this paper is available on line at ... ?· mammalian cell culture ... plant tissue culture ... in...

Download This paper is available on line at ... ?· Mammalian Cell Culture ... Plant Tissue Culture ... In Vitro…

Post on 27-Aug-2018

212 views

Category:

Documents

0 download

Embed Size (px)

TRANSCRIPT

  • Identification of Extracellular Signal-regulatedKinase 1 (ERK1) Direct Substrates using StableIsotope Labeled Kinase Assay-LinkedPhosphoproteomics*S

    Liang Xue, Pengcheng Wang, Pianpian Cao, Jian-kang Zhu,and W. Andy Tao**

    Kinase mediated phosphorylation signaling is extensivelyinvolved in cellular functions and human diseases, andunraveling phosphorylation networks requires the identi-fication of substrates targeted by kinases, which has re-mained challenging. We report here a novel proteomicstrategy to identify the specificity and direct substrates ofkinases by coupling phosphoproteomics with a sensitivestable isotope labeled kinase reaction. A whole cell ex-tract was moderately dephosphorylated and subjected toin vitro kinase reaction under the condition in which 18O-ATP is the phosphate donor. The phosphorylated proteinsare then isolated and identified by mass spectrometry, inwhich the heavy phosphate (85.979 Da) labeled phos-phopeptides reveal the kinase specificity. The in vitrophosphorylated proteins with heavy phosphates are fur-ther overlapped with in vivo kinase-dependent phospho-proteins for the identification of direct substrates withhigh confidence. The strategy allowed us to identify 46phosphorylation sites on 38 direct substrates of extracel-lular signal-regulated kinase 1, including multiple knownsubstrates and novel substrates, highlighting the abilityof this high throughput method for direct kinase substratescreening. Molecular & Cellular Proteomics 13: 10.1074/mcp.O114.038588, 31993210, 2014.

    Protein phosphorylation regulates almost all aspects of celllife, such as cell cycle, migration, and apoptosis (1), andderegulation of protein phosphorylation is one of the mostfrequent causes or consequences of human diseases includ-ing cancers, diabetes, and immune disorders (2). Up till now,however, known substrates are far from saturation for the

    majority of protein kinases (3); thus, mapping comprehensivekinase-substrate relationships is essential to understandingbiological mechanisms and uncovering new drug targets (4).

    Accompanied with advances of high-speed and high-reso-lution mass spectrometry, the technique of kinase substratescreening using proteomic strategy is quickly evolving (57).Mass spectrometry has been extensively used for kinase-substrate interaction mapping (8) and global phosphorylationprofiling (9). Although thousands of phosphorylation siteshave been detected, complex phosphorylation cascade andcrosstalk between pathways make it difficult for large-scalephosphoproteomics to reveal direct relationships betweenprotein kinases and their substrates (10, 11). Extensive sta-tistics, bioinformatics, and downstream biochemical assaysare mandatory for the substrate verification (12, 13). Anotherstrategy uses purified, active kinases to phosphorylate cellextracts in vitro, followed by mass spectrometric analysis toidentify phosphoproteins. This approach inevitably faces themajor challenge of separating real sites phosphorylated bytarget kinase and the phosphorylation triggered by endoge-nous kinases from cell lysates (14). Analog-sensitive kinaseallele (15) overcomes the issue by utilizing the engineeredkinase that can exclusively take a bulky-ATP analog under thereaction condition. Analog-sensitive kinase allele has beencoupled with -thiophosphate analog ATP to facilitate themass spectrometric analysis (1618).

    We have introduced kinase assay-linked phosphoproteom-ics (KALIP)1 to link the in vitro substrate identification and

    From the Departments of Biochemistry, Horticulture and Land-scape Architecture, Mathematics, Medicinal Chemistry and Molec-ular Pharmacology, **Chemistry, and Purdue University Center forCancer Research, Purdue University, West Lafayette, Indiana 47907

    Received February 12, 2014, and in revised form, July 7, 2014Published, MCP Papers in Press, July 14, 2014, DOI 10.1074/

    mcp.O114.038588Author contributions: L.X., J.Z., and W.A.T. designed research; L.X.

    and P.W. performed research; L.X., P.C., and W.A.T. analyzed data;L.X. and W.A.T. wrote the paper.

    1 The abbreviations used are: BAG3, BAG family molecular chap-erone regulator 3; KALIP, kinase assay-linked phosphoproteomics;regulator 3; BORG4, Cdc42 effector protein 4; EPS15, epidermalgrowth factor receptor pathway substrate 15; ERK1, extracellular sig-nal-regulated kinase 1; FASP, filter aided aided proteome preparation;FDR, false discovery rate; FSBA, adenosine-5-(4-fluorosulfonylbenzo-ate) hydrochloride; GO, gene ontology; IPA, ingenuity pathway analysis;MAPK, mitogen-activated protein kinase; PolyMAC, polymer-basedmetal-ion affinity capture; proKALIP, protein kinase assay linked withphosphoproteomics; QIKS, quantitative identification of kinasesubstrates; SILAC, stable isotope labeling by amino acids in cellculture; siKALIP, stable isotope labeling kinase assay-linkedphosphoproteomics.

    Technological Innovation and Resources 2014 by The American Society for Biochemistry and Molecular Biology, Inc.This paper is available on line at http://www.mcponline.org

    Molecular & Cellular Proteomics 13.11 3199

  • physiological phosphorylation events together in a highthroughput manner (19, 20). The strategy, however, has onlybeen applied to identify direct substrates of tyrosine kinases.In this study, we expanded the application of KALIP to serine/threonine kinases by introducing a quantitative strategytermed Stable Isotope Labeled Kinase Assay-Linked Phos-phoproteomics (siKALIP). The method was applied to identifydirect substrates of extracellular signal-regulated kinase 1(ERK1), a serine/threonine kinase acting as an essential com-ponent of the Mitogen-activated protein kinase (MAPK) signaltransduction pathway (21). A defect in the MAP/ERK pathwaycauses uncontrolled growth, which likely leads to cancer (22)and other diseases (2325). ERK1 can be activated by growthfactors such as platelet-derived growth factor (PDGF), epider-mal growth factor (EGF), and nerve growth factor (NGF) (26).Upon stimulation, ERK1 phosphorylates hundreds of sub-strates in various cellular compartments including cytoplasm,nucleus, and membrane (27). Among 38 ERK1 direct sub-strates identified by siKALIP, more than one third are previ-ously discovered by classical molecular biology approaches,highlighting high specificity and sensitivity of the strategy. Theresults also support the hypothesis that ERK1 plays complexroles in multiple pathways that are essential for the cell growthregulation.

    EXPERIMENTAL PROCEDURES

    Mammalian Cell CultureHEK293 cells (ATCC) were maintained inDulbeccos Modified Eagle Medium (DMEM) (Sigma) supplementedwith 10% heat inactivated FBS, 100 g/ml streptomycin, and 100IU/ml penicillin in 5% CO2 at 37 C. Human DG-75 B lymphoma cells(ATCC) were grown in RPMI 1640 media (Sigma) supplemented with10% heat inactivated FBS, 1 mM sodium pyruvate, 100 g/ml strep-tomycin, 100 IU/ml penicillin, and 0.05 mM 2-mercaptoethanol in 5%CO2 at 37 C. The cells were washed with PBS, trypsinized, collected,and frozen at 80 C for further use.

    Plant Tissue CultureSeedlings of A. thaliana were grown in 40 mlof half-strength Murashige and Skoog medium at 22 C in continuouslight on a rotary shaker set at 100 rpm. Twelve-day-old seedlingswere collected and frozen at 80 C for further use.

    In Vitro Kinase Reaction in siKALIPMammalian cells were lysedby sonication in lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150mM NaCl, 5 mM EDTA, and 1% Nonidet P-40 on ice. For plant cells,total protein was extracted from 2 g of seedlings by grinding in 2 mlof extraction buffer containing 100 mM Tris-HCl (pH 7.5), 250 mMNaCl, and 5 mM EDTA. The cell debris was cleared by centrifugationat 16,000 g for 10 min. The supernatant containing 400 g solubleproteins was collected. The lysate volume was adjusted to 200 lusing phosphatase buffer (Roche). 10U of rAPid alkaline phosphatase(Roche) was added and incubated at 37 C for 3 h. The phosphatasewas deactivated by heating at 75 C for 5 min. To inhibit endogenouskinases, the sample was incubated with 1 mM 5-(4-fluorosulfonyl-benzoyl)adenosine (FSBA) with 10% DMSO in Tris-HCl, pH 7.5 at30 C for 1 h. Excess FSBA is removed by Vivacon filtration units (30kDa cutoff). Samples in the filters were then incubated in buffercontaining 300 ng ERK1 (Sigma), 5 mM MgCl2, and 1 mM -[

    18O4]-ATP(Cambridge Isotope Laboratory, Andover, MA) at 30 C for 1 h. Thereaction was stopped by 8 M urea with 5 mM dithiothreitol. Filter aidedproteome preparation (FASP, San Diego, CA) digestion was per-formed according to the manufacturers specifications (Expedeon).

    In Vitro Kinase Reaction by AutoradiographyThe EPS15 andBAG3 substrates were isolated on beads by specific antibody (Anti-EPS15 from Cell Signaling, Beverly, MA and Anti-BAG3 from Protein-Tech, Chicago, IL). The BORG4 recombinant proteins were pur-chased from Abnova. Substrates were incubated in Tris-HCl, pH 7.5buffer containing 1 g ERK1 (Sigma), 5 mM MgCl2, and 25 M coldATP, 2.5 Ci [-32P] ATP at 30 C for 1 h. The reaction was quenchedby boiling the sample with 4X electrophoresis sample buffer (Invitro-gen, Carlsbad, CA). Phosphorylation signal was detected by phos-phor-imager (GE Healthcare, Pittsburgh, PA).

    Phosphopeptide EnrichmentTryptic peptides were first desaltedusing a Sep-pak C18 column (Waters, Milford, MA) and dried. Next,the peptide mixture was resuspended in 100 l of loading buffer (100mM glycolic acid, 1% trifluroacetic acid, and 50% acetonitrile) towhich 5 nmol of the PolyMAC-Ti (Tymora Analytical, IN) reagent wasadded (28). The mixture was then incubated for 5 min. 200 l of 300mM HEPES, pH 7.7, was added to the mixture to achieve a final pH of6.3. The solution was incubated with magnetic hydrazide beads tocapture the PolyMAC-Ti dendrimers. The column was gently agitatedfor 10 min and then centrifuged at 2300 g for 30 s to collect th

Recommended

View more >