Epidermal growth factor receptor juxtamembrane region regulates allosteric tyrosine kinase activation

Abstract

Structural studies of the extracellular and tyrosine kinase domains of the epidermal growth factor receptor (ErbB-1) provide considerable insight into facets of the receptor activation mechanism, but the contributions of other regions of ErbB-1 have not been ascertained. This study demonstrates that the intracellular juxtamembrane (JM) region plays a vital role in the kinase activation mechanism. In the experiments described herein, the entire ErbB-1 intracellular domain (ICD) has been expressed in mammalian cells to explore the significance of the JM region in kinase activity. Deletion of the JM region (⌬JM) results in a severe loss of ICD tyrosine phosphorylation, indicating that this region is required for maximal activity of the tyrosine kinase domain. Coexpression of ⌬JM and dimerization-deficient kinase domain ICD mutants revealed that the JM region is indispensable for allosteric kinase activation and productive monomer interactions within a dimer. Studies with the intact receptor confirmed the role of the JM region in kinase activation. Within the JM region, Thr-654 is a known protein kinase C (PKC) phosphorylation site that modulates kinase activity in the context of the intact ErbB-1 receptor; yet, the mechanism is not known. Whereas a T654A mutation promotes increased ICD tyrosine phosphorylation, the phosphomimetic T654D mutant generates a 50% reduction in ICD tyrosine phosphorylation. Similar to the ⌬JM mutants, the T654D mutant ICD failed to interact with a wild-type monomer. This study reveals an integral role for the intracellular JM region of ErbB-1 in allosteric kinase activation.A high level of homology exists among the ErbB receptors in the juxtamembrane (JM) region, which contains Ϸ37 residues (Fig. 1A). Of note is an abundance of basic residues in the N-terminal portion of this region, which is unique to the ErbB receptor family. Contained within the JM sequence are lysosomal (1, 2) and basolateral (3) sorting motifs, a nuclear localization sequence (4), a calmodulin binding site (5), and protein kinase C (PKC) (6, 7) and mitogen-activated protein kinase (8, 9) phosphorylation sites.Pretreatment of cells with the phorbol ester 12-Otetradecanoyl-phorbol-13-acetate (TPA), a PKC agonist, prevents epidermal growth factor (EGF)-induced ErbB-1 tyrosine phosphorylation (10-12). TPA treatment results in PKC phosphorylation of Thr-654 in the JM region (6, 7), and mutation of this residue abrogates TPA inhibition of EGF-induced ErbB-1 tyrosine phosphorylation (13). ErbB-1 ligands also promote phosphorylation of . However, the mechanism by which this PKC phosphorylation event promotes kinase inactivation is not understood. Complicating experiments with the intact receptor in cells is the fact that TPA also induces ErbB-1 endocytosis (15) and recycling of the internalized receptor (16).Ligand-induced dimerization of ErbB-1 is a prerequisite for receptor activation and subsequent tyrosine phosphorylation (17). Recent analysis of the ErbB-1 tyrosine kinase domain (TKD) crystal structure has provided evid...

The activating role of phospho-(Tyr)-calmodulin on the epidermal growth factor receptor

“…Thus, it is likely that both CaM species may bind to this site in living cells. The identification of P-(Tyr)-CaM as a potential intracellular ligand of the EGFR introduces further complexity to the proposed activation mechanism of this receptor [9,[29][30][31]. Thus, we suggest the existence of three fundamental states of the EGFR tyrosine kinase: (i) an inactive state in the absence of EGF in which the CaM-BD is electrostatically bound to the inner leaflet of the plasma membrane as first proposed by McLaughlin et al [25]; (ii) a partially-active state of the receptor in the presence of EGF when apo-CaM is phosphorylated and P-(Tyr)-CaM detaches the tyrosine kinase domain from the membrane; and (iii) a fully-active state in the presence of EGF when Ca 2 + /P-(Tyr)-CaM plus non-phosphorylated Ca 2 + -CaM are present, as the former plays a more efficient activating role and the latter may also accumulate after Ca 2 + /P-(Tyr)-CaM dephosphorylation.…”

“…[ [29][30][31], giving further credibility to the presumptive implication of CaM in the EGFR activation process. Although Ca 2 + -CaM is involved in ligand-dependent EGFR activation [21][22][23]25], it is not clear whether or not phospho-(Tyr)-CaM (tyrosinephosphorylated CaM) also plays an activating role in this process.…”

Receptor tyrosine kinase transmembrane domains

“…Amino acids in this region have been shown to serve as binding and phosphorylation sites for signaling molecules. [45][46][47][48][49][50] The importance of the JM domain in signaling is evident from several mutations, deletions and insertions in this domain that can lead to cancer. [51][52][53] In addition, recent studies have provided evidence for the active role of the JM domains in regulating RTK activity.…”

“…On the contrary, the JM domain has been shown to be essential in the activation of the kinase. 50 Recent studies have suggested that the role of the JM domain is to stabilize the activated asymmetric kinase dimer by forming an antiparallel dimer with the JM segment of the other receptor. 58,59 Furthermore, quantitative studies of ligand binding as a function of receptor expression have shown that the binding affinity of the ligand to the extracellular domain is affected by the JM domain.…”

Activation of the EGF Receptor by Ligand Binding and Oncogenic Mutations: The “Rotation Model”

“…This again suggests that the proposed anti-parallel helical dimer contributes to the negative cooperativity. It has long been recognized that phosphorylation of EGFR T678 (T654 in mature EGFR) leads to decreased affinity of EGF and loss of EGF-stimulated receptor autophosphorylation [78][79][80][81][82]. Phorbol 12-myristate 13-acetate-induced phosphorylation of T678 by protein kinase C leads to the complete loss of cooperativity [77].…”