Phosphoproteomics Analyses to Identify the Candidate Substrates and Signaling Intermediates of the Non-Receptor Tyrosine Kinase, SRMS

SRMS (Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylaton sites) is a non-receptor tyrosine kinase that belongs to the BRK family kinases (BFKs) and is evolutionarily related to the Src family kinases (SFKs). Like SFKs and BFKs, the SRMS protein comprises of two domains involved in protein-protein interactions, namely, the Src-homology 3 domain (SH3) and Src-homology 2 domain (SH2) and one catalytic kinase domain. Unlike members of the BFKs and SFKs, the biochemical and cellular role of SRMS is poorly understood primarily due to the lack of information on the substrates and signaling intermediates regulated by the kinase. Previous biochemical studies have shown that wild type SRMS is enzymatically active and leads to the tyrosine-phosphorylation of several proteins, when expressed exogenously in mammalian cells. These tyrosine-phosphorylated proteins represent the candidate cellular substrates of SRMS which are largely unknown. Further, previous studies have determined that the SRMS protein displays a characteristic punctate cytoplasmic localization pattern in mammalian cells. These SRMS cytoplasmic puncta are uncharacterized and may provide insights into the biochemical and cellular role of the kinase. Here, we utilized mass spectrometry-based quantitative label-free phosphoproteomics to (a) identify the candidate SRMS cellular substrates and (b) candidate signaling intermediates regulated by SRMS, in HEK293 cells expressing ectopic SRMS. Specifically, using a phosphotyrosine enrichment strategy we identified 663 candidate SRMS substrates and consensus substrate-motifs of SRMS. We used customized peptide arrays and performed the high-throughput validation of a subset of the identified candidate SRMS substrates. Further, we independently validated Vimentin and Sam68 as bonafide SRMS substrates. Next, using Titanium dioxide (TiO2)-based phosphopeptide enrichment columns, we identified multiple signaling intermediates of SRMS. Functional gene enrichment analyses revealed several common and unique cellular processes regulated by the candidate SRMS substrates and signaling intermediates. Overall, these studies led to the identification of a significant number of novel and biologically relevant SRMS candidate substrates and signaling intermediates, which mapped to a number of cellular and biological processes primarily involved in cell cycle regulation, apoptosis, RNA processing, DNA repair and protein synthesis. These findings provide an important resource for future mechanistic studies to investigate the cellular and physiological functions of the SRMS. Studies towards characterizing the SRMS cytoplasmic puncta showed that the SRMS punctate structures do not colocalize with some of the major cellular organelles investigated, such as the mitochondria, endoplasmic reticulum, golgi bodies and lysosomes. However, studies investigating the involvement of the SRMS domains in puncta-localization revealed that the SRMS SH2 domain partly regulates this localization pattern. These results highlight the potential role of the SRMS SH2 domain in the localization of SRMS to these cytoplasmic sites and lay important groundwork for future characterization studies

Developmental pathways associated with cancer metastasis: Notch, Wnt, and Hedgehog

Master developmental pathways, such as Notch, Wnt, and Hedgehog, are signaling systems that control proliferation, cell death, motility, migration, and stemness. These systems are not only commonly activated in many solid tumors, where they drive or contribute to cancer initiation, but also in primary and metastatic tumor development. The reactivation of developmental pathways in cancer stroma favors the development of cancer stem cells and allows their maintenance, indicating these signaling pathways as particularly attractive targets for efficient anticancer therapies, especially in advanced primary tumors and metastatic cancers. Metastasis is the worst feature of cancer development. This feature results from a cascade of events emerging from the hijacking of epithelial-mesenchymal transition, angiogenesis, migration, and invasion by transforming cells and is associated with poor survival, drug resistance, and tumor relapse. In the present review, we summarize and discuss experimental data suggesting pivotal roles for developmental pathways in cancer development and metastasis, considering the therapeutic potential. Emerging targeted antimetastatic therapies based on Notch, Wnt, and Hedgehog pathways are also discussed

Global phosphoproteomic analysis identifies SRMS-regulated secondary signaling intermediates

Abstract Background The non-receptor tyrosine kinase, SRMS (Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites) is a member of the BRK family kinases (BFKs) which represents an evolutionarily conserved relative of the Src family kinases (SFKs). Tyrosine kinases are known to regulate a number of cellular processes and pathways via phosphorylating substrate proteins directly and/or by partaking in signaling cross-talks leading to the indirect modulation of various signaling intermediates. In a previous study, we profiled the tyrosine-phosphoproteome of SRMS and identified multiple candidate substrates of the kinase. The broader cellular signaling intermediates of SRMS are unknown. Methods In order to uncover the broader SRMS-regulated phosphoproteome and identify the SRMS-regulated indirect signaling intermediates, we performed label-free global phosphoproteomics analysis on cells expressing wild-type SRMS. Using computational database searching and bioinformatics analyses we characterized the dataset. Results Our analyses identified 60 hyperphosphorylated (phosphoserine/phosphothreonine) proteins mapped from 140 hyperphosphorylated peptides. Bioinfomatics analyses identified a number of significantly enriched biological and cellular processes among which DNA repair pathways were found to be upregulated while apoptotic pathways were found to be downregulated. Analyses of motifs derived from the upregulated phosphosites identified Casein kinase 2 alpha (CK2α) as one of the major potential kinases contributing to the SRMS-dependent indirect regulation of signaling intermediates. Conclusions Overall, our phosphoproteomics analyses identified serine/threonine phosphorylation dynamics as important secondary events of the SRMS-regulated phosphoproteome with implications in the regulation of cellular and biological processes

BRK targets Dok1 for ubiquitin-mediated proteasomal degradation to promote cell proliferation and migration.

Breast tumor kinase (BRK), also known as protein tyrosine kinase 6 (PTK6), is a non-receptor tyrosine kinase overexpressed in more that 60% of human breast carcinomas. The overexpression of BRK has been shown to sensitize mammary epithelial cells to mitogenic signaling and to promote cell proliferation and tumor formation. The molecular mechanisms of BRK have been unveiled by the identification and characterization of BRK target proteins. Downstream of tyrosine kinases 1 or Dok1 is a scaffolding protein and a substrate of several tyrosine kinases. Herein we show that BRK interacts with and phosphorylates Dok1 specifically on Y362. We demonstrate that this phosphorylation by BRK significantly downregulates Dok1 in a ubiquitin-proteasome-mediated mechanism. Together, these results suggest a novel mechanism of action of BRK in the promotion of tumor formation, which involves the targeting of tumor suppressor Dok1 for degradation through the ubiquitin proteasomal pathway

BRK and Dok1 are differentially overexpressed in the human breast cancer cell lines.

<p>(A) Cellular proteins were detected in total cell lysates by immunoblotting analysis with anti-Dok1 and anti-BRK antibodies. β-tubulin expression served as a loading control. (B & C) SKBR3 and BT20 cells were fractionated into the cytosolic, membrane, nuclear and cytoskeleton fractions and subjected to immunoblotting analysis for the detection of BRK and Dok1. β-tubulin and Sam68 were used as controls for the cytosolic/membrane and nuclear compartments, respectively. (D) Stable BRK knockdown was performed on parental breast cancer cell lines SKBR3 using shRNA lentiviral vector plasmids against BRK and analyzed as indicated.</p

Activated BRK downregulates Dok1 by reducing its stability.

<p>(A) HEK 293 cells or HEK 293-BRK-YF stable cell line were treated with a protein synthesis inhibitor cyclohexamide (CHX: 200 µg/ml) for the indicated time points and then the cells were lysed and analyzed by immunoblotting for Dok1, BRK and β-tubulin as a loading control. (B) HEK 293 cells were stably transduced with HEK293-BRK-YF and treated with either a proteosome inhibitor MG132 (10 µM) or the vehicle DMSO as the control, at different time points (above the plot). Cellular proteins were determined in total cell lysates by immunoblotting analysis with anti-Dok1, anti-BRK, anti-phosphotyrosine antibodies. β-tubulin was used as a loading control. (C) Empty vector or V-Src was transiently transfected into HEK293 cells and the cells treated with a proteosome inhibitor MG132 (10 µM) and vehicle control DMSO for the indicated time points. Immunoblotting analysis of total cell lysates was performed to detect Dok1, v-Src, phosphotyrosines and β-tubulin served as a loading control. (D & E) HEK 293 cells were transfected with empty control vector or BRK-YF or v-Src and treated with MG132 (10 µM) and Lactacystin (5 µM) or control vehicle for 8 hours. Then the cell lysates were subjected to immunoblot analysis with anti-Dok1 antibody. β-tubulin as a loading control. (F) HEK293-BRK-YF stable cells were transiently cotransfected with Dok1 and HA-Ubiquitin plasmids and after 12 hours the cells were treated MG132 (10 µM) for an additional 8 hours. The total cell lysates were subjected to immunoprecipitation with anti-Dok1 followed by immunoblotting analysis with anti-HA and anti-Dok1 antibodies. The inputs were analysed as indicated.</p

BRK interacts with Dok1 through the SH3 domain <i>in vivo</i> and <i>in vitro</i>.

<p>(A) HEK 293 cells were transfected with empty vector, Myc-BRK-WT, Myc-BRK-YF, GFP-Dok1 or co-transfected with Myc-BRK-WT/GFP-Dok1 or Myc-BRK-YF/GFP-Dok1 and subjected to immunoprecipitation with anti-Dok1 and immunoblotted with BRK and Dok1 (top 2 panels). The expression of cellular proteins was determined in total cell lysates by immunoblotting for GFP, BRK and β-tubulin as loading control. (B) BRK was immunoprecipitated with anti-BRK and subjected to immunoblotting analysis with anti-phosphotyrosine, anti-Dok1 and anti-BRK antibodies (top panels). Total cell lysates indicate the expression of BRK and Dok1 proteins. (C &D) HEK 293 cells were transfected with GFP-Dok1 alone or cotransfected with the idicated mutants of BRK and subjected to immunoprecipitation with anti-Dok1 followed by immunoblotting analysis with anti-BRK and anti-Dok1 antibodies. The cellular proteins were determined from the total cell lysates by immunoblotting analysis with anti-BRK and anti-Dok1 antibodies. (E) Overexpressed GFP-Dok1 or GFP-Dok1-Y362F in HEK 293 cell lysates from GFP-Dok1 or GFP-Dok1-Y362F expressing cells were subjected to pull-down assays with GST alone or recombinant GST-SH3 or GST-SH2 domain of BRK and immunoblotting analysis was performed with anti-Dok1 antibody. (F) GFP-Dok1/BRK-YF or GFP-Dok1-Y362F/BRK-YF cotransfected cohorts of HEK 293 cell lysates were subjected to pull-down assays with GST alone or GST-SH3 or GST-SH2 domain of BRK followed by immunoblotting with anti-Dok1 antibody. (G) Bacterially expressed GST, GST-SH3 and GST-SH2 domain of BRK proteins were detected via Coomassie blue staining.</p

Constitutively active BRK phosphorylates Dok1 at Y362.

<p>(A) Schematic diagram of Dok1 showing different deletion and point mutants. (B) The Dok1 deletion mutants and BRK-YF were co-transfected in to HEK 293 cells, the cell were then subjected to immunoprecipitation with anti-GFP antibody followed by immunobloting analysis using anti-phosphotyrosine and anti-GFP antibodies (top panel). Lower panel shows the expression of different GFP-Dok1 deletion mutants, BRK (as input) and β-tubulin as a loading control. (C) Dok1 deletion mutants were transfected either alone or with BRK-YF into HEK 293 cells, the cell lysates were then subjected to immunobloting analysis using antibodies against Dok1, phosphotyrosine, BRK and β-tubulin as loading control. (D) HEK 293 cells were co-transfected with Dok1 point mutants and BRK-YF followed by immunoprecipitation with anti-Dok1 antibody and immunoblotting analysis using anti-phosphotyrosines and anti-Dok1 antibodies. Lower panel shows the expression of BRK, GFP-Dok1 mutants (as input) and β-tubulin as a loading control. (E) HEK 293 cells were cotransfected with BRK-YF and Dok1 point mutants or transfected with BRK-YF alone. Total cell lysates were analyzed by immunoblotting analysis with antibodies against phosphotyrosines, BRK, Dok1 and β-tubulin as loading control.</p

Dok1 inhibits BRK-induced cell proliferation and migration.

<p>(A) HEK 293 stable sub-cell lines were transduced with mCherry-Dok1 using adenoviral vector. Cellular proteins were detected in total cell lysates by immunoblotting analysis with anti-BRK, anti-Dok1, and anti-phosphotyrosine antibodies. β-tubulin served as a loading control. (B & C) HEK 293 stable cells were transduced with or without mCherry-Dok1adeno-vector and were monitored for cell proliferation. (D & E) Cell migration determined by the healing of a fixed wound area induced in the different HEK 293 stable transfectant cells. The percentage of open area at 24 h is plotted. (F & G) Cell migration analysis was performed with the indicated stable cell lines expressing mCherry-Dok1 or an empty vector. The assay was based on the rate of wound closure in the scratched cells. The percentage of open area at 24 hours is plotted. The migration assay was performed in three independent experiments. Data are means ± standard errors. Statistics: and **<i>P</i>≥0.001 and ***<i>P</i>≥0.0001.</p

Mesenchymal stromal cells’ role in tumor microenvironment: involvement of signaling pathways

International audienceMesenchymal stromal cells (MSCs) are adult multipotent stem cells residing as pericytes in various tissues and organs where they can differentiate into specialized cells to replace dying cells and damaged tissues. These cells are commonly found at injury sites and in tumors that are known to behave like " wounds that do not heal." In this article, we discuss the mechanisms of MSCs in migrating, homing, and repairing injured tissues. We also review a number of reports showing that tumor microenvironment triggers plasticity mechanisms in MSCs to induce malignant neoplastic tissue formation, maintenance, and chemoresistance, as well as tumor growth. The antitumor properties and therapeutic potential of MSCs are also discussed