Interactions between the juxtamembrane domain of the EGFR and calmodulin measured by surface plasmon resonance

One early response to epidermal growth factor receptor (EGFR) activation is an increase in intracellular calcium. We have used surface plasmon resonance (SPR) to study real-time interactions between the intracellular juxtamembrane (JM) region of EGFR and calmodulin. The EGFR-JM (Met⁶⁴⁴–Phe⁶⁸⁸) was expressed as a GST fusion protein and immobilised on a sensor chip surface. Calmodulin specifically interacts with EGFR-JM in a calcium-dependent manner with a high on and high off rate. Chemical modification of EGFR-JM by using arginine-selective phenylglyoxal or deletion of the basic segment Arg⁶⁴⁵–Arg⁶⁵⁷ inhibits the interaction. Phosphorylation of EGFR-JM by protein kinase C (PKC) or glutamate substitution of Thr⁶⁵⁴ inhibits the interaction, suggesting that PKC phosphorylation electrostatically interferes with calmodulin binding to basic arginine residues. Calmodulin binding was also inhibited by suramin. Our results suggest that EGFR-JM is essential for epidermal growth factor (EGF)-mediated calcium–calmodulin signalling and for signal integration between other signalling pathways.     

Lipid-Protein Interplay in Dimerization of Juxtamembrane Domains of Epidermal Growth Factor Receptor

Transmembrane (TM) helix and juxtamembrane (JM) domains (TM-JM) bridge the extracellular and intracellular domains of single-pass membrane proteins, including epidermal growth factor receptor (EGFR). TM-JM dimerization plays a crucial role in regulation of EGFR kinase activity at the cytoplasmic side. Although the interaction of JM with membrane lipids is thought to be important to turn on EGF signaling, and phosphorylation of Thr654 on JM leads to desensitization, the underlying kinetic mechanisms remain unclear. In particular, how Thr654 phosphorylation regulates EGFR activity is largely unknown. Here, combining single-pair FRET imaging and nanodisc techniques, we showed that phosphatidylinositol 4,5-bis phosphate (PIP₂) facilitated JM dimerization effectively. We also found that Thr654 phosphorylation dissociated JM dimers in the membranes containing acidic lipids, suggesting that Thr654 phosphorylation electrostatically prevented the interaction with basic residues in JM and acidic lipids. Based on the single-molecule experiment, we clarified the kinetic pathways of the monomer (inactive state)-to-dimer (active state) transition of JM domains and alteration in the pathways depending on the membrane lipid species and Thr654 phosphorylation.     

Diacylglycerol kinase θ counteracts protein kinase C-mediated inactivation of the EGF receptor

Epidermal growth factor receptor (EGFR) activation is negatively regulated by protein kinase C (PKC) signaling. Stimulation of A431 cells with EGF, bradykinin or UTP increased EGFR phosphorylation at Thr654 in a PKC-dependent manner. Inhibition of PKC signaling enhanced EGFR activation, as assessed by increased phosphorylation of Tyr845 and Tyr1068 residues of the EGFR. Diacylglycerol is a physiological activator of PKC that can be removed by diacylglycerol kinase (DGK) activity. We found, in A431 and HEK293 cells, that the DGKθ isozyme translocated from the cytosol to the plasma membrane, where it co-localized with the EGFR and subsequently moved into EGFR-containing intracellular vesicles. This translocation was dependent on both activation of EGFR and PKC signaling. Furthermore, DGKθ physically interacted with the EGFR and became tyrosine-phosphorylated upon EGFR stimulation. Overexpression of DGKθ attenuated the bradykinin-stimulated, PKC-mediated EGFR phosphorylation at Thr654, and enhanced the phosphorylation at Tyr845 and Tyr1068. SiRNA-induced DGKθ downregulation enhanced this PKC-mediated Thr654 phosphorylation. Our data indicate that DGKθ translocation and activity is regulated by the concerted activity of EGFR and PKC and that DGKθ attenuates PKC-mediated Thr654 phosphorylation that is linked to desensitisation of EGFR signaling.     

Intensification of growth factor receptor signalling by phorbol treatment of ligand-primed cells implies a dimer-stabilizing effect of protein kinase C-dependent juxtamembrane domain phosphorylation.

Protein kinase C (PKC) phosphorylates the juxtamembrane domain of many growth factor receptors, but the physiologic effect of this modification on ligand signalling and desensitisation is unclear. Here we show that PKC-dependent transmodulation of EGFR and ErbB2 signalling is schedule-specific: prolonged pre-treatment of A431 cells with the PKC agonist phorbol dibutyrate potently inhibits subsequent ligand-induced EGFR signalling as expected, but EGF pre-treatment reverses the inhibitory effect of phorbol. The agonist activity of PKC on receptor signalling is even more apparent when cells are treated with phorbol in the presence of a tyrosine phosphatase inhibitor. Because these findings suggested a synergistic interaction between tyrosine- and PKC-dependent phosphorylation events, we sought to define the interactions of tyrosine-phosphorylated and PKC-modified ErbB2 subsets within EGF-inducible hetero-oligomers. Growth factor-dependent PKC transphosphorylation takes place exclusively within endocytosed tyrosine-phosphorylated receptor oligomers. Moreover, phorbol differentially affects two ErbB2 C-terminal autophosphorylation sites: whereas phosphorylation of Tyr1222 is reduced, phosphorylation of Tyr1139 is increased. These results suggest that PKC-dependent phosphorylation of the juxtamembrane domain may contribute positively to both internalisation and signalling of ligand-activated receptors, simultaneously accelerating termination of growth factor action. We propose that transient PKC-dependent signal amplification results from enhanced stability of liganded receptor oligomers due to phosphorylation-dependent juxtamembrane domain interactions, analogous to the protein-protein binding now known to be induced by serine-threonine phosphorylation of CREB and SMAD.     

Epidermal growth factor receptors: function modulation by phosphorylation and glycosylation interplay

Post-translational modifications (PTMs) of proteins induce structural and functional changes that are most often transitory and difficult to follow and investigate in vivo. In silico prediction procedures for PTMs are very valuable to foresee and define such transitory changes responsible for the multifunctionality of proteins. Epidermal growth factor receptor (EGFR) is such a multifunctional transmembrane protein with intrinsic tyrosine kinase activity that is regulated primarily by ligand-stimulated transphosphorylation of dimerized receptors. In human EGFR, potential phosphorylation sites on Ser, Thr and Tyr residues including five autophosphorylation sites on Tyr were investigated using in silico procedures. In addition to phosphorylation, O-GlcNAc modifications and interplay between these two modifications was also predicted. The interplay of phosphorylation and O-GlcNAc modification on same or neighboring Ser/Thr residues is termed as Yin Yang hypothesis and the interplay sites are named as Yin Yang sites. Amongst these modification sites, one residue is localized in the juxtamembrane (Thr 654) and two are found in the catalytic domain (Ser 1046/1047) of the EGFR. We propose that, when EGFR is O-GlcNAc modified on Thr 654, EGFR may be transferred from early to late endosomes, whereas when EGFR is O-GlcNAc modified on Ser 1046/1047 desensitization of the receptor may be prevented. These findings suggest a complex interplay between phosphorylation and O-GlcNAc modification resulting in modulation of EGFR's functionality.     

Metastasis Suppressor Tetraspanin CD82/KAI1 Regulates Ubiquitylation of Epidermal Growth Factor Receptor

Ligand-induced ubiquitylation of EGF receptor (EGFR) is an important regulatory mechanism that controls endocytic trafficking of the receptor and its signaling potential. Here we report that tetraspanin CD82/KAI1 specifically suppresses ubiquitylation of EGFR after stimulation with heparin-binding EGF or amphiregulin and alters the rate of recruitment of the activated receptor to EEA1-positive endosomes. The suppressive effect of CD82 is dependent on the heparin-binding domain of the ligand. Deletion of the C-terminal cytoplasmic domain of CD82 (CD82ΔC mutant) inhibits endocytic trafficking of the tetraspanin and compromises its activity toward heparin-binding EGF-activated EGFR. Reduced ubiquitylation of EGFR is accompanied by PKC-dependent increase in serine phosphorylation of c-Cbl in cells expressing elevated levels of CD82. Furthermore, phosphorylation of threonine 654 (PKC phosphorylation site) in the juxtamembrane domain of the receptor is considerably increased in CD82-expressing cells. These results describe previously unsuspected links between tetraspanin proteins and ubiquitylation of their molecular partners (e.g., EGFR). Our data identify CD82 as a new regulator of c-Cbl, which discriminatively controls the activity of this E3 ubiquitin ligase toward heparin-binding ligand-EGFR pairs. Taken together, these observations provide an important new insight into the modulatory role of CD82 in endocytic trafficking of EGF receptor.     

Nuclear EGFR shuttling induced by ionizing radiation is regulated by phosphorylation at residue Thr654

Nuclear localisation of EGFR is associated with treatment resistance of tumor cells. The aim of this study was to identify molecular targets to block nuclear shuttling of EGFR. Mutation of Thr654, located within the putative EGFR NLS demonstrated that phosphorylation of this residue is essential for nuclear EGFR shuttling following irradiation. Deletion of Thr654 blocked nuclear transport of EGFR, whereas mutation to Glu increased shuttling. Treatment with a peptide, corresponding to the phosphorylated NLS, abolished nuclear EGFR transport and reduced radiation-induced activation of DNA-PK, essential for DNA-repair. In accordance with that, lack of nuclear EGFR increased residual DNA damage in tumor cells and reduced cellular survival following irradiation. Blockage of nuclear EGFR shuttling may be a new strategy to fight treatment resistance.

Structured summary

MINT-7987956: Karyopherin alpha (uniprotkb:P52294) physically interacts (MI:0915) with EGFR (uniprotkb:P00533) by anti bait coimmunoprecipitation (MI:0006)     

Structure of the membrane reconstituted transmembrane-juxtamembrane peptide EGFR(622-660) and its interaction with Ca2+/calmodulin

The transmembrane (TM) and juxtamembrane (JM) regions of the epidermal growth factor receptor (EGFR) couple ligand binding in the extracellular domain to activation of the kinase domain. Solid-state NMR and polarized FTIR measurements of peptides corresponding to the TM plus JM regions of EGFR (residues 622-660) reconstituted in model phospholipid membranes are presented to address the role of the short cytoplasmic JM sequence (residues 645-660) in regulating EGFR activity. We show that the TM domain is helical with a transition to non-helical structure at the TM-JM boundary. Fluorescence measurements indicate that the JM region of EGFR(622-660) binds to the membrane surface and that binding can be reversed by the addition of the complex of Ca2+ and calmodulin. Together these data support models suggesting the cytoplasmic JM region of EGFR plays an active role in regulating receptor activity.     

Role of calmodulin in the modulation of the MAPK signalling pathway and the transactivation of epidermal growth factor receptor mediated by PKC

We have recently shown that calmodulin (CaM) regulates the trafficking of epidermal growth factor receptor (EGFR) as well as the mitogen-activated protein kinase (MAPK) signalling pathway. However, the overall regulation of the MAPK pathway is achieved through a complex interplay of other several upstream effectors including G-proteins, EGF, EGFR, protein kinase C (PKC), phosphatidylinositol-3-kinase and CaM. In order to understand the role of CaM in the PKC-mediated transactivation of EGFR we have analysed the effect of a CaM antagonist, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide, on the 12-O-tetradecanoylphorbol-13-acetate-mediated activation of EGFR and the subsequent MAPK activation. The results show that CaM interferes with MAPK activation and the transactivation of EGFR mediated by PKC.     

Function of the loop residue Thr792 in human DNA topoisomerase II alpha

We studied the mutation effect of one of the putative loop residues Thr792 in human DNA topoisomerase II alpha (TOP2 alpha). Thr792 mutants were expressed from high or low copy plasmids in a temperature sensitive yeast strain deficient in TOP2 (top2-1). When expressed from a high copy plasmid, mutants with small side chains complemented the yeast defect; however, from a low copy plasmid, only wild-type, Ser, and Cys substitution mutants complemented the yeast defect. Interestingly, at the permissive temperature other mutants (e.g., Val, Gly, and Glu substitutions) showed the dominant negative effect to the top2-1 allele, which was not observed by the control alpha 4-helix mutants. T792E mutant was 10-fold less active than wild-type and the T792P had no decatenation activity in vitro. These results suggest that Thr792 in human TOP2 alpha is involved in enzyme catalysis.     

Regulation of the ligand-dependent activation of the epidermal growth factor receptor by calmodulin

Calmodulin (CaM) is the major component of calcium signaling pathways mediating the action of various effectors. Transient increases in the intracellular calcium level triggered by a variety of stimuli lead to the formation of Ca(2+)/CaM complexes, which interact with and activate target proteins. In the present study the role of Ca(2+)/CaM in the regulation of the ligand-dependent activation of the epidermal growth factor receptor (EGFR) has been examined in living cells. We show that addition of different cell permeable CaM antagonists to cultured cells or loading cells with a Ca(2+) chelator inhibited ligand-dependent EGFR auto(trans)phosphorylation. This occurred also in the presence of inhibitors of protein kinase C, CaM-dependent protein kinase II and calcineurin, which are known Ca(2+)- and/or Ca(2+)/CaM-dependent EGFR regulators, pointing to a direct effect of Ca(2+)/CaM on the receptor. Furthermore, we demonstrate that down-regulation of CaM in conditional CaM knock out cells stably transfected with the human EGFR decreased its ligand-dependent phosphorylation. Substitution of six basic amino acid residues within the CaM-binding domain (CaM-BD) of the EGFR by alanine resulted in a decreased phosphorylation of the receptor and of its downstream substrate phospholipase Cγ1. These results support the hypothesis that Ca(2+)/CaM regulates the EGFR activity by directly interacting with the CaM-BD of the receptor located at its cytosolic juxtamembrane region.     

ERK-dependent threonine phosphorylation of EGF receptor modulates receptor downregulation and signaling

Epidermal growth factor (EGF) signaling is critical in normal and aberrant cellular behavior. Extracellular signal-regulated kinase (ERK) mediates important downstream aspects of EGF signaling. Additionally, EGFR undergoes MEK1-dependent ERK consensus site phosphorylation in response to EGF or cytokines such as growth hormone (GH) and prolactin (PRL). GH- or PRL-induced EGFR phosphorylation alters subsequent EGF-induced EGFR downregulation and signal characteristics in an ERK-dependent fashion. We now use reconstitution to study mutation of the sole EGFR ERK phosphorylation consensus residue, (669)T. CHO-GHR cells, which lack EGFR and express GHR, were stably transfected to express human wild-type or T669A ((669)T changed to alanine) EGFRs at similar abundance. Treatment of cells with GH or EGF caused phosphorylation of WT, but not T669A EGFR, in an ERK activity-dependent fashion that was detected with an antibody that recognizes phosphorylation of ERK consensus sites, indicating that (669)T is required for this phosphorylation. Notably, EGF-induced downregulation of EGFR abundance was much more rapid in cells expressing EGFR T669A vs. WT EGFR. Further, pretreatment with the MEK1/ERK inhibitor PD98059 enhanced EGF-induced EGFR loss in cells expressing WT EGFR, but not EGFR T669A, suggesting that the ERK-dependent effects on EGFR downregulation required phosphorylation of (669)T. In signaling experiments, EGFR T669A displayed enhanced acute (15 min) EGFR tyrosine phosphorylation (reflecting EGFR kinase activity) compared to WT EGFR. Further, acute EGF-induced ubiquitination of WT EGFR was markedly enhanced by PD98059 pretreatment and was increased in EGFR T669A-expressing cells independent of PD98059. These signaling data suggest that ERK-mediated (669)T phosphorylation negatively modulates EGF-induced EGFR kinase activity. We furthered these investigations using a human fibrosarcoma cell line that endogenously expresses EGFR and ErbB-2 and also harbors an activating Ras mutation. In these cells, EGFR was constitutively detected with the ERK consensus site phosphorylation-specific antibody and EGF-induced EGFR downregulation was modest, but was substantially enhanced by pretreatment with MEK1/ERK inhibitor. Collectively, these data indicate that ERK activity, by phosphorylation of a threonine residue in the EGFR juxtamembrane cytoplasmic domain, modulates EGFR trafficking and signaling.     

Protein kinase C phosphorylation at Thr 654 of the unoccupied EGF receptor and EGF binding regulate functional receptor loss by independent mechanisms

To test the functional consequence of phosphorylation of the EGF receptor at Thr 654 by protein kinase C, the normal Thr 654 human EGF receptor cDNA or a mutant encoding an Ala 654 were expressed in heterologous cells. In cell lines expressing both the Thr 654 and Ala 654 receptors, functional cell-surface Thr 654 receptors were reduced or were totally lost, but were not degraded, following activation of protein kinase C by phorbol esters (TPA), whereas Ala 654 receptors were unaffected. These data suggest that protein kinase C regulates ligand-independent receptor binding and internalization via phosphorylation of Thr 654 of the EGF holoreceptor. Because EGF induces internalization and degradation of the Ala 654 EGF receptor, at least two independent mechanisms can serve to signal loss of functional EGF receptors.     

Multisite phosphorylation of the epidermal growth factor receptor. Use of site-directed mutagenesis to examine the role of serine/threonine phosphorylation

The major sites of serine and threonine phosphorylation of the human epidermal growth factor (EGF) receptor observed in intact cells are Thr654, Thr669, Ser1046, and Ser1047. Phosphorylation of the EGF receptor is increased at these sites in cells treated with platelet-derived growth factor or phorbol ester. This increase in EGF receptor phosphorylation is associated with an inhibition of the high affinity binding of EGF to cell surface receptors and an inhibition of the receptor tyrosine protein kinase activity. In order to test the hypothesis that the phosphorylation of the EGF receptor is mechanistically related to the modulation of EGF receptor function, we replaced the major sites of serine and threonine phosphorylation with alanine residues. EGF receptors containing single point mutations or multiple mutations were expressed in Chinese hamster ovary cells. Analysis of the regulation of the EGF receptor tyrosine protein kinase activity demonstrated that phorbol ester caused an inhibition of the tyrosine phosphorylation of wild-type receptors and receptors lacking Thr669, Ser1046, or Ser1047. In contrast, the inhibition of EGF receptor tyrosine phosphorylation caused by phorbol ester was not observed for any of the mutated EGF receptors that lacked Thr654. These data are consistent with the hypothesis that the phosphorylation of the EGF receptor at Thr654 is required for the inhibition of the receptor tyrosine protein kinase activity caused by phorbol ester. Investigation of the apparent affinity of the EGF receptor demonstrated that treatment with phorbol ester caused an inhibition of the high affinity binding of 125I-EGF to cells expressing wild-type EGF receptors and each of the mutated EGF receptors examined. We conclude that the regulation of the apparent affinity of the EGF receptor is independent of the major sites of serine and threonine phosphorylation of the EGF receptor.     

Sustained Activation of Protein Kinase C Induces Delayed Phosphorylation and Regulates the Fate of Epidermal Growth Factor Receptor

It is well established that acute activation of members of the protein kinase C (PKC) family induced by activation of cellular receptors can transduce extracellular stimuli to intracellular signaling. However, the functions of sustained activation of PKC are not well studied. We have previously shown that sustained activation of classical PKC isoforms over 15-60 min induced the formation of the pericentrion, a subset of recycling endosomes that are sequestered perinuclearly in a PKC- and phospholipase D (PLD)-dependent manner. In this study, we investigated the role of this process in the phosphorylation of EGFR on threonine 654 (Thr-654) and in the regulation of intracellular trafficking and fate of epidermal growth factor receptor (EGFR). Sustained stimulation of the angiotensin II receptor induced translocation of the EGFR to the pericentrion, which in turn prevents full access of EGF to the EGFR. These effects required PKC and PLD activities, and direct stimulation of PKC with phorbol esters was sufficient to reproduce these effects. Furthermore, activation of PKC induced delayed phosphorylation of EGFR on Thr-654 that coincided with the formation of the pericentrion and which was dependent on PLD and endocytosis of EGFR. Thus, Thr-654 phosphorylation required the formation of the pericentrion. On the other hand, using a T654A mutant of EGFR, we find that the phosphorylation on Thr-654 was not required for translocation of EGFR to the pericentrion but was required for protection of EGFR from degradation in response to EGF. Taken together, these results demonstrate a novel role for the pericentrion in the regulation of EGFR phosphorylation, which in turn is important for the fates of EGFR.     

Calcium-sensitive interaction between calmodulin and modified forms of rat brain neurogranin/RC3

Neurogranin (NG) binding of calmodulin (CaM) at its IQ domain is sensitive to Ca(2+) concentration and to modifications by protein kinase C (PKC) and oxidants. The PKC phosphorylation site of NG is within the IQ domain whereas the four oxidant-sensitive Cys residues are outside this region. These Cys residues were oxidized forming two pairs of intramolecular disulfides, and could also be glutathiolated by S-nitrosoglutathione resulting in the incorporation of four glutathiones per NG. Circular dichroism (CD) showed that modification of NG by phosphorylation, oxidation forming intramolecular disulfides, or glutathiolation did not affect the alpha-helical content of this protein. Mutation of the four Cys residues [Cys(-)-NG] to Gly and Ser did not affect the alpha-helical content either. Interaction of CaM with the reduced (red)-, glutathiolated (GS)-, or Cys(-)-NG in the Ca(2+)-free solution resulted in an increase in the alpha-helicity determined by their CD spectra, but relatively little change was seen with the oxidized NG (ox-NG) or phosphorylated NG (PO(4)-NG). The binding affinities between the various modified forms of NG and CaM were determined by CD spectrometry and sedimentation equilibrium: their affinities were Cys(-)-NG > red-NG, GS-NG > ox-NG > PO(4)-NG. Unlike Cys(-)-, red-, and GS-NG, neither ox- nor PO(4)-NG bound to a CaM-affinity column. Thus, both oxidation of NG to form intramolecular disulfides and phosphorylation of NG by PKC are effective in modulating the intracellular level of CaM. These results indicate that modification of NG to form intramolecular disulfides outside the IQ domain provides an alternative mechanism for regulation of its binding affinity to CaM.     

The cytoplasmic domain of Alzheimer's amyloid precursor protein is phosphorylated at Thr654, Ser655, and Thr668 in adult rat brain and cultured cells.

BACKGROUND: The cytoplasmic domain of the Alzheimer's disease amyloid precursor protein (APP) is phosphorylated in vitro at Thr654 and Ser655, and both in vitro and in intact cells at Thr668 (numbering for APP695 isoform). MATERIALS AND METHODS: We have developed phosphorylation state-specific antibodies to each of the sites, and we have used these to analyze the phosphorylation of APP in adult rat brain and in cultured cell lines. RESULTS: We demonstrate that all three sites in APP are phosphorylated in adult rat brain. Phosphorylation at Thr654, Ser655, and Thr668 was also observed in several cultured cell lines. In PC12 cells, phosphorylation at Ser655 was increased more than 10-fold by treatment with okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, but was not affected by activators of protein kinase C. In HeLa cells, phosphorylation at Thr668 was regulated in a cell cycle-dependent manner with near-stoichiometric phosphorylation being observed at the G2/M phase of the cell cycle. In general, phosphorylation at Ser655 was found to be highest in mature APP isoforms, whereas phosphorylation of Thr668 was highest in immature APP isoforms in cultured cells. CONCLUSIONS: The results demonstrate that phosphorylation of the cytoplasmic domain of APP occurs at Thr654, Ser655, and Thr668 under physiological conditions. The further characterization of APP phosphorylation using phosphorylation-specific antibodies may help in the elucidation of the biological function of APP.     

Importance of conserved Thr214 in domain A of the Na+,K+ -ATPase for stabilization of the phosphoryl transition state complex in E2P dephosphorylation

Thr(214) of the highly conserved (214)TGES sequence in domain A of the Na(+),K(+)-ATPase was replaced with alanine, and the mutant was compared functionally with the previously characterized domain A mutant Gly(263) --> Ala. Thr(214) --> Ala displayed a conspicuous 150-fold reduction of the apparent vanadate affinity for inhibition of ATPase activity, which could not simply be explained by the observed shifts of the conformational equilibria in favor of E(1) and E(1)P. The intrinsic vanadate affinity of the E(2) form and the effect on the apparent vanadate affinity of displacement of the E(1)-E(2) equilibrium were determined in a phosphorylation assay that allows the enzyme-vanadate complex to be formed under equilibrium conditions. When the E(2) form prevailed, Thr(214) --> Ala retained a reduced vanadate affinity relative to wild type, whereas the affinity of Gly(263) --> Ala became wild type-like. Thus, mutation of Thr(214) affected the intrinsic affinity of E(2) for vanadate. Furthermore, Thr(214) --> Ala showed at least a 5-fold reduced E(2)P dephosphorylation rate relative to wild type in the presence of saturating concentrations of K(+) and Mg(2+). Because vanadate is a phosphoryl transition state analog, it is proposed that defective binding of the phosphoryl transition state complex (transition state destabilization) causes the inability to catalyze E(2)P dephosphorylation properly. By contrast, the phosphorylation site in the E(1) form was unaffected in Thr(214) --> Ala. Replacement of the glutamate, Glu(216), of (214)TGES with alanine was incompatible with cell viability, indicating a very low transport activity or expression level. Our results support the hypothesis that domain A is isolated in the E(1) form, but contributes to make up the catalytic site in the E(2) and E(2)P conformations.     

Conformational regulation of the EGFR kinase core by the juxtamembrane and C-terminal tail: a molecular dynamics study

The catalytic domain of epidermal growth factor receptor (EGFR) is activated by dimerization, which requires allosteric coupling between distal dimerization and catalytic sites. Although crystal structures of EGFR kinases, solved in various conformational states, have provided important insights into EGFR activation by dimerization, the atomic details of how dimerization signals are dynamically coupled to catalytic regions of the kinase core are not fully understood. In this study, we have performed unrestrained and targeted molecular dynamics simulations on the active and inactive states of EGFR, followed by principal component analysis on the simulated trajectories, to identify correlated motions in the EGFR kinase domain upon dimerization. Our analysis reveals that the conformational changes associated with the catalytic functions of the kinase core are highly correlated with motions in the juxtamembrane (JM) and C-terminal tail, two flexible structural elements that play an active role in EGFR kinase activation and dimerization. In particular, the opening and closing of the ATP binding lobe relative to the substrate binding lobe is highly correlated with motions in the JM and C-terminal tail, suggesting that ATP and substrate binding can be coordinated with dimerization through conformational changes in the JM and C-terminal tail. Our study pinpoints key residues involved in this conformational coupling, and provides new insights into the role of the JM and C-terminal tail segments in EGFR kinase functions.