Phosphoproteomic analysis of Syk kinase signaling in human cancer cells reveals its role in cell–cell adhesion

Larive, Romain M.; Urbach, Serge; Poncet, Joël; Jouin, Patrick; Mascré, Guilhem; Sahuquet, Alain; Mangeat, Paul; Coopman, Peter J.; Bettache, Nadir

doi:10.1038/onc.2009.99

Cited by 40 publications

(51 citation statements)

References 33 publications

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“…In contrast, Syk appears to play an unusual role in a subset of cancers by restricting their metastatic behavior, but the full repertoire of substrates for Syk, especially in epithelial cells, is not known. A previous study identified a number of proteins potentially phosphorylated on tyrosine in Syk-expressing breast cancer cells, but sites of phosphorylation were not determined (21). Interestingly, the majority of the phosphorylation sites identified in this study were characterized by a high abundance of acidic amino acid residues surrounding the phosphorylated tyrosine, which is consistent with the known substrate specificity of Syk (supplemental Figs.…”

Section: Discussionsupporting

confidence: 71%

In-depth Analyses of Kinase-dependent Tyrosine Phosphoproteomes Based on Metal Ion-functionalized Soluble Nanopolymers

Iliuk

Martin

Alicie

et al. 2010

Molecular & Cellular Proteomics

154

180

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The ability to obtain in-depth understanding of signaling networks in cells is a key objective of systems biology research. Such ability depends largely on unbiased and reproducible analysis of phosphoproteomes. We present here a novel proteomics tool, polymer-based metal ion affinity capture (PolyMAC), for the highly efficient isolation of phosphopeptides to facilitate comprehensive phosphoproteome analyses. This approach uses polyamidoamine dendrimers multifunctionalized with titanium ions and aldehyde groups to allow the chelation and subsequent isolation of phosphopeptides in a homogeneous environment. Compared with current strategies based on solid phase micro-and nanoparticles, PolyMAC demonstrated outstanding reproducibility, exceptional selectivity, fast chelation times, and high phosphopeptide recovery from complex mixtures. Using the PolyMAC method combined with antibody enrichment, we identified 794 unique sites of tyrosine phosphorylation in malignant breast cancer cells, 514 of which are dependent on the expression of Syk, a protein-tyrosine kinase with unusual properties of a tumor suppressor. The superior sensitivity of PolyMAC allowed us to identify novel components in a variety of major signaling networks, including cell migration and apoptosis. PolyMAC offers a powerful and widely applicable tool for phosphoproteomics and molecular signaling. Molecular & Cellular Proteomics 9:2162-2172, 2010.Reversible phosphorylation of proteins is a major mechanism for the regulation of multiple cellular processes (1, 2). Mass spectrometry-based phosphoproteomics provides a method for the global analysis of protein phosphorylation and molecular signaling in cells (3,4). Despite the great progress that has been made over the past few years, the isolation of phosphopeptides and their analysis by mass spectrometry are still a considerable challenge because of the typically low stoichiometry of protein phosphorylation and the resulting low abundance of phosphopeptides. An early step in any phosphoproteome analysis is the isolation of phosphopeptides, preferably with high efficiency, selectivity, sensitivity, and reproducibility. Currently, there are three major strategies for the isolation of phosphopeptides: antibody-based affinity capture, chemical derivatization of phosphoamino acids, and metal ion-based affinity capture. Antibody-based methods are used mainly for the selective isolation of phosphotyrosinecontaining proteins or peptides (5-8). Chemical derivatization methods begin with the ␤-elimination of phosphates from phosphoserine and phosphothreonine (9) or the formation of phosphoramidates by reactions with amines (10) to selectively immobilize phosphopeptides. Metal ion-based affinity capture techniques use immobilized metal affinity chromatography (IMAC) with Fe(III) (11,12) or Ga(III) (13) and, for the past a few years, more successful metal oxide approaches (i.e. TiO 2 (14, 15) and ZrO 2 (16, 17)) for the selective binding of phosphorylated peptides. Almost all of the current isolation methods are b...

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Section: Discussionsupporting

confidence: 71%

In-depth Analyses of Kinase-dependent Tyrosine Phosphoproteomes Based on Metal Ion-functionalized Soluble Nanopolymers

Iliuk

Martin

Alicie

et al. 2010

Molecular & Cellular Proteomics

154

180

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“…Thus, phosphorylation at Thr526 appears to provide a means of regulating proteolysis mediated by the nearby PEST sequences. Since PEST sequences are a conserved feature of Abp1p homologs in fungal and mammalian species and phosphorylation within the PRR is seen in mouse (Larbolette et al 1999;Han et al 2003;Zanivan et al 2008;Larive et al 2009;Boateng et al 2012) and human Abp1 (Olsen et al 2006;Molina et al 2007), we propose that this regulation of Abp1p stability may be a conserved feature of this family. It is possible that the putative intramolecular interaction between the SH3 domain and the PRR, which is supported by our NMR data, is affected by phosphorylation at Thr526 since this site lies between the SH3 domain and the PRR ( Figure 5B).…”

Section: Discussionmentioning

confidence: 99%

“…Since mAbp1 is phosphorylated (Larbolette et al 1999;Han et al 2003;Larive et al 2009;Boateng et al 2012) and several phosphorylated peptides from yeast Abp1p have been identified in large-scale studies (Ficarro et al 2002;Ubersax et al 2003;Holt et al 2009), we sought to further investigate the possible phosphorylation of Abp1p. Interestingly, Abp1p migrates as a double-protein band on SDS-PAGE gels (Drubin et al 1988).…”

Section: Yeast Abp1p Is Phosphorylated Within the Prrmentioning

confidence: 99%

“…A sequence logo, which quantifies conservation at each position in the alignment, is also shown. et al 1999; Han et al 2003;Larive et al 2009;Boateng et al 2012) and phosphorylated peptides from Abp1p have been identified in several global studies of yeast protein phosphorylation (Ficarro et al 2002;Ubersax et al 2003;Chi et al 2007;Holt et al 2009), a role for phosphorylation in the function of Abp1p might be expected.…”

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confidence: 99%

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The Importance of Conserved Features of Yeast Actin-Binding Protein 1 (Abp1p): The Conditional Nature of Essentiality

Garcı́a¹,

Stollar

Davidson

2012

Genetics

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Saccharomyces cerevisiae Actin-Binding Protein 1 (Abp1p) is a member of the Abp1 family of proteins, which are in diverse organisms including fungi, nematodes, flies, and mammals. All proteins in this family possess an N-terminal Actin Depolymerizing Factor Homology (ADF-H) domain, a central Proline-Rich Region (PRR), and a C-terminal SH3 domain. In this study, we employed sequence analysis to identify additional conserved features of the family, including sequences rich in proline, glutamic acid, serine, and threonine amino acids (PEST), which are found in all family members examined, and two motifs, Conserved Fungal Motifs 1 and 2 (CFM1 and CFM2), that are conserved in fungi. We also discovered that, similar to its mammalian homologs, Abp1p is phosphorylated in its PRR. This phosphorylation is mediated by the Cdc28p and Pho85p kinases, and it protects Abp1p from proteolysis mediated by the conserved PEST sequences. We provide evidence for an intramolecular interaction between the PRR region and SH3 domain that may be affected by phosphorylation. Although deletion of CFM1 alone caused no detectable phenotype in any genetic backgrounds or conditions tested, deletion of this motif resulted in a significant reduction of growth when it was combined with a deletion of the ADF-H domain. Importantly, this result demonstrates that deletion of highly conserved domains on its own may produce no phenotype unless the domains are assayed in conjunction with deletions of other functionally important elements within the same protein. Detection of this type of intragenic synthetic lethality provides an important approach for understanding the function of individual protein domains or motifs.

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“…13,15 Focal adhesions are specific types of large protein complexes through which the cytoskeleton of a cell interacts with proteins of the extracellular matrix. In breast cancer cells, SYK inhibits cell motility, while promoting the expression of cell adhesion molecules, such as vinculin, 15 E-cadherin, 16 and tensin2. 17 Although studies have shown that SYK is associated with the actin filament network and focal adhesion kinase (FAK) in hematopoietic cells, 18,19 in non-hematopoietic cells, the relationship between SYK and focal adhesion molecules remains poorly understood.…”

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confidence: 99%

Downregulation of spleen tyrosine kinase in hepatocellular carcinoma by promoter CpG island hypermethylation and its potential role in carcinogenesis

Shin

Kim

Lee

et al. 2014

Laboratory Investigation

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Spleen tyrosine kinase (SYK) has predominantly been studied in hematopoietic cells, where it is involved in immunoreceptor-mediated signaling. However, SYK expression has been shown in numerous non-hematopoietic cells, and its downregulation has been shown to be involved in tumor formation and progression. SYK methylation has been demonstrated to identify a subset of hepatocellular carcinoma (HCC) cases with poor prognosis, but little is known regarding the biological role of SYK in HCC. We found that SYK methylation is a common event in HCC, and is inversely associated with its expression. We established stable HCC cell lines with inducible SYK expression vectors, and compared the differential RNA expression profiles of HCC cell lines with or without the induction of SYK. Gene ontology analysis revealed that the SYK-regulated genes were enriched for genes involved in cell adhesion. Accordingly, we found that the induction of SYK expression increased the adhesion of cells to fibronectin and decreased cell migration and invasion, and that cessation of SYK overexpression increased cell migration and invasion. Our findings suggest that SYK is involved in regulating cell to matrix adhesions, and that SYK loss affects the migration, and invasion of HCC cells. (2014) 94, 1396-1405 doi:10.1038/labinvest.2014 published online 13 October 2014 Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. In men, it ranks the fifth most common cancer worldwide and is the second leading cause of cancer death, with an estimate of 4520 000 new cases each year. 1 The major risk factors associated with the incidence of HCC are well established, and include infection by hepatitis B and hepatitis C viruses, chronic alcoholism, and aflatoxin exposure. 2 However, the molecular carcinogenesis pathways involving the development and progression of HCC remain largely unclear. Like most solid tumors, it has been believed that the progression of HCC occurs as a consequence of a series of genetic and epigenetic alterations. 2 Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that is widely expressed in hematopoietic cells. Recently, SYK expression has been demonstrated in numerous nonhematopoietic cells, and its downregulation has been shown to be involved in tumor formation and progression. [3][4][5][6] The transfection of SYK into an SYK-negative cancer cell line dramatically inhibited its cell growth, migration, and invasion. [3][4][5][6] Conversely, the knockdown of SYK in SYK-positive breast cancer cells increased proliferation and invasion. 7 Several researchers have also reported that the loss of SYK expression correlates with poor survival and tumor metastasis in patients with breast, 8 bladder, 9 liver, 10 pancreas, 5 or stomach cancer. 11 Epigenetic silencing through hypermethylation of promoter CpG islands has been proposed to be involved in the loss of SYK expression in these tumors. [3][4][5][6] Although SYK was shown to affect cell proliferation, motility, and invasion in several types of c...

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Phosphoproteomic analysis of Syk kinase signaling in human cancer cells reveals its role in cell–cell adhesion

Cited by 40 publications

References 33 publications

In-depth Analyses of Kinase-dependent Tyrosine Phosphoproteomes Based on Metal Ion-functionalized Soluble Nanopolymers

In-depth Analyses of Kinase-dependent Tyrosine Phosphoproteomes Based on Metal Ion-functionalized Soluble Nanopolymers

The Importance of Conserved Features of Yeast Actin-Binding Protein 1 (Abp1p): The Conditional Nature of Essentiality

Downregulation of spleen tyrosine kinase in hepatocellular carcinoma by promoter CpG island hypermethylation and its potential role in carcinogenesis

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