An Integrated Phosphoproteomics Work Flow Reveals Extensive Network Regulation in Early Lysophosphatidic Acid Signaling*

Lysophosphatidic acid (LPA) induces a variety of cellular signaling pathways through the activation of its cognate G protein-coupled receptors. To investigate early LPA responses and assess the contribution of epidermal growth factor (EGF) receptor transactivation in LPA signaling, we performed phosphoproteomics analyses of both total cell lysate and protein kinase-enriched fractions as complementary strategies to monitor phosphorylation changes in A498 kidney carcinoma cells. Our integrated work flow enabled the identification and quantification of more than 5,300 phosphorylation sites of which 224 were consistently regulated by LPA. In addition to induced phosphorylation events, we also obtained evidence for early dephosphorylation reactions due to rapid phosphatase regulation upon LPA treatment. Phosphorylation changes induced by direct heparin-binding EGF-like growth factor-mediated EGF receptor activation were typically weaker and only detected on a subset of LPA-regulated sites, indicating signal integration among EGF receptor transactivation and other LPA-triggered pathways. Our results reveal rapid phosphoregulation of many proteins not yet implicated in G protein-coupled receptor signaling and point to various additional mechanisms by which LPA might regulate cell survival and migration as well as gene transcription on the molecular level. Moreover, our phosphoproteomics analysis of both total lysate and kinase-enriched fractions provided highly complementary parts of the LPA-regulated signaling network and thus represents a useful and generic strategy toward comprehensive signaling studies on a system-wide level.

[1]  Jens Timmer,et al.  Systems-level interactions between insulin–EGF networks amplify mitogenic signaling , 2009, Molecular systems biology.

[2]  J. Westendorf,et al.  Bone Morphogenic Protein 2 Activates Protein Kinase D to Regulate Histone Deacetylase 7 Localization and Repression of Runx2* , 2009, Journal of Biological Chemistry.

[3]  Christian von Mering,et al.  STRING 8—a global view on proteins and their functional interactions in 630 organisms , 2008, Nucleic Acids Res..

[4]  R. Gniadecki,et al.  Internalization of EGF receptor following lipid rafts disruption in keratinocytes is delayed and dependent on p38 MAPK activation , 2008, Journal of cellular physiology.

[5]  M. Mann,et al.  MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification , 2008, Nature Biotechnology.

[6]  Ronald J. Moore,et al.  Quantitative phosphoproteome analysis of lysophosphatidic acid induced chemotaxis applying dual-step (18)O labeling coupled with immobilized metal-ion affinity chromatography. , 2008, Journal of proteome research.

[7]  J. Aoki,et al.  Two pathways for lysophosphatidic acid production. , 2008, Biochimica et biophysica acta.

[8]  M. Mann,et al.  Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle. , 2008, Molecular cell.

[9]  S. Gibson,et al.  Lysophosphatidic Acid Protects Cancer Cells from Histone Deacetylase (HDAC) Inhibitor-induced Apoptosis through Activation of HDAC* , 2008, Journal of Biological Chemistry.

[10]  Jian Zhang,et al.  Membrane association and functional regulation of Sec3 by phospholipids and Cdc42 , 2008, The Journal of cell biology.

[11]  V. Olkkonen,et al.  Characteristics of oxysterol binding proteins. , 2008, International review of cytology.

[12]  E. Rozengurt.  Mitogenic signaling pathways induced by G protein‐coupled receptors , 2007, Journal of cellular physiology.

[13]  Michael L. Creech,et al.  Integration of biological networks and gene expression data using Cytoscape , 2007, Nature Protocols.

[14]  Brad T. Sherman,et al.  The DAVID Gene Functional Classification Tool: a novel biological module-centric algorithm to functionally analyze large gene lists , 2007, Genome Biology.

[15]  Bernhard Kuster,et al.  Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors , 2007, Nature Biotechnology.

[16]  S. Iovino,et al.  Phorbol Esters Induce Intracellular Accumulation of the Anti-apoptotic Protein PED/PEA-15 by Preventing Ubiquitinylation and Proteasomal Degradation* , 2007, Journal of Biological Chemistry.

[17]  Guido Dieterich,et al.  Proteomics Analysis of Protein Kinases by Target Class-selective Prefractionation and Tandem Mass Spectrometry *S , 2007, Molecular & Cellular Proteomics.

[18]  W. Moolenaar,et al.  Regulation and biological activities of the autotaxin-LPA axis. , 2007, Progress in lipid research.

[19]  M. Mann,et al.  In-gel digestion for mass spectrometric characterization of proteins and proteomes , 2006, Nature Protocols.

[20]  M. Mann,et al.  Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips , 2007, Nature Protocols.

[21]  Matthias Mann,et al.  Innovations: Functional and quantitative proteomics using SILAC , 2006, Nature Reviews Molecular Cell Biology.

[22]  C. Isacke,et al.  Directional sensing of a phorbol ester gradient requires CD44 and is regulated by CD44 phosphorylation , 2006, Oncogene.

[23]  M. Mann,et al.  Global, In Vivo, and Site-Specific Phosphorylation Dynamics in Signaling Networks , 2006, Cell.

[24]  G. Mills,et al.  Phosphorylation of TNF-alpha converting enzyme by gastrin-releasing peptide induces amphiregulin release and EGF receptor activation. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[25]  S. Mohammed,et al.  Quantitative proteomics identifies Gemin5, a scaffolding protein involved in ribonucleoprotein assembly, as a novel partner for eukaryotic initiation factor 4E. , 2006, Journal of proteome research.

[26]  P. Altevogt,et al.  A role for exosomes in the constitutive and stimulus-induced ectodomain cleavage of L1 and CD44. , 2006, The Biochemical journal.

[27]  A. Dubin,et al.  Emerging medicinal roles for lysophospholipid signaling. , 2006, Trends in molecular medicine.

[28]  Y. Pekarsky,et al.  Akt phosphorylates and regulates Pdcd4 tumor suppressor protein. , 2005, Cancer research.

[29]  M. Mann,et al.  Parts per Million Mass Accuracy on an Orbitrap Mass Spectrometer via Lock Mass Injection into a C-trap*S , 2005, Molecular & Cellular Proteomics.

[30]  A. Ullrich,et al.  GPCR-induced migration of breast carcinoma cells depends on both EGFR signal transactivation and EGFR-independent pathways , 2005, Biological chemistry.

[31]  Steven P Gygi,et al.  Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations , 2005, Nature Methods.

[32]  M. Mifune,et al.  G Protein Coupling and Second Messenger Generation Are Indispensable for Metalloprotease-dependent, Heparin-binding Epidermal Growth Factor Shedding through Angiotensin II Type-1 Receptor* , 2005, Journal of Biological Chemistry.

[33]  P. Roepstorff,et al.  Highly Selective Enrichment of Phosphorylated Peptides from Peptide Mixtures Using Titanium Dioxide Microcolumns* , 2005, Molecular & Cellular Proteomics.

[34]  Wei Zhang,et al.  Protein Kinase D Specifically Mediates Apoptosis Signal-regulating Kinase 1-JNK Signaling Induced by H2O2 but Not Tumor Necrosis Factor* , 2005, Journal of Biological Chemistry.

[35]  E. Olson,et al.  Protein Kinase D1 Phosphorylates HDAC7 and Induces Its Nuclear Export after T-cell Receptor Activation* , 2005, Journal of Biological Chemistry.

[36]  F. Dequiedt,et al.  Phosphorylation of histone deacetylase 7 by protein kinase D mediates T cell receptor–induced Nur77 expression and apoptosis , 2005, The Journal of experimental medicine.

[37]  György Kéri,et al.  Cellular targets of gefitinib. , 2005, Cancer research.

[38]  J. Brown,et al.  G protein mediated signaling pathways in lysophospholipid induced cell proliferation and survival , 2004, Journal of cellular biochemistry.

[39]  B. de Strooper,et al.  Cell–matrix interaction via CD44 is independently regulated by different metalloproteinases activated in response to extracellular Ca2+ influx and PKC activation , 2004, The Journal of cell biology.

[40]  M. Mandai,et al.  Requirement for Tec Kinases in Chemokine-Induced Migration and Activation of Cdc42 and Rac , 2004, Current Biology.

[41]  J. Shabanowitz,et al.  A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry. , 2004, Analytical chemistry.

[42]  P. Borchmann,et al.  Depletion of Cellular Cholesterol and Lipid Rafts Increases Shedding of CD301 , 2004, The Journal of Immunology.

[43]  N. Hay,et al.  Inhibition of Chk1 by Activated PKB/Akt , 2004, Cell cycle.

[44]  A. Ullrich,et al.  Mutual Regulation of Protein-tyrosine Phosphatase 20 and Protein-tyrosine Kinase Tec Activities by Tyrosine Phosphorylation and Dephosphorylation* , 2004, Journal of Biological Chemistry.

[45]  D. Toksoz,et al.  Role of Lbc RhoGEF in Gα12/13-induced signals to Rho GTPase , 2004 .

[46]  A. Ullrich,et al.  Multiple G-protein-coupled receptor signals converge on the epidermal growth factor receptor to promote migration and invasion , 2004, Oncogene.

[47]  D. Toksoz,et al.  Role of Lbc RhoGEF in Galpha12/13-induced signals to Rho GTPase. , 2004, Cellular signalling.

[48]  A. Ullrich,et al.  EGFR signal transactivation in cancer cells. , 2003, Biochemical Society transactions.

[49]  G. Mills,et al.  The emerging role of lysophosphatidic acid in cancer , 2003, Nature Reviews Cancer.

[50]  G. Condorelli,et al.  Protein Kinase B/Akt Binds and Phosphorylates PED/PEA-15, Stabilizing Its Antiapoptotic Action , 2003, Molecular and Cellular Biology.

[51]  V. Kiermer,et al.  HDAC7, a thymus-specific class II histone deacetylase, regulates Nur77 transcription and TCR-mediated apoptosis. , 2003, Immunity.

[52]  E. Rozengurt,et al.  Cooperation of Gq, Gi, and G12/13 in protein kinase D activation and phosphorylation induced by lysophosphatidic acid. , 2003, The Journal of biological chemistry.

[53]  T. Hunter,et al.  The Protein Kinase Complement of the Human Genome , 2002, Science.

[54]  Z. Werb,et al.  The metalloprotease Kuzbanian (ADAM10) mediates the transactivation of EGF receptor by G protein–coupled receptors , 2002, The Journal of cell biology.

[55]  L. Sherman,et al.  CD44 Associates With EGFR and erbB2 in Metastasizing Mammary Carcinoma Cells , 2002, Applied immunohistochemistry & molecular morphology : AIMM.

[56]  Hiroshi Asanuma,et al.  Cardiac hypertrophy is inhibited by antagonism of ADAM12 processing of HB-EGF: Metalloproteinase inhibitors as a new therapy , 2002, Nature Medicine.

[57]  R. Lefkowitz,et al.  Epidermal Growth Factor (EGF) Receptor-dependent ERK Activation by G Protein-coupled Receptors A CO-CULTURE SYSTEM FOR IDENTIFYING INTERMEDIATES UPSTREAM AND DOWNSTREAM OF HEPARIN-BINDING EGF SHEDDING* , 2001 .

[58]  Cherrington,et al.  SU6668 is a potent antiangiogenic and antitumor agent that induces regression of established tumors. , 2000, Cancer research.

[59]  M. Rudnicki,et al.  Caspase 3 Cleavage of the Ste20-Related Kinase SLK Releases and Activates an Apoptosis-Inducing Kinase Domain and an Actin-Disassembling Region , 2000, Molecular and Cellular Biology.

[60]  A. Ullrich,et al.  EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF , 1999, Nature.

[61]  R. Desnick,et al.  Regulation of rat hepatic 3beta-hydroxysterol delta7-reductase: substrate specificity, competitive and non-competitive inhibition, and phosphorylation/dephosphorylation. , 1998, Journal of lipid research.

[62]  H. Mano,et al.  Tec/Bmx non‐receptor tyrosine kinases are involved in regulation of Rho and serum response factor by Gα12/13 , 1998, The EMBO journal.

[63]  P C Sternweis,et al.  Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Galpha13. , 1998, Science.

[64]  G. Schultz,et al.  The G-protein G13 but Not G12 Mediates Signaling from Lysophosphatidic Acid Receptor via Epidermal Growth Factor Receptor to Rho* , 1998, The Journal of Biological Chemistry.

[65]  A. Ullrich,et al.  Signal characteristics of G protein‐transactivated EGF receptor , 1997, The EMBO journal.

[66]  Anne J. Ridley,et al.  The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors , 1992, Cell.

[67]  Kees Jalink,et al.  Lysophosphatidate-induced cell proliferation: Identification and dissection of signaling pathways mediated by G proteins , 1989, Cell.