The epidermal growth factor receptor (ErbB1/EGFR) is a prototypical receptor tyrosine kinase (RTK) that activates multikinase phosphorylation cascades and regulates diverse cellular processes including proliferation, migration, and differentiation (Citri and Yarden, 2006
). Differential binding of 13 known extracellular ligands to ErbB1–4 receptors induces formation of homo- and hetero-oligomers. In the case of ErbB1, whose structure has been studied in detail, ligand binding is thought to promote a conformational switch that positions the C-terminal cytoplasmic tail of one receptor near the activation loop of the other, thereby facilitating phosphorylation in trans
(Zhang et al., 2006
). Receptor dimers can form in the absence of ligand but the switch to an active conformation probably occurs only upon ligand binding (Chung et al., 2010
). In solid tumors ErbB receptors are frequently mutated, overexpressed or activated by autocrine or paracrine ligands (Holbro and Hynes, 2004
), and multiple small molecule kinase inhibitors and therapeutic antibodies targeting ErbB receptors are in clinical use (Tables S1 and S2
). In many cases, the reasons for the differential effectiveness of these drugs are not well understood.
Active ErbB receptors phosphorylate each other on four to 12 tyrosine residues that serve as docking sites for recruitment of diverse adaptor proteins containing Src homology domain 2 (SH2) and phosphotyrosine binding (PTB) domains (Jones et al., 2006
; Kaushansky et al., 2008
; Schulze et al., 2005
). Adaptors, and the proteins that bind to them, are often themselves targets for phosphorylation by ErbB receptors or by cytoplasmic kinases. This leads to assembly of large multiprotein “signalosomes” that transmit signals to downstream pathways including the Raf-MEK-ERK (MAPK) and PI3K-Akt kinase cascades (Yarden and Sliwkowski, 2001
) and the actin cytoskeleton (Hirsch et al., 2006
) (). In cells exposed to exogenous ligand, phosphorylation of receptors and adaptor proteins usually peaks within 10 min and then declines to prestimulus levels ~1–2 hr later, thereby driving the immediate-early response. Endocytosis and degradation of activated ErbB1 in the lysosome plays the primary role in receptor adaptation (Sorkin and Goh, 2009
), but internalization is less important for ErbB2–4 (Baulida et al., 1996
). Extensive evidence also points to a regulatory role for protein tyrosine phosphatases (PTPs) in ErbB biology (Table S3
) (Tiganis, 2002
), but it remains poorly understood how receptors are controlled by a combination of changes in receptor conformation, oligomerization, phosphorylation/dephosphorylation, and localization. The classical view is that conformational changes triggered by ligand binding drive the rapid formation of tyrosine phosphorylated ErbB1 (ErbB1-pY) and that the subsequent slower fall in ErbB1-pY levels involves relocalization of receptors to phosphatase-rich intracellular compartments, and attenuation of signaling via endocytic degradation and the action of transcriptional feedback loops (Avraham and Yarden, 2011
). However, several experiments suggest a more dynamic balance between activation and inactivation. For example, treatment of cells with the potent pan-specific tyrosine phosphatase inhibitor pervanadate causes large and immediate increases in ErbB1-pY (and increased phosphotyrosine levels on many other proteins) in the absence of added ligand (Ruff et al., 1997
), implying a requirement for phosphatases in opposing receptor autoactivation. In addition, sequential exposure of cells to ligand and then to a small molecule kinase inhibitor causes phosphorylation to rise and then fall rapidly (Böhmer et al., 1995
; Offterdinger et al., 2004
). These data on turnover of tyrosine phosphates on ErbB1 motivated us to perform a more quantitative and extensive study.
Overview of Relevant RTK Signaling and Mathematical Models
Here, we use a series of mathematical models () and detailed time course data to address five unanswered questions about RTK phosphorylation: (1) What is the rate at which tyrosine phosphorylation turns over on active ErbB1 receptors under various conditions? (2) Is the rapid dephosphorylation an artifact of drug binding? (3) Is the pool of ErbB1 subject to rapid dephosphorylation the same pool that is active in signal transduction, and, if so, what are the consequences of rapid phospho-turnover for downstream signaling? (4) Do other ErbB receptors and unrelated RTKs also exhibit rapid phospho-turnover? (5) What are the consequences of ErbB phospho-turnover for the mechanisms of action of small molecule drugs that bind ErbB receptors? The latter question seemed particularly interesting because in vitro studies on anti-ErbB drugs have been performed in the absence of phosphatases. We report that ErbB1–3, the insulin-like growth factor 1 receptor (IGF1R), and the fibroblast growth factor receptor 1 (FGFR1) cycle between phosphorylated and unphosphorylated states on the time scale of seconds and that phosphorylated forms of downstream kinases such as ERK, Akt, JNK, and p38 also turn over rapidly. We argue this is unlikely to be an artifact of drug binding. This implies that a single RTK molecule is phosphorylated and dephosphorylated at least 100–1000 times over the course of an ~1 hr immediate-early response. Rapid phosphorylation and dephosphorylation of RTKs has significant implications for signalosome assembly and mechanisms of kinase inhibition.