Characterization of Kinase Expression Related to Increased Migration of PC-3M Cells Using Global Comparative Phosphoproteome Analysis

Abstract

Background/aim: Prostate cancer (PCa) is the second-most commonly occurring cancer among men, worldwide. Although the mechanisms associated with the progression of castration-resistant prostate cancer (CRPC) have been widely studied, the mechanism associated with more distant metastases from the bone remains unknown. This study aimed to characterize potential pathogenic kinases associated with highly metastatic PCa, that may regulate phosphorylation in extensively involved and diverse signaling pathways that are associated with the development of various cancers.

Materials and methods: A mass spectrometry (MS)-based comparative phosphoproteome strategy was utilized to identify differentially expressed kinases between the highly aggressive PCa cell-lines PC-3 and PC-3M.

Results: Among 2,968 phosphorylation sites in PCa cells, 151 differently expressed phosphoproteins were identified. Seven motifs: -SP-, -SxxE-, -PxS-, -PxSP-, -SxxK-, -SPxK-, and -SxxxxxP- were found to be highly expressed in PC-3M cells. Based on these motifs, the kinases p21-activated kinase (PAK)2, Ste20-like kinase (SLK), mammalian Ste20-like kinase (MST)4, mitogen-activated kinase kinase (MAP2K)2, and A-Raf proto-oncogene serine/threonine kinase (ARAF) were up-regulated in PC-3M cells.

Conclusion: PAK2, SLK, MST4, MAP2K2, and ARAF are kinases that are potentially associated with the progression of increased migration in PC-3M cells and may represent molecule regulators or drug targets for highly metastatic PCa therapy.

Keywords: Metastatic prostate cancer; kinases; phosphorylation; proteomics.

Figure 1. Screening of phosphorylation expression patterns in PCa cells by western blotting analysis. (A) Protein expression profiling in PC-3 and PC-3M cells by western blotting analysis. Ten micrograms of protein were loaded into lanes 1-4. Lanes 1-2: duplicate loading of protein extracts from PC-3 cells, lanes 3-4: duplication loading from PC-3M cells. (B) Phosphorylation validation in PC-3 and PC-3M cells, by western blotting analysis. Phosphorylation patterns for the amino acids serine (Ser), threonine (Thr), tyrosine (Tyr), and histidine (His) were detected using corresponding antibodies.

Figure 2. MS-based quantitative proteomic profiling of phosphorylation in PCa. (A) Experimental scheme for quantitative proteomic analysis. PC- 3 (K4R6) and PC-3M (K0R0) were lysed and mixed at equal protein amounts, and tryptic digestion was performed. Phosphorylated peptides were affinity purified using a TiO2 column, and eluted peptides were injected into the nano-LC-MS/MS system. MS spectra were searched in MaxQuant (version 1.5). (B) Overview of the frequency of differentially expressed phosphorylation from proteomics analysis. (C) Correlation of technically replication of MS data.

Figure 3. DAVID-generated GO enrichment and KEGG pathway analysis of DEPs. (A) Up-regulated phosphoproteins and (B) down-regulated phosphoproteins. The –log of Fisher’s exact test was used to represent the enrichment index. GOBP: Gene Ontology Biological Process, CC: cellular component, MF: molecular function, KEGG: Kyoto Encyclopedia of Genes and Genomes.

Figure 4. Differentially expressed motifs in PCa. (A) Twenty-four extensively enriched motifs were identified among the phosphosites, including 18 pSer and 6 pThr motifs. Percentage of foreground matches was used as the enrichment index. (B) Differentially expressed motifs between PC-3 and PC-3M cells. Seven motifs -SxxE-, -PxS-, -PxSP-, -SxxK-, -SPxK-, -SxxxxxP- and -SP-, were up-regulated in PC-3M cells. The motifs -RxxS- and - SP- were down-regulated. (Motif -SP- was enriched among both up- and down-regulated phosphosites).

Figure 5. Protein–-protein network (PPN) of the upstream kinases in highly metastatic PCa cell. (A) A total of 195 kinase-substrate interactions were predicted from the up-regulated phosphosites. (B) A total of 16 overlapping kinases between phosphosite-predicated and the global proteome experiment were detected. Five kinases (*) are identified as novel kinases regulators during the progression to highly metastatic PCa. (C) PPN of the 16 overlapping kinases. PPN was mapped through the STRING database, with a high confidence value of 0.9.