Kinase enzymes phosphorylate protein substrates in a highly ordered manner to control cell signaling. Unregulated kinase activity is associated with a variety of disease states, most notably cancer, making the characterization of kinase activity in cellulo critical to understand disease formation. However, the paucity of available tools has prevented a full mapping of the substrates and interacting proteins of kinases involved in cellular function. Recently we developed kinase-catalyzed crosslinking to covalently connect substrate and kinase in a phosphorylation-dependent manner. Here, we report a new method combining kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP) to identify kinase-substrate pairs and kinase-associated proteins. K-CLIP was applied to the substrate p53, which is robustly phosphorylated. Both known and unknown kinases of p53 were isolated from cell lysates using K-CLIP. In follow-up validation studies, MRCKbeta was identified as a new p53 kinase. Beyond kinases, a variety of p53 and kinase-associated proteins were also identified using K-CLIP, which provided a snapshot of cellular interactions. The K-CLIP method represents an immediately useful chemical tool to identify kinase-substrate pairs and multi-proteins complexes in cells, which will embolden cell signaling research and enhance our understanding of kinase activity in normal and disease states.
Phosphorylation is a key post-translational modification in cell signaling, which is regulated by the equilibrium activities of kinases and phosphatases. The biological significance of many phosphorylation events remains poorly characterized due to the scarcity of tools to discover phosphatases substrates. In prior work, we established kinase-catalyzed biotinylation where kinases accept the γ-modified ATP analog, ATP-biotin, to label phosphoproteins. Here, we developed a novel method to study substrates of phosphatases using kinase-catalyzed biotinylation termed K-BIPS (Kinase-catalyzed Biotinylation to Identify Phosphatase Substrates). In a proof-of-concept experiment, K-BIPS was initially used to explore the substrates of phosphatases inhibited by okadaic acid. Many known phosphatase substrates were observed, confirming K-BIPS as a valid phosphatase substrate identification tool. Then, as a further application, K-BIPS was used to discover the substrates of the PP1-Gadd34 phosphatase complex in the context of unfolded protein response (UPR). In addition to the known substrate eIF2α, K-BIPS revealed several novel substrates, suggesting a more prominent role for the PP1-Gadd34 complex in UPR than previously appreciated. Overall, the two studies establish K-BIPS as a powerful tool to discover the cellular substrates of phosphatases.
Few methods are available to discover the cellular kinase that phosphorylates a specific amino acid, or phosphosite, on a protein. In addition, identifying the associated proteins bound near a phosphosite during phosphorylation would provide insights into cell biology and signaling. Here, we report K-CLASP (Kinase Catalyzed CrossLinking And Streptavidin Purification) as a method for both phosphosite-specific kinase identification and the discovery of kinase interacting proteins. K-CLASP offers a powerful tool to discover unanticipated protein–protein interactions in phosphorylation-mediated biological events.
We present the functional characterization of a pseudogene associated recurrent gene fusion in prostate cancer. The fusion gene KLK4-KLKP1 is formed by the fusion of the protein coding gene KLK4 with the noncoding pseudogene KLKP1 . Screening of a cohort of 659 patients (380 Caucasian American; 250 African American, and 29 patients from other races) revealed that the KLK4-KLKP1 is expressed in about 32% of prostate cancer patients. Correlative analysis with other ETS gene fusions and SPINK1 revealed a concomitant expression pattern of KLK4-KLKP1 with ERG and a mutually exclusive expression pattern with SPINK1, ETV1, ETV4, and ETV5. Development of an antibody specific to KLK4-KLKP1 fusion protein confirmed the expression of the full-length KLK4-KLKP1 protein in prostate tissues. The in vitro and in vivo functional assays to study the oncogenic properties of KLK4-KLKP1 confirmed its role in cell proliferation, cell invasion, intravasation, and tumor formation. Presence of strong ERG and AR binding sites located at the fusion junction in KLK4-KLKP1 suggests that the fusion gene is regulated by ERG and AR . Correlative analysis of clinical data showed an association of KLK4-KLKP1 with lower preoperative PSA values and in young men (<50 years) with prostate cancer. Screening of patient urine samples showed that KLK4-KLKP1 can be detected noninvasively in urine. Taken together, we present KLK4-KLKP1 as a class of pseudogene associated fusion transcript in cancer with potential applications as a biomarker for routine screening of prostate cancer.
27 Analysis of next generation transcriptome sequencing data of prostate cancer 28 identified a novel gene fusion formed by the fusion of a protein coding gene (KLK4) with a 29 non-coding pseudogene (KLKP1) and expression of its cognate protein. Screening of 659 30 prostate cancer TMA showed about 32% of positive cases predominantly expressed in 31 higher Gleason grade tumors. Concomitant expression with ERG but not with SPINK1 and 32 other ETS fusion positive tumors. Fusion gene expression potentially regulated by AR and 33 ERG. Antibody specific to the KLK4-KLKP1 fusion protein was validated by 34 immunohistochemistry and western blot methods. Oncogenic properties were validated by 35 in vitro and in vivo functional studies. Clinical data analysis shows significant association 36 with prostate cancer in young men and overall survival analysis indicate favorable prognosis. 37 Non-invasive detection in urine samples has been confirmed. Taken together, we present a 38 novel biomarker for routine screening of high Gleason grade prostate cancer at diagnosis. 39 40 SIGNIFICANCE 41We discovered and validated a novel prostate cancer (PCa) specific fusion gene involving 42 a protein coding (KLK4) and a pseudogene (KLKP1) and its cognate protein. The unique feature 43 of this fusion gene is the conversion of the noncoding pseudogene into a protein coding gene and 44 its unique expression only in about 30% of high Gleason grade PCa. Expression of this gene is 45 found to be concomitant in ERG fusion positive prostate cancer but mutually exclusive with 46 SPINK1, ETV1, ETV4 and ETV5 positive tumors. Like other ETS family gene fusions, KLK4-47
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