Comparisons of tyrosine phosphorylated proteins in cells expressing lung cancer-specific alleles of EGFR and KRAS.
We have used unbiased phosphoproteomic approaches, based on quantitative mass spectrometry using stable isotope labeling with amino acids in cell culture (SILAC), to identify tyrosine phosphorylated proteins in isogenic human bronchial epithelial cells (HBECs) and human lung adenocarcinoma cell lines, expressing either of the two mutant alleles of EGFR (L858R and Del E746-A750), or a mutant KRAS allele, which are common in human lung adenocarcinomas. Tyrosine phosphorylation of signaling molecules was greater in HBECs expressing the mutant EGFRs than in cells expressing WT EGFR or mutant KRAS. Receptor tyrosine kinases (such as EGFR, ERBB2, MET, and IGF1R), and Mig-6, an inhibitor of EGFR signaling, were more phosphorylated in HBECs expressing mutant EGFR than in cells expressing WT EGFR or mutant RAS. Phosphorylation of some proteins differed in the two EGFR mutant-expressing cells; for example, some cell junction proteins (beta-catenin, plakoglobin, and E-cadherin) were more phosphorylated in HBECs expressing L858R EGFR than in cells expressing Del EGFR. There were also differences in degree of phosphorylation at individual tyrosine sites within a protein; for example, a previously uncharacterized phosphorylation site in the nucleotide-binding loop of the kinase domains of EGFR (Y727), ERBB2 (Y735), or ERBB4 (Y733), is phosphorylated significantly more in HBECs expressing the deletion mutant than in cells expressing the wild type or L858R EGFR. Signaling molecules not previously implicated in ERBB signaling, such as polymerase transcript release factor (PTRF), were also phosphorylated in cells expressing mutant EGFR. Bayesian network analysis of these and other datasets revealed that PTRF might be a potentially important component of the ERBB signaling network.
Guha
Udayan
U
Program in Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. guhau@mskcc.org
Chaerkady
Raghothama
R
Marimuthu
Arivusudar
A
Patterson
A Scott
AS
Kashyap
Manoj K
MK
Harsha
H C
HC
Sato
Mitsuo
M
Bader
Joel S
JS
Lash
Alex E
AE
Minna
John D
JD
Pandey
Akhilesh
A
Varmus
Harold E
HE
D000483:Alleles; D000595:Amino Acid Sequence; D001980:Bronchi / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism; D066246:ErbB Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D009154:Mutation / Q000235:genetics; D019000:Phosphotyrosine / Q000378:metabolism*; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras); D015398:Signal Transduction; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D019000:Phosphotyrosine; D066246:ErbB Receptors; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins
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