Temporal Profiling of Lapatinib-suppressed Phosphorylation Signals in EGFR/HER2 Pathways

Abstract

Lapatinib is a clinically potent kinase inhibitor for breast cancer patients because of its outstanding selectivity for epidermal growth factor receptor (EGFR) and EGFR2 (also known as HER2). However, there is only limited information about the in vivo effects of lapatinib on EGFR/HER2 and downstream signaling targets. Here, we profiled the lapatinib-induced time-and dose-dependent phosphorylation dynamics in SKBR3 breast cancer cells by means of quantitative phosphoproteomics. Among 4953 identified phosphopeptides from 1548 proteins, a small proportion (5-7%) was regulated at least twofold by 1-10 M lapatinib. We obtained a comprehensive phosphorylation map of 21 sites on EGFR/HER2, including nine novel sites on HER2. Among them, serine/threonine phosphosites located in a small region of HER2 (amino acid residues 1049 -1083) were up-regulated by the drug, whereas all other sites were down-regulated. We show that cAMP-dependent protein kinase is involved in phosphorylation of this particular region of HER2 and regulates HER2 tyrosine kinase activity. Computational analyses of quantitative phosphoproteome data indicated for the first time that protein-protein networks related to cytoskeletal organization and transcriptional/translational regulation, such as RNP complexes (i.e. hnRNP, snRNP, telomerase, ribosome), are linked to EGFR/ HER2 signaling networks. To our knowledge, this is the first report to profile the temporal response of phosphorylation dynamics to a kinase inhibitor. The results provide new insights into EGFR/HER2 regulation through region-specific phosphorylation, as well as a global view of the cellular signaling networks associated with the anti-breast cancer action of lapatinib. Molecular & Cellular

Biotinylated Phosphoproteins from Kinase‐Catalyzed Biotinylation are Stable to Phosphatases: Implications for Phosphoproteomics

“…These experiments indicate that kinase inhibitors similarly reduce phosphorylation or biotinylation. Because monitoring changes in phosphorylation after inhibitor treatment has been used as a drug characterization tool, [29] a future direction is use of ATP-biotin labeling to characterize kinase inhibitor activities.…”

Phosphoproteomics Reveals HMGA1, a CK2 Substrate, as a Drug-Resistant Target in Non-Small Cell Lung Cancer

“…Based on kinase-motif analysis using a linear motif atlas12, reported that three kinases (ATM, ATR, and DNA-dependent protein kinases) were highly activated during mitotic S phase of the DNA damage response network. Imami, et al 13. described the temporal response of phosphorylation dynamics of the kinase inhibitor lapatinib.…”

Large-scale profiling of protein kinases for cellular signaling studies by mass spectrometry and other techniques

“…These techniques have been used in combination with other in vivo experiments to discover novel kinase-substrate pairs [114][115][116]. Furthermore, results obtained by kinome profiling have made it possible to modify the substrate preferences of a kinase by mutation at the activation loop [117].…”