53 is located in the cytoplasm, perhaps as a means of downregulating its activity. We recently showed that hsp90 forms a complex with the cytoplasmically localized mutant p53 (TSp53vall35) within transformed cells (Sepehrnia et al., J. Biol. Chem. 271, 15084, 1996). The present study was undertaken to determine the p53 conformation bound to hsp90 and the role of hsp90 in p53 nuclear translocation. We show that hsp90 binds both a native and a denatured form of p53 as determined by conformation-specific antibodies. hsp90 does not bind p53 in a spatial-specific manner because it remains bound to p53 when induced to translocate to the nucleus by the protein synthesis inhibitor cycloheximide (CHX). Treatment of transformed cells with geldanamycin (GA), a small molecule that binds hsp90, causes a rapid destabilization of p53 by 50%. Residual p53 that survives GA treatment is incapable of translocating to the nucleus. GA does not destabilize p53 in cells where p53 is genotypically wild type. Although GA appears to dramatically alter the translocating properties of mutant p53 it does not dissociate the p53-hsp90 complex. We suggest that a second chaperone protein, called p23, which we show also binds p53, may play an important role in these GA-mediated effects.

Background: The peptide segment surrounding residues Leu22 and Trp23 of the p53 transactivation domain plays a critical role in the transactivation activity of p53. This region binds basal transcriptional components such as the TATA-box binding protein associated factors TAF II 40 and TAF II 60 as well as the mdm-2 and adenovirus type 5 E1B 55 kDa oncoproteins. Results: The structure of residues 14–28 of p53 was studied by nuclear magnetic resonance spectroscopy and found to prefer a two- β -turn structure stabilized by a hydrophobic cluster consisting of residues known to be important for transactivation and binding to p53-binding proteins. A peptide segment in which Leu22 and Trp23 were replaced by Gln and Ser displays a random structure. Conclusions: This structural propensity observed in the wild-type p53 peptide is important for understanding the mechanism of transcriptional activation, because very few structural data are available on transactivation domains to date. These results should aid in the design of therapeutics that could competitively inhibit binding of p53 to the oncogene product mdm-2.

Background: We have previously shown that levels of the 32kDa dopamine and cAMP-regulated phosphoprotein Darpp-32 (Dp32) and its amino-truncated isoform t-Darpp (tDp) are altered in trastuzumab-resistant HER2+ breast cancer cells. Specifically, tDp is upregulated in models of trastuzumab resistance and over-expression of the protein is sufficient to confer trastuzumab resistance. Dp32 antagonizes the effect of tDp, exhibiting overall growth inhibitory properties in breast cancer cell lines. In neuronal cells, Dp32 is regulated by phosphorylation at two key threonine residues, T34 and T75. The T34 site is present only in Dp23 as tDp is truncated by the first 39 amino acids. The latter site is phosphorylated predominantly by cdk5 in vivo and by either cdk5 or cdk1 in vitro. It is not known whether tDp is similarly phosphorylated or if the same kinases act in breast cancer cells as in neuronal cells. Because we have preliminary data suggesting an interaction between tDp and cdk1 (but not cdk5) in HER2+ breast cancer cells, we investigated whether cdk1 or cdk5 could phosphorylate tDp at the T75 site. Methods: We performed in vitro kinase assays using recombinant tDp and commercial cdk1 and cdk5 enzymes. Phosphorylation was also examined in cells that overexpress endogenous tDp (SK/HerR and BT/HerR cells), that overexpress exogenous tDp (SK.tDp cells) or that express exogenous tDp and Dp32 (SK.dDp). Cells were treated with either RO-3306, a cdk1 inhibitor, or Roscovitine, a pan-cdk inhibitor with preference for cdk5, and assayed by Western blotting for T75 phosphorylation. All experiments were repeated three times and calculated for statistical significance. Results: In vitro kinase assays demonstrated that both cdk1 and cdk5 were capable of phosphorylating tDp at the T75 residue, with an apparent preferential activity by cdk1. In cell lines, RO-3306 led to decreased T75 phosphorylation within one hour. The effect of RO-3306 was also confirmed in a dose-dependent manner with optimal inhibition at a concentration of 10 μM. Roscovitine also led to decreased T75 phosphorylation at a concentration of 20 μM. In cells that express both Dp32 and tDp, the cdk inhibitors led to decreased phosphorylation of both proteins to similar extents. Conclusion: Our study provides new insight into cdk1-mediated regulation of tDp and Dp32. We demonstrate that both cdk1 and cdk5 are able to phosphorylate these proteins but that cdk1 appears to be the predominant mediator of phosphorylation in breast cancer cell lines. A possible competition might occur between Dp32 and tDp, given their similar propensity for phosphorylation by cdk1. Further examination of the relationship between the two kinases and their activities toward tDp and Dp32 will help us delineate the mechanism by which tDp mediates trastuzumab resistance and Dp32 antagonizes this phenotype. Citation Format: Erin Denny, Arianna Celis, Jamil Momand, Susan E. Kane. t-Darpp is phosphorylated by cdk1 and cdk5 in HER2+ breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A058.