6 research outputs found
Development Of Tools For Phosphosite-Specific Kinase Identification And Discovery Of Phosphatase Substrates
Phosphorylation is a ubiquitous post translational modification implicated in many diseases, such as cancer. The phosphorylation status of cellular proteins is regulated by the activity of kinases and phosphatases. The biological significance of many phosphorylation events remain unknown because the methods to determine which kinase or phosphatase is responsible for phosphorylation are limited. Previously, we established kinase-catalyzed labeling where kinases accept Îł-modified ATP analogs, such as ATP-arylazide and ATP-biotin, to label phosphoproteins. To study substrates of kinases and phosphatases, here we developed two new methods using kinase-catalyzed labeling. As one application, we developed K-CLASP (Kinase-catalyzed CrossLinking And Streptavdin Purification) to identify the in-cellulo kinase of a phosphorylated site on a protein. In this case, we used ATP-arylazide to mediate crosslinking between a biotin tagged peptide carrying a phosphosite of interest and the respective kinase. Using Protein kinase A (PKA) and its known peptide substrate kemptide, we demonstrated that K-CLASP is capable of identifying PKA as the kinase responsible for kemptide phosphorylation in cell lysates. Then we used K-CLASP to identify the kinases that phosphorylate S178 of the Miz1 protein in a collaboration project. For phosphatase substrate identification, we developed K-BIPS (Kinase-catalyzed Biotinylation to Identify Phosphatase Substrates). In prior work, we observed that labeling of phosphoproteins by ATP-biotin is reduced when phosphatases are inactive. The phosphatase dependency of biotinylation is due to the presence of already existing phosphorylation, which prevents ATP-biotin labeling. Therefore, in K-BIPS, ATP-biotin labeling is carried out after the inactivation of a particular phosphatase. The loss in biotinylation can then be analyzed to reveal substrates. To establish K-BIPS as a viable method, we carried out ATP-biotin labeling in lysates treated with the general phosphatase inhibitor okadaic acid. Many known phosphatase substrates were observed validating our method. Then as further applications, we used K-BIPS to explore substrates of PP1-Gadd34 and PP1-MYPT1 phosphatase complexes. The results demonstrate that K-BIS-Phos is a feasible method for phosphatase substrate identification. In summary, we have developed two chemical tools based on kinase-catalyzed labeling that will enhance our understanding of phosphorylation events mediated by kinases and phosphatases
K‑CLASP: A Tool to Identify Phosphosite Specific Kinases and Interacting Proteins
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
Clonal evaluation of prostate cancer molecular heterogeneity in biopsy samples by dual immunohistochemistry and dual RNA in situ hybridization
Prostate cancer is frequently multifocal. Although there may be morphological variation, the genetic underpinnings of each tumor are not clearly understood. To assess the inter and intra tumor molecular heterogeneity in prostate biopsy samples, we developed a combined immunohistochemistry and RNA in situ hybridization method for the simultaneous evaluation of ERG, SPINK1, ETV1, and ETV4. Screening of 601 biopsy cores from 120 consecutive patients revealed multiple alterations in a mutually exclusive manner in 37% of patients, suggesting multifocal tumors with considerable genetic differences. Furthermore, the incidence of molecular heterogeneity was higher in African Americans patients compared with Caucasian American patients. About 47% of the biopsy cores with discontinuous tumor foci showed clonal differences with distinct molecular aberrations. ERG positivity occurred in low-grade cancer, whereas ETV4 expression was observed mostly in high-grade cancer. Further studies revealed correlation between the incidence of molecular markers and clinical and pathologic findings, suggesting potential implications for diagnostic pathology practice, such as defining dominant tumor nodules and discriminating juxtaposed but molecularly different tumors of different grade patterns
Pseudogene Associated Recurrent Gene Fusion in Prostate Cancer.
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 (\u3c50 \u3eyears) 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