Large scale phosphoproteome analysis of LNCaP human prostate cancer cells

Abstract

Prostate cancer is the most frequently diagnosed cancer among men in the western world. The androgen receptor, a phosphoprotein, is suspected to be involved in all stages of the prostate cancer. Androgen receptor activity can be modulated by various kinases such as PKA, MAPK, AKT, and Src. Phosphorylation is an important post-translational modification and serves as a molecular on/off switch to regulate signaling. Disruptions of cellular phosphorylation are associated with various diseases such as cancer and kinases provide important drug targets. Here we present an analysis of the phosphoproteome in LNCaP human prostate cancer cells. The analytical strategy employed used proteomics based methodologies with a combination of detergent and chaotropic reagent during trypsin digestion followed by titanium dioxide enrichment of phosphopeptides. Over the course of multiple analyses by mass spectrometry we identified a total of 746 phosphorylation sites in 540 phosphopeptides corresponding to 116 phosphoproteins, of which 56 have not been previously reported. Phosphoproteins identified included transcription factors, co-regulators of the androgen receptor, and cancer-related proteins that include β-catenin, USP10, and histone deacetylase-2. The information of signaling pathways, motifs of phosphorylated peptides, biological processes, molecular functions, cellular components, and protein interactions from the identified phosphoproteins established a map of phosphoproteome and signaling pathways in LNCaP cells.

Structure of Phosphorylated SF1 Bound to U2AF65 in an Essential Splicing Factor Complex

“…Subsequent phospho-proteome analyses of HeLa (Beausoleil et al, 2004), lymphoma (Shu et al, 2004), and prostate cancer cells (Myung and Sadar, 2012) confirm the prevalence of SF1 SPSP phosphorylation and suggest a role in cancer initiation or progression. A kinase called Kinase Interacting with Stathmin (KIS), or UHMK1, specifically phosphorylates SF1 serines at position 80 and 82 within the SPSP motif (Manceau et al, 2008; Manceau et al, 2006).…”

Fibronectin induces MMP2 expression in human prostate cancer cells

“…Taken together, all these reports highlight the importance of the cell-ECM interactions for studying transcriptomes and proteomes from both normal and tumoral cells, at in vitro systems [3][4][5][8][9][10]13,[16][17][18]. In this sense, future investigators should be aware that RWPE-1, LNCaP and PC-3 cells, and most likely other tumor cells, require cell-ECM interaction in order to express detectable levels of MMPs on their CM.…”

“…Castration-resistant prostate cancer (CRPC) is the most advanced form of prostate cancer exhibiting reduced survival, with reported values varying between 9 and 30 months [2]. In this sense, in vitro culture systems are widely employed in prostate cancer research and, more recently, for transcriptome and proteomic analysis because the cultures reproduce rapidly and readily [3]. Although these monolayer cultures provide rapid and important results, they recreate only a fraction of a complex scenario [4].…”

Impact of Phosphoproteomics in the Era of Precision Medicine for Prostate Cancer

“…Other studies using the parental LNCaP PrCa cell line identified phosphopeptides corresponding to several actionable kinase targets. These included BRAF, PIK3C2G, STK39, CDK1, MAPK2, AKT1, PRKD1, casein kinase 1 and 2 (CK2A1, CK2A2), and glycogen synthase kinase 3 (GSK3B) ( 58 – 60 ).…”