Androgen receptor phosphorylation. Regulation and identification of the phosphorylation sites

Activation of signal transduction kinase cascades has been shown to alter androgen receptor (AR) activity. Although it has been suggested that changes in AR phosphorylation might be directly responsible, the basal and regulated phosphorylations of the AR have not been fully determined. We have identified the major sites of AR phosphorylation on ARs expressed in COS-1 cells using a combination of peptide mapping, Edman degradation, and mass spectrometry. We describe the identification of seven AR phosphorylation sites, show that the phosphopeptides seen with exogenously expressed ARs are highly similar to those seen with endogenous ARs in LNCaP cells and show that specific agonists differentially regulate the phosphorylation state of endogenous ARs in LNCaP prostate cancer cells. Treatment of LNCaP cells with the synthetic androgen, R1881, elevates phosphorylation of serines 16, 81, 256, 308, 424, and 650. Ser-94 appears constitutively phosphorylated. Forskolin, epidermal growth factor, and phorbol 12-myristate 13-acetate increase the phosphorylation of Ser-650. The kinetics of phosphorylation of most sites in response to hormone or forskolin is temporally delayed, reaching a maximum at 2 h post-stimulation. The exception is Ser-81, which continues to display increasing phosphorylation at 6 h. These data provide a basis for analyzing mechanisms of cross-talk between growth factor signaling and androgen in prostate development, physiology, and cancer.     

A multidimensional chromatography technology for in-depth phosphoproteome analysis

Protein phosphorylation is a post-translational modification widely used to regulate cellular responses. Recent studies showed that global phosphorylation analysis could be used to study signaling pathways and to identify targets of protein kinases in cells. A key objective of global phosphorylation analysis is to obtain an in-depth mapping of low abundance protein phosphorylation in cells; this necessitates the use of suitable separation techniques because of the complexity of the phosphoproteome. Here we developed a multidimensional chromatography technology, combining IMAC, hydrophilic interaction chromatography, and reverse phase LC, for phosphopeptide purification and fractionation. Its application to the yeast Saccharomyces cerevisiae after DNA damage led to the identification of 8764 unique phosphopeptides from 2278 phosphoproteins using tandem MS. Analysis of two low abundance proteins, Rad9 and Mrc1, revealed that approximately 50% of their phosphorylation was identified via this global phosphorylation analysis. Thus, this technology is suited for in-depth phosphoproteome studies.     

Characterization of the phosphoproteome in androgen-repressed human prostate cancer cells by Fourier transform ion cyclotron resonance mass spectrometry

Androgen-repressed human prostate cancer, ARCaP, grows and is highly metastatic to bone and soft tissues in castrated mice. The molecular mechanisms underlying the aberrant responses to androgen are not fully understood. Here, we apply state-of-the-art mass spectrometry methods to investigate the phosphoproteome profiles in ARCaP cells. Because protein biological phosphorylation is always substoichiometric and the ionization efficiency of phosphopeptides is low, selective enrichment of phosphorylated proteins/peptides is required for mass spectrometric analysis of phosphorylation from complex biological samples. Therefore, we compare the sensitivity, efficiency, and specificity for three established enrichment strategies: calcium phosphate precipitation (CPP), immobilized metal ion affinity chromatography (IMAC), and TiO(2)-modified metal oxide chromatography. Calcium phosphate precipitation coupled with the TiO(2) approach offers the best strategy to characterize phosphorylation in ARCaP cells. We analyzed phosphopeptides from ARCaP cells by LC-MS/MS with a hybrid LTQ/FT-ICR mass spectrometer. After database search and stringent filtering, we identified 385 phosphoproteins with an average peptide mass error of 0.32 ± 0.6 ppm. Key identified oncogenic pathways include the mammalian target of rapamycin (mTOR) pathway and the E2F signaling pathway. Androgen-induced proliferation inhibitor (APRIN) was detected in its phosphorylated form, implicating a molecular mechanism underlying the ARCaP phenotype.     

Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle

Protein kinases are pivotal regulators of cell signaling that modulate each other's functions and activities through site-specific phosphorylation events. These key regulatory modifications have not been studied comprehensively, because low cellular abundance of kinases has resulted in their underrepresentation in previous phosphoproteome studies. Here, we combine kinase-selective affinity purification with quantitative mass spectrometry to analyze the cell-cycle regulation of protein kinases. This proteomics approach enabled us to quantify 219 protein kinases from S and M phase-arrested human cancer cells. We identified more than 1000 phosphorylation sites on protein kinases. Intriguingly, half of all kinase phosphopeptides were upregulated in mitosis. Our data reveal numerous unknown M phase-induced phosphorylation sites on kinases with established mitotic functions. We also find potential phosphorylation networks involving many protein kinases not previously implicated in mitotic progression. These results provide a vastly extended knowledge base for functional studies on kinases and their regulation through site-specific phosphorylation.     

Analysis of the Molecular Networks in Androgen Dependent and Independent Prostate Cancer Revealed Fragile and Robust Subsystems

Androgen ablation therapy is currently the primary treatment for metastatic prostate cancer. Unfortunately, in nearly all cases, androgen ablation fails to permanently arrest cancer progression. As androgens like testosterone are withdrawn, prostate cancer cells lose their androgen sensitivity and begin to proliferate without hormone growth factors. In this study, we constructed and analyzed a mathematical model of the integration between hormone growth factor signaling, androgen receptor activation, and the expression of cyclin D and Prostate-Specific Antigen in human LNCaP prostate adenocarcinoma cells. The objective of the study was to investigate which signaling systems were important in the loss of androgen dependence. The model was formulated as a set of ordinary differential equations which described 212 species and 384 interactions, including both the mRNA and protein levels for key species. An ensemble approach was chosen to constrain model parameters and to estimate the impact of parametric uncertainty on model predictions. Model parameters were identified using 14 steady-state and dynamic LNCaP data sets taken from literature sources. Alterations in the rate of Prostatic Acid Phosphatase expression was sufficient to capture varying levels of androgen dependence. Analysis of the model provided insight into the importance of network components as a function of androgen dependence. The importance of androgen receptor availability and the MAPK/Akt signaling axes was independent of androgen status. Interestingly, androgen receptor availability was important even in androgen-independent LNCaP cells. Translation became progressively more important in androgen-independent LNCaP cells. Further analysis suggested a positive synergy between the MAPK and Akt signaling axes and the translation of key proliferative markers like cyclin D in androgen-independent cells. Taken together, the results support the targeting of both the Akt and MAPK pathways. Moreover, the analysis suggested that direct targeting of the translational machinery, specifically eIF4E, could be efficacious in androgen-independent prostate cancers.     

Quantitative phosphoproteomics of transforming growth factor-β signaling in colon cancer cells

The transforming growth factor‐β (TGF‐β) signaling pathway progresses through a series of protein phosphorylation regulated steps. Smad4 is a key mediator of the classical TGF‐β signaling pathway; however, reports suggest that TGF‐β can activate other cellular pathways independent of Smad4. By investigating the TGF‐β‐regulated phosphoproteome, we aimed to uncover new functions controlled by TGF‐β. We applied titanium dioxide to enrich phosphopeptides from stable isotope labeling with amino acids in cell culture (SILAC)‐labeled SW480 cells stably expressing Smad4 and profiled them by mass spectrometry. TGF‐β stimulation for 30 min resulted in the induction of 17 phosphopeptides and the repression of 8 from a total of 149 unique phosphopeptides. Proteins previously not known to be phosphorylated by TGF‐β including programmed cell death protein 4, nuclear ubiquitous casein and cyclin‐dependent kinases substrate, hepatoma‐derived growth factor and cell division kinases amongst others were induced following TGF‐β stimulation, while the phosphorylation of TRAF2 and NCK‐interacting protein kinase are examples of proteins whose phosphorylation status was repressed. This phosphoproteomic screen has identified new TGF‐β‐modulated phosphorylation responses in colon carcinoma cells.     

Ursolic acid overcomes Bcl‐2‐mediated resistance to apoptosis in prostate cancer cells involving activation of JNK‐induced Bcl‐2 phosphorylation and degradation

Androgen‐independent prostate cancers express high levels of Bcl‐2, and this over‐expression of Bcl‐2 protects prostate cancer cells from undergoing apoptosis. Ursolic acid (UA) has demonstrated an anti‐proliferative effect in various tumor types. The aim of this study is to evaluate the difference between UA‐induced apoptosis in androgen‐dependent prostate cancer cell line LNCaP cells and androgen‐independent prostate cancer cell line LNCaP‐AI cells and to reveal the molecular mechanisms underlying the apoptosis. We found that UA treatment in vitro can effectively induce apoptosis in LNCaP and LNCaP‐AI cells. UA can overcome Bcl‐2‐mediated resistance to apoptosis in LNCaP‐AI cells. Intrinsic apoptotic pathways can be triggered by UA treatment because c‐Jun N‐terminal kinase (JNK) is activated and subsequently provokes Bcl‐2 phosphorylation and degradation, inducing activation of caspase‐9. Although further evaluation is clearly needed, the present results suggest the potential utility of UA as a novel therapeutic agent in advanced prostate cancer. J. Cell. Biochem. 109: 764–773, 2010. © 2009 Wiley‐Liss, Inc.     

15N-labeled brain enables quantification of proteome and phosphoproteome in cultured primary neurons

Terminally differentiated primary cells represent a valuable in vitro model to study signaling events associated within a specific tissue. Quantitative proteomic methods using metabolic labeling in primary cells encounter labeling efficiency issues hindering the use of these cells. Here we developed a method to quantify the proteome and phosphoproteome of cultured neurons using (15)N-labeled brain tissue as an internal standard and applied this method to determine how an inhibitor of an excitatory neural transmitter receptor, phencyclidine (PCP), affects the global phosphoproteome of cortical neurons. We identified over 10,000 phosphopeptides and made accurate quantitative measurements of the neuronal phosphoproteome after neuronal inhibition. We show that short PCP treatments lead to changes in phosphorylation for 7% of neuronal phosphopeptides and that prolonged PCP treatment alters the total levels of several proteins essential for synaptic transmission and plasticity and leads to a massive reduction in the synaptic strength of inhibitory synapses. The results provide valuable insights into the dynamics of molecular networks implicated in PCP-mediated NMDA receptor inhibition and sensorimotor deficits.     

Analysis of the phosphoproteome of Chlamydomonas reinhardtii provides new insights into various cellular pathways

The unicellular flagellated green alga Chlamydomonas reinhardtii has emerged as a model organism for the study of a variety of cellular processes. Posttranslational control via protein phosphorylation plays a key role in signal transduction, regulation of gene expression, and control of metabolism. Thus, analysis of the phosphoproteome of C. reinhardtii can significantly enhance our understanding of various regulatory pathways. In this study, we have grown C. reinhardtii cultures in the presence of an inhibitor of Ser/Thr phosphatases to increase the phosphoprotein pool. Phosphopeptides from these cells were enriched by immobilized metal-ion affinity chromatography and analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS-MS as well as neutral-loss-triggered MS-MS-MS spectra. In this way, we were able to identify 360 phosphopeptides from 328 different phosphoproteins of C. reinhardtii, thus providing new insights into a variety of cellular processes, including metabolic and signaling pathways. Comparative analysis of the phosphoproteome also yielded new functional information on proteins controlled by redox regulation (thioredoxin target proteins) and proteins of the chloroplast 70S ribosome, the centriole, and especially the flagella, for which 32 phosphoproteins were identified. The high yield of phosphoproteins of the latter correlates well with the presence of several flagellar kinases and indicates that phosphorylation/dephosphorylation represents one of the key regulatory mechanisms of eukaryotic cilia. Our data also provide new insights into certain cilium-related mammalian diseases.     

Motif-specific sampling of phosphoproteomes

Phosphoproteomics, the targeted study of a subfraction of the proteome which is modified by phosphorylation, has become an indispensable tool to study cell signaling dynamics. We described a methodology that linked phosphoproteome and proteome analysis based on Ba2+ binding properties of amino acids. This technology selected motif-specific phosphopeptides independent of the system under analysis. MudPIT (Multidimensional Identification Technology) identified 1037 precipitated phosphopeptides from as little as 250 microg of proteins. To extend coverage of the phosphoproteome, we sampled the nuclear extract of HeLa cells with three values of Ba2+ ions molarity. The presence of more than 70% of identified phosphoproteins was further substantiated by their nonmodified peptides. Upon isoproterenol stimulation of HEK cells, we identified an increasing number of phosphoproteins from MAPK cascades and AKAP signaling hubs. We quantified changes in both protein and phosphorylation levels of 197 phosphoproteins including a critical kinase, MAPK1. Integration of differential phosphorylation of MAPK1 with knowledge bases constructed modules that correlated well with its role as node in cross-talk of canonical pathways.     

Neuropeptide-Induced Androgen Independence in Prostate Cancer Cells: Roles of Nonreceptor Tyrosine Kinases Etk/Bmx, Src, and Focal Adhesion Kinase

The bombesin/gastrin-releasing peptide (GRP) family of neuropeptides has been implicated in various in vitro and in vivo models of human malignancies including prostate cancers. It was previously shown that bombesin and/or neurotensin (NT) acts as a survival and migratory factor(s) for androgen-independent prostate cancers. However, a role in the transition from an androgen-dependent to -refractory state has not been addressed. In this study, we investigate the biological effects and signal pathways of bombesin and NT on LNCaP, a prostate cancer cell line which requires androgen for growth. We show that both neurotrophic factors can induce LNCaP growth in the absence of androgen. Concurrent transactivation of reporter genes driven by the prostate-specific antigen promoter or a promoter carrying an androgen-responsive element (ARE) indicate that growth stimulation is accompanied by androgen receptor (AR) activation. Furthermore, neurotrophic factor-induced gene activation was also present in PC3 cells transfected with the AR but not in the parental line which lacks the AR. Given that bombesin does not directly bind to the AR and is known to engage a G-protein-coupled receptor, we investigated downstream signaling events that could possibly interact with the AR pathway. We found that three nonreceptor tyrosine kinases, focal adhesion kinase (FAK), Src, and Etk/BMX play important parts in this process. Etk/Bmx activation requires FAK and Src and is critical for neurotrophic factor-induced growth, as LNCaP cells transfected with a dominant-negative Etk/BMX fail to respond to bombesin. Etk's activation requires FAK, Src, but not phosphatidylinositol 3-kinase. Likewise, bombesin-induced AR activation is inhibited by the dominant-negative mutant of either Src or FAK. Thus, in addition to defining a new G-protein pathway, this report makes the following points regarding prostate cancer. (i) Neurotrophic factors can activate the AR, thus circumventing the normal growth inhibition caused by androgen ablation. (ii) Tyrosine kinases are involved in neurotrophic factor-mediated AR activation and, as such, may serve as targets of future therapeutics, to be used in conjunction with current antihormone and antineuropeptide therapies.     

5alpha-androstane-3alpha,17beta-diol selectively activates the canonical PI3K/AKT pathway: a bioinformatics-based evidence for androgen-activated cytoplasmic signaling

5alpha-Androstane-3alpha,17beta-diol (3alpha-diol) is reduced from the potent androgen, 5alpha-dihydrotestosterone (5alpha-DHT), by reductive 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs) in the prostate. 3alpha-diol is recognized as a weak androgen with low affinity toward the androgen receptor (AR), but can be oxidized back to 5alpha-DHT. However, 3alpha-diol may have potent effects by activating cytoplasmic signaling pathways, stimulating AR-independent prostate cell growth, and, more importantly, providing a key signal for androgen-independent prostate cancer progression. A cancer-specific, cDNA-based membrane array was used to determine 3alpha-diol-activated pathways in regulating prostate cancer cell survival and/or proliferation. Several canonical pathways appeared to be affected by 3alpha-diol-regulated responses in LNCaP cells; among them are apoptosis signaling, PI3K/AKT signaling, and death receptor signaling pathways. Biological analysis confirmed that 3alpha-diol stimulates AKT activation; and the AKT pathway can be activated independent of the classical AR signaling. These observations sustained our previous observations that 3alpha-diol continues to support prostate cell survival and proliferation regardless the status of the AR. We provided the first systems biology approach to demonstrate that 3alpha-diol-activated cytoplasmic signaling pathways are important components of androgen-activated biological functions in human prostate cells. Based on the observations that levels of reductive 3alpha-HSD expression are significantly elevated in localized and advanced prostate cancer, 3alpha-diol may, therefore, play a critical role for the transition from androgen-dependent to androgen-independent prostate cancer in the presence of androgen deprivation.     

5α-androstane-3α,17β-diol selectively activates the canonical PI3K/AKT pathway: a bioinformatics-based evidence for androgen-activated cytoplasmic signaling

5α-Androstane-3α,17β-diol (3α-diol) is reduced from the potent androgen, 5α-dihydrotestosterone (5α-DHT), by reductive 3α-hydroxysteroid dehydrogenases (3α-HSDs) in the prostate. 3α-diol is recognized as a weak androgen with low affinity toward the androgen receptor (AR), but can be oxidized back to 5α-DHT. However, 3α-diol may have potent effects by activating cytoplasmic signaling pathways, stimulating AR-independent prostate cell growth, and, more importantly, providing a key signal for androgen-independent prostate cancer progression. A cancer-specific, cDNA-based membrane array was used to determine 3α-diol-activated pathways in regulating prostate cancer cell survival and/or proliferation. Several canonical pathways appeared to be affected by 3α-diol-regulated responses in LNCaP cells; among them are apoptosis signaling, PI3K/AKT signaling, and death receptor signaling pathways. Biological analysis confirmed that 3α-diol stimulates AKT activation; and the AKT pathway can be activated independent of the classical AR signaling. These observations sustained our previous observations that 3α-diol continues to support prostate cell survival and proliferation regardless the status of the AR. We provided the first systems biology approach to demonstrate that 3α-diol-activated cytoplasmic signaling pathways are important components of androgen-activated biological functions in human prostate cells. Based on the observations that levels of reductive 3α-HSD expression are significantly elevated in localized and advanced prostate cancer, 3α-diol may, therefore, play a critical role for the transition from androgen-dependent to androgen-independent prostate cancer in the presence of androgen deprivation.