Phosphotyrosine signaling analysis of site-specific mutations on EGFRvIII identifies determinants governing glioblastoma cell growth.
To evaluate the role of individual EGFR phosphorylation sites in activating components of the cellular signaling network we have performed a mass spectrometry-based analysis of the phosphotyrosine network downstream of site-specific EGFRvIII mutants, enabling quantification of network-level effects of site-specific point mutations. Mutation at Y845, Y1068 or Y1148 resulted in diminished receptor phosphorylation, while mutation at Y1173 led to increased phosphorylation on multiple EGFRvIII residues. Altered phosphorylation at the receptor was recapitulated in downstream signaling network activation levels, with Y1173F mutation leading to increased phosphorylation throughout the network. Computational modeling of GBM cell growth as a function of network phosphorylation levels highlights the Erk pathway as crucial for regulating EGFRvIII-driven U87MG GBM cell behavior, with the unexpected finding that Erk1/2 is negatively correlated to GBM cell growth. Genetic manipulation of this pathway supports the model, demonstrating that EGFRvIII-expressing U87MG GBM cells are sensitive to Erk activation levels. Additionally, we developed a model describing glioblastoma cell growth based on a reduced set of phosphoproteins, which represent potential candidates for future development as therapeutic targets for EGFRvIII-positive glioblastoma patients.
Huang
Paul H
PH
Protein Networks Team, Section for Cell and Molecular Biology, Institute of Cancer Research, London, UK.
Miraldi
Emily R
ER
Xu
Alexander M
AM
Kundukulam
Vibin A
VA
Del Rosario
Amanda M
AM
Flynn
Ryan A
RA
Cavenee
Webster K
WK
Furnari
Frank B
FB
White
Forest M
FM
D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000235:genetics* / Q000378:metabolism; D053263:Gene Regulatory Networks / Q000502:physiology; D005909:Glioblastoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D010766:Phosphorylation; D019000:Phosphotyrosine; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D014443:Tyrosine / Q000235:genetics* / Q000378:metabolism
C410723:epidermal growth factor receptor VIII; D019000:Phosphotyrosine; D014443:Tyrosine; D066246:ErbB Receptors; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3
Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):6981-5
7542783
Nat Genet. 2007 Apr;39(4):503-12
17322878
J Proteome Res. 2005 Jul-Aug;4(4):1339-46
16083285
Cancer Res. 2006 Aug 1;66(15):7473-81
16885344
Genomics. 1995 May 1;27(1):165-73
7665165
Mol Cell Biol. 1994 Apr;14(4):2777-85
7511210
Oncogene. 2007 May 14;26(22):3291-310
17496923
Mol Cell Biol. 1994 Aug;14(8):5192-201
7518560
Proc Natl Acad Sci U S A. 1997 Nov 11;94(23):12479-84
9356475
Nat Genet. 2008 May;40(5):600-8
18372904
J Biol Chem. 2007 Jul 20;282(29):20925-32
17507376
Mol Pharmacol. 2006 Jun;69(6):1938-44
16505154
PLoS Comput Biol. 2007 Jan 5;3(1):e4
17206861
Mol Cell Biol. 2000 May;20(10):3482-96
10779338
Mol Syst Biol. 2006;2:54
17016520
Proc Natl Acad Sci U S A. 2007 Jul 31;104(31):12867-72
17646646
Mol Syst Biol. 2005;1:2005.0008
16729043
Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7727-31
8052651
Mol Biosyst. 2009 Jan;5(1):59-67
19081932
J Biol Chem. 1998 Sep 18;273(38):24839-46
9733788
Cell. 1994 Jun 17;77(6):841-52
7911739
Nature. 2008 Mar 13;452(7184):181-6
18337815
Nature. 2006 Jan 12;439(7073):168-74
16273093
J Biol Chem. 1997 Jan 31;272(5):2927-35
9006938
Am J Pathol. 2007 May;170(5):1445-53
17456751
Nat Rev Drug Discov. 2007 May;6(5):391-403
17473844
Genes Dev. 2007 Nov 1;21(21):2683-710
17974913
J Biol Chem. 1995 Dec 22;270(51):30562-6
8530489
Cell Cycle. 2007 Nov 15;6(22):2750-4
17986864
N Engl J Med. 2005 Nov 10;353(19):2012-24
16282176
Nat Rev Mol Cell Biol. 2006 Nov;7(11):820-8
17057752
EMBO J. 1994 Jul 1;13(13):3020-32
7518772
Mol Carcinog. 2005 Dec;44(4):262-73
16167350
Nature. 1993 May 6;363(6424):85-8
8479541
J Biol Chem. 1996 Oct 11;271(41):25639-45
8810340
Mol Syst Biol. 2007;3:151
18059446