title: Profiling the dynamics of a human phosphorylome reveals new components in HGF/c-Met signaling.
abstract: Protein phosphorylation is a dynamic and reversible event that greatly influences cellular function. Identifying the key regulatory elements that determine cellular phenotypes during development and oncogenesis requires the ability to dynamically monitor proteome-wide events. Here, we report the development of a new strategy to monitor dynamic changes of protein phosphorylation in cells and tissues using functional protein microarrays as the readout. To demonstrate this technology's ability to identify condition-dependent phosphorylation events, human protein microarrays were incubated with lysates from cells or tissues under activation or inhibition of c-Met, a receptor tyrosine kinase involved in tissue morphogenesis and malignancy. By comparing the differences between the protein phosphorylation profiles obtained using the protein microarrays, we were able to recover many of the proteins that are known to be specifically activated (i.e., phosphorylated) upon c-Met activation by the hepatocyte growth factor (HGF). Most importantly, we discovered many proteins that were differentially phosphorylated by lysates from cells or tissues when the c-Met pathway was active. Using phosphorylation-specific antibodies, we were able to validate several candidate proteins as new downstream components of the c-Met signaling pathway in cells. We envision that this new approach, like its DNA microarray counterpart, can be further extended toward profiling dynamics of global protein phosphorylation under many different physiological conditions both in cellulo and in vivo in a high-throughput and cost-effective fashion.
authors.affiliation: Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Crystal L
authors.initials: CL
authors.lastname: Woodard
authors.forename: C Rory
authors.initials: CR
authors.lastname: Goodwin
authors.forename: Jun
authors.initials: J
authors.lastname: Wan
authors.forename: Shuli
authors.initials: S
authors.lastname: Xia
authors.forename: Robert
authors.initials: R
authors.lastname: Newman
authors.forename: Jianfei
authors.initials: J
authors.lastname: Hu
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
authors.forename: S Diane
authors.initials: SD
authors.lastname: Hayward
authors.forename: Jiang
authors.initials: J
authors.lastname: Qian
authors.forename: John
authors.initials: J
authors.lastname: Laterra
authors.forename: Heng
authors.initials: H
authors.lastname: Zhu
mesh_terms: D017228:Hepatocyte Growth Factor / Q000378:metabolism*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D010766:Phosphorylation / Q000502:physiology; D040081:Protein Array Analysis; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology
chemical_list: D017228:Hepatocyte Growth Factor; C491743:MET protein, human; D019859:Proto-Oncogene Proteins c-met
references.citation: Bioinformatics. 2011 Feb 1;27(3):431-2
references.pmid: 21149340
references.citation: Cell. 2009 Mar 20;136(6):1073-84
references.pmid: 19303850
references.citation: Mol Biol Cell. 2004 Jun;15(6):2771-81
references.pmid: 15047863
references.citation: Cancer Chemother Pharmacol. 1989;24(3):148-54
references.pmid: 2544306
references.citation: Anal Chem. 2008 Mar 1;80(5):1448-58
references.pmid: 18254611
references.citation: Brief Funct Genomics. 2010 Jan;9(1):32-42
references.pmid: 20056723
references.citation: Trends Cell Biol. 2003 Jun;13(6):328-35
references.pmid: 12791299
references.citation: Nature. 1998 Oct 15;395(6703):713-6
references.pmid: 9790194
references.citation: Cell Host Microbe. 2011 Oct 20;10(4):390-400
references.pmid: 22018239
references.citation: Mol Cell. 2006 Mar 3;21(5):701-10
references.pmid: 16507367
references.citation: Nat Biotechnol. 2006 Oct;24(10):1253-4
references.pmid: 17013375
references.citation: Biochim Biophys Acta. 2010 Apr;1802(4):396-405
references.pmid: 20079433
references.citation: J Virol. 2009 May;83(10):5219-31
references.pmid: 19244323
references.citation: Genomics Proteomics Bioinformatics. 2013 Feb;11(1):18-28
references.pmid: 23395178
references.citation: Methods Enzymol. 1991;200:62-81
references.pmid: 1956339
references.citation: PLoS One. 2010 Dec 31;5(12):e15123
references.pmid: 21217812
references.citation: Nat Methods. 2007 Oct;4(10):798-806
references.pmid: 17901869
references.citation: Mol Cell Proteomics. 2008 Jan;7(1):35-45
references.pmid: 17951556
references.citation: Nat Methods. 2008 Jan;5(1):69-74
references.pmid: 18084297
references.citation: Proc Natl Acad Sci U S A. 1997 May 13;94(10):4884-9
references.pmid: 9144159
references.citation: Eur J Biochem. 2002 Nov;269(22):5360-8
references.pmid: 12423334
references.citation: Neuro Oncol. 2005 Oct;7(4):436-51
references.pmid: 16212809
references.citation: Methods Mol Biol. 2013;981:151-65
references.pmid: 23381860
references.citation: Curr Biol. 1995 Dec 1;5(12):1394-1403
references.pmid: 8749392
references.citation: Stem Cell Rev. 2007 Jan;3(1):7-17
references.pmid: 17873377
references.citation: Nat Biotechnol. 2003 Mar;21(3):255-61
references.pmid: 12610572
references.citation: Genes Dev. 2003 Nov 1;17(21):2698-708
references.pmid: 14597664
references.citation: J Biol Chem. 1998 Jan 16;273(3):1496-505
references.pmid: 9430688
references.citation: Mech Ageing Dev. 2005 Jan;126(1):171-5
references.pmid: 15610776
references.citation: Cell. 2011 Sep 16;146(6):969-79
references.pmid: 21906795
references.citation: J Proteome Res. 2011 Jul 1;10(7):3200-11
references.pmid: 21609022
references.citation: Clin Cancer Res. 2006 Feb 15;12(4):1292-8
references.pmid: 16489086
references.citation: Cancer Res. 2001 Nov 1;61(21):8022-31
references.pmid: 11691828
references.citation: Clin Cancer Res. 2005 Jun 15;11(12):4479-86
references.pmid: 15958633
references.citation: FEBS J. 2009 Dec;276(23):6871-9
references.pmid: 19860827
references.citation: Nat Rev Mol Cell Biol. 2010 Dec;11(12):834-48
references.pmid: 21102609
references.citation: Cancer Metastasis Rev. 2010 Dec;29(4):751-9
references.pmid: 20922461
references.citation: Cell. 2009 Oct 30;139(3):610-22
references.pmid: 19879846
references.citation: Annu Rev Biochem. 1997;66:807-22
references.pmid: 9242925
references.citation: Mol Cancer Res. 2008 Jan;6(1):139-50
references.pmid: 18234969
references.citation: Nat Rev Cancer. 2009 Jan;9(1):28-39
references.pmid: 19104514
references.citation: Proteomics Clin Appl. 2012 Dec;6(11-12):548-62
references.pmid: 23027439
references.citation: Curr Protoc Protein Sci. 2013 Apr;Chapter 27:Unit 27.1
references.pmid: 23546620
references.citation: Respir Med. 2010 Jan;104(1):61-6
references.pmid: 19726173
references.citation: Nucleic Acids Res. 2008 Jan;36(Database issue):D240-4
references.pmid: 17962309
references.citation: J Proteome Res. 2010 May 7;9(5):2734-42
references.pmid: 20222723
references.citation: Mol Syst Biol. 2013;9:655
references.pmid: 23549483
references.citation: Nature. 2005 Dec 1;438(7068):679-84
references.pmid: 16319894
references.citation: Mol Cell Proteomics. 2010 Dec;9(12):2719-28
references.pmid: 20923971
references.citation: Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350-4
references.pmid: 388439
references.citation: Science. 2001 Sep 14;293(5537):2101-5
references.pmid: 11474067
references.citation: Bioessays. 2003 Jan;25(1):17-24
references.pmid: 12508278
references.citation: Nat Rev Mol Cell Biol. 2003 Dec;4(12):915-25
references.pmid: 14685170
references.citation: Exp Cell Res. 2007 Jan 1;313(1):121-32
references.pmid: 17055484
references.citation: Mol Syst Biol. 2007;3:116
references.pmid: 17551511