Báo cáo khoa học: Calmodulin-mediated regulation of the epidermal growth factor receptor doc

REVIEW ARTICLE Calmodulin-mediated regulation of the epidermal growth factor receptor ´ ´ Pablo Sanchez-Gonzalez, Karim Jellali* and Antonio Villalobo ´ ´ Instituto de Investigaciones Biomedicas, Consejo Superior de Investigaciones Cientıficas, Madrid, Spain Keywords calcium; calmodulin; capacitative calcium entry; channels; epidermal growth factor receptor; ErbB receptors; G protein-coupled receptor; membranes; metalloprotease; tyrosine kinase Correspondence A Villalobo, Instituto de Investigaciones ´ Biomedicas, Consejo Superior de ´ Investigaciones Cientıficas, Arturo Duperier 4, E-28029 Madrid, Spain Fax: +34 91 585 4401 Tel: +34 91 585 4424 E-mail: antonio.villalobo@iib.uam.es In this review, we first describe the mechanisms by which the epidermal growth factor receptor generates a Ca2+ signal and, subsequently, we compile the available experimental evidence regarding the role that the Ca2+ ⁄ calmodulin complex, formed after the rise in cytosolic free Ca2+ concentration, exerts on the receptor We focus not only on the indirect action that Ca2+ ⁄ calmodulin exerts on the epidermal growth factor receptor, as a result of the activation of distinct calmodulin-dependent kinases, but also, and more extensively, on the direct interaction of Ca2+ ⁄ calmodulin with the receptor We also describe several mechanistic models that could account for the Ca2+ ⁄ calmodulin-mediated regulation of epidermal growth factor receptor activity The control exerted by calmodulin on distinct epidermal growth factor receptor-mediated cellular functions is also discussed Finally, the phosphorylation of this Ca2+ sensor by the epidermal growth factor receptor is highlighted *Present address Centre of Biotechnology of Sfax, Sfax, Tunisia (Received 20 August 2009, revised 30 September 2009, accepted 29 October 2009) doi:10.1111/j.1742-4658.2009.07469.x Introduction The calcium ion is enormously important for regulating multiple cellular functions Its role as a second messenger, based on its low free cytosolic concentration under basal conditions ( 10 nm) and its transient increase ( 0.1–1 lm) upon cellular activation by multiple agonists following defined and distinct pathways, has been extensively studied This includes the distribution of its oscillatory patterns, the transport systems intervening in its management, its segregation in defined pools within intracellular organelles, the dynamic exchanges among these intracellular pools, and its vivid cross-talk with other second messengers [1–7] An important player participating in many Ca2+mediated cellular functions is calmodulin (CaM), a multifunctional omnipresent regulator in eukaryotic cells, which, by acting as an intracellular Ca2+ sensor, takes part in the generation, dynamics and fate of the Ca2+ signal by decoding its meaning, thus participat- Abbreviations BD, binding domain; CaM, calmodulin; EGFR, epidermal growth factor receptor; ER, endoplasmic reticulum; GPCR, G protein-coupled receptor; IP3, inositol-1,4,5-trisphosphate; Jak2, Janus kinase 2; JM, juxtamembrane; LD, like domain; NCX, Na+ ⁄ Ca2+ exchanger; NLS, nuclear localization sequence; PKC, protein kinase C; PMCA, plasma membrane Ca2+-ATPase; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase; siRNA, small interfering RNA; STIM, stromal interaction molecule; TM, transmembrane; W-13, N-(4-aminobutyl)-5-chloro1-naphthalenesulfonamide; W-7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide FEBS Journal 277 (2010) 327–342 ª 2009 The Authors Journal compilation ª 2009 FEBS 327 ´ ´ P Sanchez-Gonzalez et al Calmodulin and the EGFR ing in the ensuing outcome of multiple Ca2+-controlled cellular responses [8–15] Among the multiple systems controlled by the Ca2+ ⁄ CaM complex is the epidermal growth factor receptor (EGFR) This tyrosine kinase receptor belongs to the ErbB family, which comprises four members: EGFR ⁄ ErbB1 ⁄ HER1, ErbB2 ⁄ Neu ⁄ HER2, ErbB3 ⁄ HER3 and ErbB4 ⁄ HER4 These receptors enrol a large family of peptidic ligands that induce the formation of active auto(trans)phosphorylated receptor homo ⁄ heterodimers The active dimers, upon recruitment of adaptor and signalling proteins, initiate multiple signalling events [16–20] The EGFR is implicated in the control of cell proliferation and differentiation, cell survival, apoptosis and cellular migration The EGFR and other ErbB receptors are prone to undergo multiple mutations, gene amplification and ⁄ or overexpression processes in a variety of human cancers, thus contributing to their pathogenesis [16–19] The Ca2+ signal generated by the EGFR The activation of the EGFR generates a Ca2+ signal, broadly defined as the transient rise of the intracellular concentration of Ca2+ This is followed by the formation of the Ca2+ ⁄ CaM complex and the initiation of elaborated mechanisms pertaining to the control of the receptor at multiple levels [21,22] This Ca2+ signal occurs in multiple cell types [23–28] The cytosolic Ca2+ rise is followed by an increase in the concentration of free Ca2+ in the nucleus [28–30], and this process appears to be relevant for Ca2+-regulated gene transcription after the decoding of the amplitude and frequency of the Ca2+ signal, although the mechanism involved in the Ca2+ translocation process is not yet fully understood Complex oscillatory changes in the cytosolic concentration of Ca2+ in response to different concentrations of EGF, and hence the number of occupied receptors, have been observed [30–32] Contributing to this complexity, the EGF-induced Ca2+ signal has two components: a Ca2+ release from intracellular stores and a net Ca2+ influx from the outer medium [24,26,33–35] Moreover, both processes might occur sequentially because of the implication of store-operated (capacitative) Ca2+ channels (Fig 1), which start to work in EGF-stimulated cells after the depletion of intracellular Ca2+ stores [36] The EGFR-mediated activation of both phospholipases Cc and A2, together with the subsequent synthesis of a series of messengers that act as effectors of Ca2+-channels, is responsible for the cytosolic Ca2+ rise Phospholipase Cc (EC 3.1.4.11) hydrolyzes 328 phosphatidylinositol 4,5-bisphosphate yielding inositol1,4,5-trisphosphate (IP3), and phospholipase A2 (EC 3.1.1.4) releases arachidonic acid, which is transformed thereafter to leukotriene C4 IP3 releases Ca2+ from the endoplasmic reticulum (ER) [6] (Fig 1), whereas leukotriene C4 opens plasma membrane voltage-insensitive Ca2+-channels [37,38] Ca2+ influx into the cytosol activates small conductance Ca2+-activated K+ channels, which enhances the transmembrane electrical potential [39,40] This activates hyperpolarizationsensitive Ca2+-channels, contributing to an enhancement of the Ca2+ influx [40] Below a cytosolic Ca2+ concentration of 0.2 lm, the Ca2+ ⁄ CaM complex is undetectable in intact cells [41] Therefore, this extracellular Ca2+ influx, occurring with a delay of approximately 20–30 s [40], should contribute greatly to the formation of the Ca2+ ⁄ CaM complex EGFR activation also engages store-operated (capacitative) Ca2+ channels, which start to become functional upon exhaustion of the ER Ca2+ pool [36] This mechanism implicates the stromal interaction molecule (STIM) ⁄ Orai system [7,42,43], where STIM1 or STIM2, acting as a Ca2+ sensor, located in the ER membrane, are translocated to the plasma membrane and clustered at the ER-plasma membrane junctions after the detection of a shortage of Ca2+ in the ER lumen Subsequently, STIM engages Ca2+ channels denoted Orai, also called calcium-released-activated calcium modulator 1, located at the plasma membrane, allowing the entry of Ca2+ into the cytosol (Fig 1) The fast increasing cytosolic Ca2+ concentration is brought to a halt and, eventually, returns to its basal level within a few minutes as a result of the operation of several Ca2+ transport systems that remove Ca2+ from the cytosol, including the sarco(endo)plasmic reticulum Ca2+-ATPase (EC 3.6.3.8) (SERCA), the CaM-dependent plasma membrane Ca2+-ATPase (PMCA) and the Na+ ⁄ Ca2+ exchanger (NCX), thus ensuring the transient nature of the Ca2+ signal (Fig 1) Indirect regulation of the EGFR by CaM-dependent kinases The Ca2+ ⁄ CaM complex indirectly controls the functionality of the EGFR by activating CaM-dependent protein kinases (EC 2.7.11.17), which in turn phosphorylate the receptor In this context, the EGFR is phosphorylated by CaM-dependent protein kinase II (CaMKII) at S744, S1046, S1047, S1057 and S1142, with the first one being located in its tyrosine kinase domain [44,45] EGF-dependent phosphorylation of the EGFR by CaMKII down-regulates its tyrosine FEBS Journal 277 (2010) 327–342 ª 2009 The Authors Journal compilation ª 2009 FEBS ´ ´ P Sanchez-Gonzalez et al Calmodulin and the EGFR Fig EGFR-mediated capacitative Ca2+ entry The EGFR-induced release of Ca2+ from the ER (as described in the text) results in the eventual depletion of Ca2+ from its lumen The low luminal Ca2+ concentration is sensed by the STIM (e.g its isoform STIM1), inducing its clustering and translocation to the plasma membrane, and its association with a member of the Ca2+ channels denoted Orai, which is also known as calcium-released-activated calcium modulator (CRACM) [e.g Orai1 (CRACM1)] This process occurs at the peripheral ER in proximity to the plasma membrane STIM proteins also participate in the microtubule-induced pulling of the ER to the vicinity of the plasma membrane (not shown) The Orai channels therefore take over the role of augmenting the cytosolic Ca2+ concentration when the ER store is depleted The transport systems SERCA, PMCA and NCX subsequently operate to return the cytosolic Ca2+ concentration to its basal level Additional details are provided in the text kinase activity and increases the rate of endocytosis [44,45] Replacement of either S1046 and ⁄ or S1047 to alanine yields EGFR mutants with a very low endocytosis rate and decreased down-regulation, but without impaired EGF binding capacity or decreased tyrosine kinase activity [44,46] By contrast, the S1046A ⁄ S1047A mutations enhance the EGFR tyrosine kinase and the capacity to transform fibroblasts, as measured by foci formation by transfected cells, and these effects are further increased by introducing additional mutations at S1057 and S1142, particularly at the latter residue [45] The S744A substitution also results in a mutant EGFR with close to double tyrosine kinase activity compared to its wild-type counterpart [45] S744 is located at the C a-helix in the N-lobe of the tyrosine kinase domain, close to residues K721 and E738, which are known to interact when the receptor is in its active conformation [47] In addition, this S744 is exposed to the interface of the C-lobe of the tyrosine kinase domain of the apposed monomer during dimerization [47,48] Thus, phosphorylation of S744 could disrupt the electrostatic K721–E738 interaction and ⁄ or avert the correct contact between apposed tyrosine kinase domains, thus preventing EGFR activation This could explain why the S744A mutation activates (and the phosphomimetic S744D mutation inhibits) the receptor [45] CaMKII also targets the leukemogenic truncated chicken erbB oncogene product at S477 ⁄ S478 The relevant gene encodes for an EGFR homologue lacking its extracellular domain S477 ⁄ S478 are homologue residues of S1046 ⁄ S1047 in the human EGFR Mutation of these residues enhances its oncogenic potential, as demonstrated in vitro by anchorageindependent growth of chicken embryos and murine fibroblasts, and by the formation of wing web tumours in vivo [49] Moreover, it has been shown that the overexpression of CaMKIb2 also negatively regulates the EGFR, and hence its downstream signalling, by inducing ligandindependent internalization and the subsequent degradation of the receptor in transfected human embryonic kidney cells [50] Direct regulation of the EGFR by CaM The direct regulation of the EGFR upon binding of the Ca2+ ⁄ CaM complex to the receptor has been extensively studied This binding process plays a FEBS Journal 277 (2010) 327–342 ª 2009 The Authors Journal compilation ª 2009 FEBS 329 ´ ´ P Sanchez-Gonzalez et al Calmodulin and the EGFR prominent role in EGF-dependent activation and the fate of this receptor The EGFR CaM-binding domain The first report demonstrating that the EGFR is a CaM-binding protein arise from studies performed by our group, in which the detergent-solubilized receptor was isolated from rat liver by Ca2+-dependent CaMaffinity chromatography [51] In this early work, it was suggested that the cytosolic juxtamembrane (JM) region of the receptor, more precisely the sequence (645)RRRHIVRKRTLRRLLQ(660) containing eight positively-charged amino acids distributed in three basic clusters (shown underlined), was implicated in Ca2+-dependent CaM binding, as subsequently demonstrated experimentally [52–54] Another study concluded that the R645–R657 segment was the relevant part involved in Ca2+ ⁄ CaM binding, and indicated the important but not exclusive relevance of the R647 residue [53] Moreover, the Ca2+-dependent interaction of CaM with the full-length EGFR was also demonstrated, employing both cross-linkage reagents followed by immunoprecipitation of the CaM ⁄ EGFR complex and overlay techniques using biotinylated CaM [55] One characteristic of the detergent-solubilized rat liver EGFR isolated by CaM-affinity chromatography was that the binding of EGF induces the phosphorylation of not only tyrosine residues, but also serine residues to some extent, suggesting the presence of some serine ⁄ threonine-kinase(s) in the preparations [51] In these less-than-ideal detergent-solubilized EGFR preparations, the addition of exogenous CaM inhibited the tyrosine kinase activity of the receptor in a manner that was partially dependent on the presence of Ca2+ [51] Intriguingly, the detergent-solubilized receptor presents high tyrosine kinase activity in the absence of ligands [51] This basal activity was further activated (up to two- to three-fold) by the presence of EGF or transforming growth factor-a in preparations isolated from rat liver [51], but not at all in preparations isolated from murine fibroblasts that were stably transfected with the human EGFR, where the detergent-solubilized receptor appears to be fully active in the absence of ligands [52] This suggests that membrane integrity could be a prerequisite for maintaining the tyrosine kinase of the EGFR in an auto-inhibited state in the absence of ligands This observation agrees perfectly with a model in which an auto-inhibitory role was ascribed to the positively-charged cytosolic JM region and part of the tyrosine kinase domain, with both electrostatically interacting with the negatively330 charged inner leaflet of the plasma membrane in the absence of ligands [54] The cytosolic JM sequence R645–Q660 was predicted to form a basic amphiphilic a-helix [52], as usually occurs in distinct CaM binding sites from other proteins [56] The organization of the cytosolic JM region of the EGFR in three helical segments, in which the first segment comprises the CaM-binding domain (BD), has been determined by NMR spectroscopy using the recombinant R645–G697 peptide bound to phospholipid micelles [57] By contrast, this peptide presents a mostly unstructured conformation in aqueous solution, even though a nascent helix, including the segment containing a di-leucine motif at residues 679 ⁄ 680, was detected [57] The helical conformation of the CaM-BD was also confirmed by solid-state NMR using a peptide (I622–Q660) corresponding to the transmembrane (TM) region plus the first part of the cytosolic JM segment containing the CaM-BD of the receptor reconstituted into phospholipid vesicles, except for a nonhelical structure detected just at the TM ⁄ JM boundary [58] More recently, the X-ray crystallographic structure of the intracellular region of the EGFR lacking the C-terminal tail (residues R645–G998) has been obtained [48] In this crystal structure, the segment T654–Q660, corresponding to the distal part of the CaM-BD, clearly forms an a-helix, although insufficient resolution was achieved to allow visualization of the proximal part of the CaM-BD comprising the R645–R653 segment [48] The functional importance of the CaM-binding domain The functional importance of the CaM-BD was demonstrated upon deletion of residues R645–L657 ⁄ L658, resulting in mutant receptors with no detectable EGFdependent tyrosine kinase activity but maintaining intact ligand-binding capacity [59–61] Significantly, no apparent aberrant intracellular localization of the deleted receptor was detected [61] Moreover, this deletion also inhibits the tyrosine kinase activity of a truncated receptor lacking its extracellular region [61] The deletion of the CaM-BD prevents the binding of the EGFR to agarose-immobilized CaM [62] Furthermore, the substitution of some positively-charged amino acids in the cytosolic JM region of the receptor to neutral amino acids (asparagine or alanine) also results in tyrosine kinase-mute receptors [48,59] A detailed analysis by performing alanine-scanning mutagenesis of each one of the CaM-BD residues shows that the R646A and R647A mutations are the most FEBS Journal 277 (2010) 327–342 ª 2009 The Authors Journal compilation ª 2009 FEBS ´ ´ P Sanchez-Gonzalez et al Calmodulin and the EGFR disruptive for the tyrosine kinase activity [48] As in the case of the JM deletion mutants, no significant difference in EGF binding affinity was detected in all the JM basic-to-neutral substitution mutants tested [59] Deletion of the Q660–P667 segment, however, does not alter the affinity of the receptor for its ligand or its intrinsic tyrosine kinase activity, but dramatically decreases EGF-dependent proliferation [63] Overall, these data suggest that prevention of CaM binding to the CaM-BD impairs EGFR activation The insertion of a 23 amino acid segment containing eight net negative charges into the cytosolic JM region between the first and second basic clusters of the CaM-BD results in a receptor with a slight increment in EGF binding capacity [64] The 658 ⁄ 659 di-leucine motif within the CaM-BD, and the previously mentioned distally located di-leucine motif at residues 679 ⁄ 680, play an important role in the normal expression and turnover of the EGFR [65] Further studies with the A679 ⁄ A680 mutant confirmed that the 679 ⁄ 680 di-leucine motif facilitates the sequestration of the ligand-occupied EGFR into multivesicular endosomes, which direct the receptor to lysosomal degradation [66] When a peptide corresponding to the JM segment R645–R657 of the EGFR was added either to the purified full-length receptor, a C-terminal deleted receptor (D1022–1186) or a constitutively active receptor lacking the extracellular ligand-binding site, the tyrosine phosphorylation stoichiometry of those receptors was enhanced in all cases, although to a different degree [67] The resulting tyrosine phosphorylated residues in the receptor were identified as those usually targeted by c-Src [67] Because the activating effect of the R645–R657 peptide was also observed in a constitutively active EGFR lacking the ligand-binding site, it was concluded that the R645–R657 peptide competes to disrupt the interaction of the JM region with another non-identified region in the receptor [67] The non-identified region of the receptor to which the JM region might bind could correspond to the tyrosine kinase domain [68,69] or the acidic CaM-like domain (LD) [22,61,70] (Fig 2) The R645–R657 segment of the EGFR also appears to be relevant for the binding and phosphorylation of the a subunit of a trimeric stimulatory G protein [60] Moreover, it is important to note that the cytosolic JM region of the EGFR also interacts with other EGF EGF EGF EGF EGF EGF EGF EGF + + + + + + + + + - +++++ - +++++ +++++ - +++++ - - - + (Quasi-stable dimer) - CaM CaM Inactive (monomer) - CaM CaM - CaM +++++ Cyt +++++ Ext CaM Active (stable dimer) (Quasi-stable dimer) Inactive (monomer) Fig The CaM-BD ⁄ CaM-LD and the CaM-BD ⁄ membrane electrostatic interaction models The first model (from left to centre) proposes that the positively-charged CaM-BD interacts with the negatively-charged CaM-LD, thus maintaining the unoccupied receptor monomers (and ⁄ or unoccupied receptor oligomers; not shown) in an auto-inhibited state Upon EGF binding, the receptor is activated and the formed Ca2+ ⁄ CaM complex actively undoes the intra-molecular CaM-BD ⁄ CaM-LD electrostatic interaction, although the formed EGFR dimer is maintained in a quasi-stable conformation The subsequent occurrence of inter-molecular CaM-BD ⁄ CaM-LD electrostatic interaction between apposed monomers further stabilizes the active dimer On the basis, in part, of previously proposed models [22,61,70] The second model (from right to centre) proposes that the positively-charged CaM-BD of the EGFR interacts with the negatively-charged inner leaflet of the plasma membrane, thus maintaining the unoccupied receptor monomers (and ⁄ or unoccupied receptor oligomers; not shown) in an autoinhibited state Upon EGF binding, the receptor is activated with the help of the Ca2+ ⁄ CaM complex that actively pulls off the CaM-BD from the membrane, thus undoing the auto-inhibitory CaM-BD ⁄ membrane electrostatic interaction We propose that the quasi-stable dimer is thereafter stabilized by an inter-molecular CaM-BD ⁄ CaM-LD electrostatic interaction between apposed monomers This is based, in part, on the electrostatic engine model previously proposed [54] The positively-charged CaM-BD and the negatively-charged CaM-LD are highlighted, respectively, as boxes with plus (+) and minus ()) signs The lengths of the CaM-BD and CaM-LD, in comparison with the total length of the EGFR, are not drawn to scale, and the presented conformational changes in the receptor chain are arbitrarily assigned Additional details are provided in the text FEBS Journal 277 (2010) 327–342 ª 2009 The Authors Journal compilation ª 2009 FEBS 331 ´ ´ P Sanchez-Gonzalez et al Calmodulin and the EGFR proteins containing Src homology domains and 3, such as the adaptors p97eps8 [71] and Nck [72] The binding of these proteins could prevent the interaction of the Ca2+ ⁄ CaM complex to the receptor if the CaMBD were occluded at least in part Nevertheless, the docking of these adaptor proteins to the JM could initiate additional sustained signalling events unrelated to the canonical docking of signalling proteins to the autophosphorylated tyrosine residues in the C-terminal tail of the receptor Protein kinase C (EC 2.7.11.1) (PKC)-mediated phosphorylation of the CaM-BD PKC phosphorylates the EGFR at T654 located at its cytosolic JM region [73] Besides blocking the EGFR tyrosine kinase activity and the associated mitogenic response [46,73–77], this PKC-mediated phosphorylation slows both ligand-induced internalization and degradation of the receptor within the lysosomal ⁄ proteasomal pathways [76,78,79], favouring the recycling back of internalized receptors from early endosomes to the cell surface [77] It has been proposed, however, that T654 phosphorylation by PKC transiently enhances signalling by the ligand-activated receptor before inactivation takes place, possibly because of the stabilization of receptor dimers ⁄ oligomers [80] Although treatment with a phorbol ester results in a decreased exposure of high affinity EGFbinding sites [46,79,81], this effect appears to be independent, or at least not exclusively dependent, on T654 phosphorylation [46,81] The use of cells transfected with EGFR mutants with either the phosphorylation-negative substitutions T654A [46,79,80] or T654Y (not phosphorylatable by PKC) [75], and the phospho-mimetic substitution T654E [81], supports the above conclusions Because T654 is located within the CaM-BD of the EGFR [52], this suggests that CaM could play a role regulating the intracellular traffic of the EGFR upon phosphorylation of this residue and ligand-induced internalization Thus, binding of the Ca2+ ⁄ CaM complex to this site prevents PKC-mediated phosphorylation of T654 and, conversely, phosphorylation of T654 by PKC prevents CaM binding [52,53,82] This effect was mimicked by the T654E substitution [53,82], suggesting that the ionized phosphate in phosphorylated T654 and the negative charge of glutamic acid both prevent CaM binding by electrostatic repulsion [52,53,82] Because phosphorylation of a glutathione S-transferase (EC 2.5.1.18)-JM(T654G) mutant peptide by PKC also inhibited CaM binding, it was concluded that an additional nonspecified phosphorylation site(s) 332 besides T654 could be involved in the process [53] However, the phosphorylation of T669 was excluded with respect to affecting CaM binding [82] Mechanistic models for CaM-regulated EGFR activation The current data strongly favour the view that a highaffinity form of unoccupied receptors is present at the plasma membrane in an inactive but pre-dimerized ⁄ oligomerized state, which is subsequently activated after ligand binding by inducing the rotational reorganization of both monomers, with such an activation mechanism being termed the twist model [83] An asymmetric allosteric model accounts for the EGF-dependent activation of the EGFR, where the C-terminal lobe of the kinase domain of one of the monomers forming the dimeric receptor interacts with the N-terminal lobe of the apposed monomer [19,47,84] Of relevance for the implication of CaM in this model, it has been shown that the intracellular JM region of the EGFR, which contains the CaM-BD, plays an indispensable role in the operation of this allosteric activation mechanism [85], and also exerts an allosteric control of ligand binding [86] This is a result of the interaction of the distal segment of the JM, particularly the E663–S671 residues, with the C-terminal lobe of the kinase domain of the apposed monomer [48,69], as well as the stabilizing role that the dimerization of the proximal segment of the JM, comprising the CaM-BD, exerts on the receptor [69] An auto-inhibitory role for the CaM-BD of the EGFR on its activity in the absence of ligand was first proposed by our group based on the potential electrostatic interaction between the positively-charged R645– Q660 segment with the negatively-charged segment (979)DEEDMDDVVDADEY(992), containing four acidic clusters (underlined), located distal from the tyrosine kinase domain of the human receptor [22] We denoted this segment the CaM-LD because of its partial similarity to a region in human CaM, with the sequence (118)DEEVDEMIREADI(130) [22] The putative CaM-DB ⁄ CaM-LD electrostatic interaction was initially modelled to occur intramolecularly within a single unoccupied monomer [22] (Fig 2, left to centre) This model was later modified and refined, based on in silico structural modelling studies, when the occurrence of an intermolecular electrostatic interaction was suggested between apposed EGFR monomers in which the positively-charged R645–R657 segment and the negatively-charged D979–E991 segment facilitate the formation of dimers after EGF binding [53,70] (Fig 2, left to centre) It was also suggested in these FEBS Journal 277 (2010) 327–342 ª 2009 The Authors Journal compilation ª 2009 FEBS ´ ´ P Sanchez-Gonzalez et al studies that, in the absence of ligand, the receptor is maintained in an inactive conformation by CaM, by which the T654 residue in the EGFR interacts with the E120 residue in CaM [82] By contrast, after EGF binding, it was proposed that T654 in the CaM-BD forms a hydrogen bond with an aspartic acid within the CaM-LD of the apposed monomer, thus stabilizing the EGFR dimer [82] This latter model, which is purported to explain the auto-inhibited state of the EGFR in the absence of ligand, is reminiscent of another previously proposed model, which was based on the electrostatic interaction of the sequence corresponding to the CaM-LD with the tyrosine kinase domain of the apposed monomer [68,69] Nevertheless, experiments performed with a truncated EGFR lacking the CaM-LD suggest that this region might not be involved in the allosteric regulation of ligand binding affinity in the receptor [86] Interestingly, the occurrence of in-frame tandem duplication of exons 18–25 ⁄ 18–26 in the EGFR gene results in mutant receptors with duplication of the CaM-LD, as detected in a set of human gliomas [87– 89] The functional consequences, if any, of these mutations with respect to the proposed CaMBD ⁄ CaM-LD electrostatic interaction model are unknown and therefore are worthy of being studied further An alternative model that might explain the role of Ca2+ ⁄ CaM on EGFR activation suggests that, in the absence of ligands, the positively-charged CaM-BD and a positively-charged segment of the tyrosine kinase domain both electrostatically bind to the negativelycharged inner leaflet of the plasma membrane [54] This maintains the receptor in an auto-inhibited conformation and, upon binding of the Ca2+ ⁄ CaM complex to this site, the auto-inhibition is released [54] (Fig 2, right to centre) This mechanism, dubbed ‘the electrostatic engine model’, results in the sequestration of polyvalent acidic lipids such as phosphatidylinositol 4,5-bisphosphate, but not of monovalent acidic lipids such as phosphatidylserine, by the CaM-BD embedded in the inner leaflet of the plasma membrane [54,90,91] This also occurs with other basic amino acid segments in peripheral and other integral membrane proteins [92] This CaM-BD ⁄ membrane electrostatic interaction model has attained further experimental support as a result of studies demonstrating that the distal part of an I622–Q660 peptide, corresponding to the TM ⁄ JM segment, or a derivative of the free R645–Q660 peptide, bind to the outer leaflet of phospholipid vesicles by electrostatic interaction, and that the addition of CaM in the presence of Ca2+ efficiently releases those peptides from the membrane [58,93,94] Furthermore, Calmodulin and the EGFR structural models suggest that the proximal region of the JM segment (essentially formed by the CaM-BD) of apposed monomers could form an antiparallel helical dimer, and that the side chains of the basic amino acids could interact with the negatively-charged inner leaflet of the plasma membrane [69] Kinetics measurements, using stop-flow techniques with a fluorescent probe-labelled peptide corresponding to the CaM-BD (R645–Q660) of the EGFR bound to phospholipid vesicles, strongly support the idea that the Ca2+ ⁄ CaM complex actively and very rapidly pulls the JM domain out of the membrane, instead of passively binding once it spontaneously detaches from the membrane [94] Moreover, it has been shown that the introduction of palmitoylation consensus sites in the cytosolic JM region of the EGFR (substitutions R647C and V650C) yields mutant palmitoylated receptors in transfected cells The cytosolic JM region of the palmitoylated EGFR is linked to the membrane, thus restricting the helical rotation or tilt of the CaM-BD with respect to the membrane plane during EGFdependent activation [95] This produces a receptor exhibiting only low-affinity EGF binding sites and significantly lower autophosphorylation and internalization capacities [95] The electrostatic interaction of the R645–Q660 peptide with the membrane was also disrupted by the presence of weak bases, such as different CaM antagonists [93], suggesting that the results derived from experiments in living cells with these widely used compounds should be interpreted cautiously when studying their action on different CaM-dependent systems because of the possible existence of unwanted side effects as a result of the potential detachment of autoinhibitory sites of the protein under study from cell membranes In this context, a dual action of the CaM inhibitor N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide (W-13) on the activity of the EGFR in living cells was observed: a stimulatory action when assayed in the absence of EGF [93,96,97], most likely a result of the disruption of the auto-inhibitory CaMBD ⁄ membrane interaction [93]; and an inhibitory action when assayed in the presence of the ligand, interpreted as a consequence of CaM inhibition, suggesting that the Ca2+ ⁄ CaM complex could be required for EGF-dependent EGFR activation in living cells [54,93,98] Regulation of the EGFR by CaM in living cells We have co-immunoprecipitated EGFR and CaM from two distinct cell lines overexpressing the receptor FEBS Journal 277 (2010) 327–342 ª 2009 The Authors Journal compilation ª 2009 FEBS 333 ´ ´ P Sanchez-Gonzalez et al Calmodulin and the EGFR [98], suggesting the occurrence of this complex in living cells Moreover, the cell-permeable high affinity CaM antagonists W-13 and N-(6-aminohexyl)-5-chloro-1naphthalenesulfonamide (W-7) and, to a much lesser extent, the low affinity analogue N-(4-aminobutyl)-1naphthalenesulfonamide (W-12), prevent in part the EGF-dependent activation of the receptor in cultured cells [54,93,98] This phenomenon could explain the inhibitory action of W-7 on the EGF-dependent proliferation of cells [99] The inhibitory effect of W-13 was not observed, however, in an insertional EGFR mutant in which the CaM-BD was split in two by an intervening sequence rich in acidic amino acids, which was expected to disrupt CaM binding [98] The inhibitory action of W-13 was strongly enhanced upon treating the cells with the Ca2+ ionophore A23187 [93], suggesting that Ca2+ favours the interaction of W-13 with CaM It is important to note that the inhibitory effect of these CaM antagonists was not observed in assays performed in vitro using a detergent-solubilized EGFR preparation [100], in contrast to living cells Moreover, this inhibition was observed even when both PKC and CaMKII activities were abolished by cell permeable specific inhibitors [93] These experiments exclude the participation of interfering Ca2+-dependent and ⁄ or Ca2+ ⁄ CaM-dependent regulatory systems of the EGFR during the testing of the CaM antagonists in living cells As noted above, W-13 was shown to enhance tyrosine-phosphorylation of the EGFR in the absence of ligand in distinct cell lines [93,96,97] This observation was first ascribed to the activation of metalloproteases (EC 3.4.24), which appears to induce the shedding of heparin-binding-EGF, but not of amphiregulin or transforming growth factor-a, thus activating the receptor and its downstream signalling pathways as a result of the recruitment of the SH2 containing adaptor protein Shc [96,97] An alternative explanation, however, is that W-13 releases the positivelycharged CaM-BD of the EGFR from the negativelycharged inner leaflet of the plasma membrane because this agent is a weak base [93] In any event, the action of CaM on the downstream pathways of the EGFR, such as the Ras ⁄ mitogen-activated protein kinase (EC 2.7.11.24) pathway [96,97,101–103] and the IP3 kinase ⁄ Akt [104] axis, have also been demonstrated, independent of its action on the receptor In this latter study, CaM expression was significantly down-regulated using a mixture of small interfering (si)RNAs targeting the three gene transcripts coding CaM in mammalian cells [104] However, the potential effect of these siRNAs on EGF-dependent autophosphorylation was not tested 334 The implication of CaM on EGFR-mediated cellular functions Different upstream and downstream signalling pathways controlling or affecting EGFR-mediated cellular functions are modulated by CaM (Fig 3) In this context, it was demonstrated in an early study that CaM antagonists decreased the binding of [125I]EGF to the cell surface of simian virus 40-transformed fibroblasts [105] This process was correlated with a decrease in the affinity of the EGFR for its ligand but not a decrease in the number of receptors present at the cell surface [105] Furthermore, an intriguing observation shows that, in skeletal muscle cells, activation of EGFR results in the association of the glycolytic enzymes phosphofructokinase (EC 2.7.1.11) and aldolase (EC 4.1.2.13) to the cytoskeleton, and that CaM antagonists prevent this association both in vitro and in vivo [106] The molecular mechanism responsible for (as well as the possible physiological significance of) these effects nevertheless remains unclear The role of CaM in intracellular EGFR traffic Inhibition of CaM by W-13 does not appear to block EGFR internalization but interferes with intracellular EGFR traffic by favouring the sequestration of the receptor in early endosomes, thus preventing either its recycling back to the plasma membrane or its onward transport to the lysosomal degradation pathway [96] (Fig 3) This process appears to be controlled by PKCd because the inhibition of this kinase by rottlerin or decreasing its expression by siRNA technology restores EGFR traffic [107] The mechanism underlying PKCdmediated EGFR sequestration in endosomes appears to be a result of the formation of an F-actin coat surrounding these intracellular vesicles [108] In this context, the JM region distal from residue R651 up to residue L723 was first implicated in intracellular EGFR sorting [109] More precisely, the L652–A674 segment, which partially overlaps the CaM-BD (R645–Q660), was subsequently demonstrated to constitute the sorting determinant because it was able to direct the migration of the EGFR from the trans-Golgi network to the basolateral plasma membrane in polarized cells [110] This suggests that CaM could play a regulatory role in EGFR sorting Involvement of CaM in G protein-coupled receptor (GPCR)-mediated EGFR transactivation Transactivation of the EGFR mediated by different GPCRs has been shown to occur either by: (a) the shedding of EGFR ligands after the proteolytic pro- FEBS Journal 277 (2010) 327–342 ª 2009 The Authors Journal compilation ª 2009 FEBS ´ ´ P Sanchez-Gonzalez et al Calmodulin and the EGFR Fig CaM and the regulation of EGFR-mediated cellular functions The regulatory role of CaM on the functionality of the EGFR is exerted at multiple levels Thus, CaM modulates the following functions (in a clockwise order): (a) the control of the EGFR mediated by CaM-dependent kinases, such as CaMK-II, which phosphorylates the receptor; (b) the direct activation of the EGFR and the regulation of downstream signalling pathways; (c) intracellular EGFR traffic within endosomes (endo), which is either destined to lysosomes (lyso) for degradation or its recycling back to the plasma membrane; (d) the transactivation of the EGFR by GPCRs, either controlling the shedding of mature receptor ligands [e.g the heparin-binding (HB)-EGF-like growth factor] from the membrane-bound precursor (HB-EGF-pre) after its proteolysis by a matrix metalloprotease (MMP) and ⁄ or the activation of the nonreceptor tyrosine kinase Src, which directly phosphorylates the EGFR; and (e) putatively regulating the translocation of the EGFR into the nucleus, although the latter is a hypothetical mechanism inferred from the overlapping sequences of the CaM-BD and the NLS located at the cytosolic juxtamembrane region of the receptor The positively-charged CaM-BD ⁄ NLS region is highlighted as a box with a plus sign (+) The lengths of the CaM-BD ⁄ NLS, in comparison with the total length of the EGFR, is not drawn to scale, and the presented conformational changes in the receptor chain entering the nuclear pore are arbitrarily assigned Additional details are provided in the text cessing of membrane-bound ligand precursors by matrix metalloproteases, comprising ligands that would dimerize and activate the EGFR, or (b) by GPCRinduced activation of Src, which thereafter phosphorylates the EGFR and ⁄ or the SH2 containing adaptor protein bound Shc to the receptor, thus inducing downstream signalling [111,112] (Fig 3) Of relevance, crosstalk between distinct GPCRs and the EGFR appears to play a role in tumour cell resistance to therapeutic agents targeting the EGFR [113] CaM has been implicated in the transactivation of the EGFR arbitrated by GPCRs (Fig 3) Hence, in cardiac fibroblasts, the Ca2+ ⁄ CaM complex is involved in angiotensin II type receptor-mediated transactivation of the EGFR and activation of their downstream signalling pathways, as demonstrated upon abrogation of these phenomena by CaM antagonists such as W-7 and calmidazolium, or by loading the cells with 1,2bis(o-aminophenoxy)ethane-N,N,N¢,N¢-tetraacetic acid tetra(acetoxymethyl) ester [114] By contrast, the Ca2+ ionophore A23187 induces EGFR activation, a process that was fully abrogated by W-7, although this CaM inhibitor exerts, in this particular case, a lesser effect on the EGF-dependent activation of the receptor [114] EGFR transactivation by angiotensin II stimulation was not mediated by the shedding of EGFR ligands [114] Hence, the mechanism of action of Ca2+ ⁄ CaM in this process remains obscure Nevertheless, increasing the cytosolic concentration of free Ca2+ upon inhibiting SERCA with thapsigargin also stimulates EGFR phosphorylation and downstream mitogenactivated protein kinase signalling in intestinal epithelial cells [115] This suggests that a Ca2+-dependent mechanism could be involved Further confirmation was obtained during EGFR transactivation using carbachol, a muscarinic GPCR ligand that induces Ca2+ mobilization [115] Thus, it was demonstrated that loading cells with 1,2-bis(o-aminophenoxy)ethaneN,N,N¢,N¢-tetraacetic acid tetra(acetoxymethyl) ester or inhibiting CaM with an antagonist blocks the associa- FEBS Journal 277 (2010) 327–342 ª 2009 The Authors Journal compilation ª 2009 FEBS 335 ´ ´ P Sanchez-Gonzalez et al Calmodulin and the EGFR tion of the Ca2+-dependent tyrosine kinase PYK2 to the EGFR [115] Carbachol also induces the association of Src to the EGFR [115] The implication of CaM in the transactivation of the EGFR by other members of the GPCR family has also been reported Thus, lysophosphatidic acid receptors in myometrial smooth muscle cells activate the EGFR via the shedding of receptor ligands, and this process appears to be controlled by a CaM-dependent kinase [116] CaM action during EGFR transactivation is not mediated by a universal mechanism operative simply as a result of the entry of Ca2+ into the cell Thus, in rat pheocromocytoma PC12 cells, when Ca2+entry was stimulated by two distinct mechanisms, the CaM dependency of EGFR transactivation was dissected In this context, the KCl-mediated, but not the bradykinin-mediated, transactivation of the EGFR via the implication of a CaM-dependent kinase was pinpointed to the phosphorylation of the cytosolic tyrosine kinase PYK2 after Ca2+ entry into the cell as a result of cell membrane depolarization [117] Although no mechanistic information on the actual role of the CaM-dependent kinase was provided in these studies [116,117], more recently, the direct interaction of the Ca2+ ⁄ CaM complex with PYK2, by inducing its activation upon formation of a dimer, was reported [118] The action of CaM on GPCR-mediated EGFR transactivation could also be a result of the direct binding of CaM to the GPCR, as demonstrated with the l-opioid receptor [119] Potential implication of CaM in EGFR nuclear translocation An unanticipated finding currently under close scrutiny is the observation that the EGFR translocates to the nucleus in an EGF-dependent manner [120] as well as the identification of its nuclear localization sequence (NLS) as the R645–R657 segment [120,121] We have noted the overlap of the described NLS at R645–R657 with the CaM-BD at R645–Q660 [22,122], suggesting that the Ca2+ ⁄ CaM complex could regulate the translocation of the receptor to the nucleus (Fig 3) The phosphorylation of EGFR at T654, located within the CaM-BD, regulates the radiation- and phosphotyrosine-induced translocation of the receptor to the nucleus, as this process was demonstrated to be impaired by the specific down-regulation of PKCe by siRNA [123] Although not yet demonstrated, if CaM were to play a role in the translocation of the EGFR into the nucleus, the above-mentioned implication of PKC suggests that an additional regulatory crosstalk 336 between this kinase and CaM might exist during the nuclear translocation process Phosphorylation of CaM by the EGFR Multiple kinases phosphorylate CaM at serine, threonine or tyrosine residues, modifying different CaMdependent target systems [14] The first demonstration that the EGFR phosphorylates CaM was obtained in vitro using a detergent-solubilized EGFR preparation isolated by CaM-affinity chromatography [51,124, 125] or in detergent-permeabilized EGFR-overexpressing cells [126] This phosphorylation was dependent on the presence of histone or other basic polypeptide, which act as co-factors, and was inhibited by low concentrations of free Ca2+ [51,124–127] The phosphorylation of CaM by the EGFR not only occurs at Y99 [124], but also at Y138, as demonstrated using recombinant CaM mutants in which either of the two tyrosine residues were replaced with phenylalanine [127] Tyrosine-phosphorylated CaM could exert a stimulatory effect on the EGF-dependent activation of the EGFR [14] The functional importance of tyrosinephosphorylated CaM on EGFR-mediated activation of the downstream Na+ ⁄ H+ exchanger was demonstrated [128] Two alternative routes were proposed to account for these observations: in the first pathway, activation of Janus kinase (Jak2) by the EGF-activated receptor (independent of its tyrosine kinase activity) results in the phosphorylation of CaM at tyrosine residues by Jak2, and phospho(Y)-CaM binds and activates the Na+ ⁄ H+ exchanger [128] In the second pathway, the EGF-activated EGFR somehow promotes the association of CaM to the Na+ ⁄ H+ exchanger (independent of Jak2) thus inducing its activation [128] These studies suggest that the EGFR is unlikely to phosphorylate CaM in this system because an EGFR inhibitor, in contrast to a Jak2 inhibitor, has no significant effect on CaM phosphorylation However, the direct phosphorylation of CaM by the EGFR cannot be rigorously excluded because, in the presence of the Jak2 inhibitor, a residual EGF-dependent phosphorylation of CaM was clearly detected [128] CaM and other ErbB receptors We have also shown that ErbB2 directly interacts with CaM in a Ca2+-dependent fashion [100] Furthermore, in living cells, the permeable CaM antagonist W-7 also inhibits the heregulin b1-induced phosphorylation of ErbB2 [100] The Ca2+ ⁄ CaM complex also negatively regulates the tyrosine kinase activity of ErbB2 by an FEBS Journal 277 (2010) 327–342 ª 2009 The Authors Journal compilation ª 2009 FEBS ´ ´ P Sanchez-Gonzalez et al indirect mechanism consisting of the phosphorylation of its T1172 by CaMKII [129] Activation of ErbB4 also generates a Ca2+ signal, and the subsequent formation of the Ca2+ ⁄ CaM complex appears to regulate this receptor [130] It is relevant to highlight the phylogenetic similarities of both the CaM-BD and the CaM-LD orthologue sequences across species in the EGFR as well as other ErbB receptors [22,52,70] Nevertheless, a higher divergence in both CaM-BD and CaM-LD sequences in ErbB3 is apparent, suggesting a nonfunctional role of these segments in this tyrosine kinase-mute receptor [22,52,70] This also coincides with the lower affinity of the Ca2+ ⁄ CaM complex for a peptide corresponding to the CaM-BD of ErbB3 compared to the binding to homologous peptides from other ErbB receptors [54] Future perspectives The regulation of the EGFR by CaM is an emerging research topic that requires additional and innovative attention to clarify the mechanisms of action of this modulator when operating at distinct levels with respect to controlling the functionality and fate of this receptor We consider that a high-resolution crystallographic structure of the full-length receptor might help to explain the regulation exerted by CaM on the activation of the EGFR If the structure of distinct EGFR ⁄ CaM complexes, corresponding to different stages of the EGFR activation cycle, were obtained, this would provide great insight into the actual role that the direct binding of CaM to the receptor could play with regard to its activation mechanism Furthermore, the identification of naturally-occurring EGFR mutants potentially affected at the CaM-BD and ⁄ or CaM-LD in tumours could be of high medical importance Another area of interest is to determine whether the action of CaM on the different functions controlled by the EGFR is mediated by nonphosphorylated and ⁄ or phosphorylated CaM Acknowledgements Research in the authors’ laboratory was financed by ´ grants (to A.V.) from the Direccion General de Inves´ ´ tigacion, Ministerio de Ciencia e Innovacion ´ (SAF2008-00986), the Consejerı´ a de Educacion de la Comunidad de Madrid (S-BIO-0170-2006), the Agen´ cia Espanola de Cooperacion Internacional para el ˜ Desarrollo (A ⁄ 019018 ⁄ 08) and the European Commission (MRTN-CT-2005-19561) P.S.G was supported by a predoctoral fellowship from the Junta de Ampli- Calmodulin and the EGFR ´ acion de Estudios, CSIC, and K.J was supported by an AECID grant References Carafoli E (1987) Intracellular calcium homeostasis Annu Rev Biochem 56, 395–433 Carafoli E (2002) Calcium signaling: a tale for all seasons Proc Natl Acad Sci USA 99, 1115–1122 Berridge MJ, Bootman MD & Roderick HL (2003) Calcium: signalling: dynamics, homeostasis and remodelling Nat Rev Mol Cell Biol 4, 517–529 Guerini D, Coletto L & Carafoli E (2005) Exporting calcium from cells Cell Calcium 38, 281–289 Berridge MJ (2006) Calcium microdomains: organization and function Cell Calcium 40, 405–412 Berridge MJ (2007) Inositol trisphosphate and calcium oscillations Biochem Soc Symp 74, 1–7 ´ Varnai P, Hunyady L & Balla T (2009) STIM and Orai: the 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Nck [72] The binding of these proteins could prevent the interaction of the Ca2+ ⁄ CaM complex to the receptor if the CaMBD were occluded at least in part Nevertheless, the docking of these adaptor... internalization and the subsequent degradation of the receptor in transfected human embryonic kidney cells [50] Direct regulation of the EGFR by CaM The direct regulation of the EGFR upon binding of the Ca2+... activation of the EGFR, where the C-terminal lobe of the kinase domain of one of the monomers forming the dimeric receptor interacts with the N-terminal lobe of the apposed monomer [19,47,84] Of relevance