Docking protein FRS2 links the protein tyrosine kinase RET and its oncogenic forms with the mitogen-activated protein kinase signaling cascade

The receptor tyrosine kinase RET functions as the signal transducing receptor for the GDNF (for "glial cell-derived neurotrophic factors") family of ligands. Mutations in the RET gene were implicated in Hirschsprung disease (HSCR), multiple endocrine neoplasia type 2 (MEN 2), and thyroid carcinomas. In this report we demonstrate that the docking protein FRS2 is tyrosine phosphorylated by ligand-stimulated and by constitutively activated oncogenic forms of RET. Complex formation between RET and FRS2 is mediated by binding of the phosphotyrosine-binding domain of FRS2 to pY1062, a residue in RET that also functions as a binding site for Shc. However, overexpression of FRS2 but not Shc potentiates mitogen-activated protein (MAP) kinase activation by RET oncoproteins. We demonstrate that oncogenic RET-PTC proteins are associated with FRS2 constitutively, leading to tyrosine phosphorylation of FRS2, MAP kinase stimulation, and cell proliferation. However, loss-of-function HSCR-associated RET mutants exhibit impaired FRS2 binding and reduced MAP kinase activation. These experiments demonstrate that FRS2 couples both ligand-regulated and oncogenic forms of RET, with the MAP kinase signaling cascade as part of the response of RET under normal biological conditions and pathological conditions, such as MEN 2 and papillary thyroid carcinomas.     

RET/PTC (rearranged in transformation/papillary thyroid carcinomas) tyrosine kinase phosphorylates and activates phosphoinositide-dependent kinase 1 (PDK1): an alternative phosphatidylinositol 3-kinase-independent pathway to activate PDK1

Thyroid cancers are a leading cause of death due to endocrine malignancies. RET/PTC (rearranged in transformation/papillary thyroid carcinomas) gene rearrangements are the most frequent genetic alterations identified in papillary thyroid carcinoma. Although the oncogenic potential of RET/PTC is related to intrinsic tyrosine kinase activity, the substrates for this enzyme are yet to be identified. In this report, we show that phosphoinositide-dependent kinase 1 (PDK1), a pivotal serine/threonine kinase in growth factor-signaling pathways, is a target of RET/PTC. RET/PTC and PDK1 colocalize in the cytoplasm. RET/PTC phosphorylates a specific tyrosine (Y9) residue located in the N-terminal region of PDK1. Y9 phosphorylation of PDK1 by RET/PTC requires an intact catalytic kinase domain. The short (iso 9) and long forms (iso 51) of the RET/PTC kinases (RET/PTC1 and RET/PTC3) induce Y9 phosphorylation of PDK1. Moreover, Y9 phosphorylation of PDK1 by RET/PTC does not require phosphatidylinositol 3-kinase or Src activity. RET/PTC-induced phosphorylation of the Y9 residue results in increased PDK1 activity, decrease of cellular p53 levels, and repression of p53-dependent transactivation. In conclusion, RET/PTC-induced tyrosine phosphorylation of PDK1 may be one of the mechanisms by which it acts as an oncogenic tyrosine kinase in thyroid carcinogenesis.     

Protein-tyrosine Phosphatase SHP2 Contributes to GDNF Neurotrophic Activity through Direct Binding to Phospho-Tyr687 in the RET Receptor Tyrosine Kinase

The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr⁶⁸⁷ in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr⁶⁸⁷ and association with components of the Tyr¹⁰⁶² signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser⁶⁹⁶, a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr⁶⁸⁷ as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions.     

Phosphorylation-dependent C-terminal binding of 14-3-3 proteins promotes cell surface expression of HIV co-receptor GPR15

Membrane trafficking is dictated by dynamic molecular interactions involving discrete determinants in the cargo proteins and the intracellular transport machineries. We have previously reported that cell surface expression of GPR15, a G protein-coupled receptor (GPCR) that serves as a co-receptor for HIV, is correlated with the mode III binding of 14-3-3 proteins to the receptor C terminus. Here we provide a mechanistic basis for the role of 14-3-3 in promoting the cell surface expression of GPR15. The Ala mutation of penultimate phospho-Ser (S359A) that abolishes 14-3-3 binding resulted in substantially reduced O-glycosylation and the cell surface expression of GPR15. The surface membrane protein CD8 fused with the C-terminal tail of GPR15(S359A) mutant was re-localized in the endoplasmic reticulum (ER). In the context of S359A mutation, the additional mutations in the upstream stretch of basic residues (RXR motif) restored O-glycosylation and the cell surface expression. The RXR motif was responsible for the interaction with coatomer protein I (COPI), which was inversely correlated with the 14-3-3 binding and cell surface expression. These results suggest that 14-3-3 binding promotes cell surface expression of GPR15 by releasing the receptor from ER retrieval/retention pathway that is mediated by the interaction of RXR motif and COPI. Moreover, 14-3-3 binding substantially increased the stability of GPR15 protein. Thus 14-3-3 proteins play multiple roles in biogenesis and trafficking of an HIV co-receptor GPR15 to control its cell surface density in response to the phosphorylation signal.     

Central role of the threonine residue within the p+1 loop of receptor tyrosine kinase in STAT3 constitutive phosphorylation in metastatic cancer cells

The receptor tyrosine kinases (RTKs) RET, MET, and RON all carry the Met(p+1loop)-->Thr point mutation (i.e., 2B mutation), leading to the formation of tumors with high metastatic potential. Utilizing a novel antibody array, we identified constitutive phosphorylation of STAT3 in cells expressing the 2B mutation but not wild-type RET. MET or RON with the 2B mutation also constitutively phosphorylated STAT3. Members of the EPH, the only group of wild-type RTK that carry Thr(p+1loop) residue, are often expressed unexpectedly in different types of cancers. Ectopic expression of wild-type but not Thr(p+1loop)-->Met substituted EPH family members constitutively phosphorylated STAT3. In both RTK(Metp+1loop) with 2B mutation and wild-type EPH members the Thr(p+1loop) residue is required for constitutive kinase autophosphorylation and STAT3 recruitment. In multiple endocrine neoplasia 2B (MEN-2B) patients expressing RET(M918T), nuclear enrichment of STAT3 and elevated expression of CXCR4 was detected in metastatic thyroid C-cell carcinoma in the liver. In breast adenocarcinoma cell lines expressing multiple EPH members, STAT3 constitutively bound to the promoters of MUC1, MUC4, and MUC5B genes. Inhibiting STAT3 expression resulted in reduced expression of these metastasis-related genes and inhibited mobility. These findings provide insight into Thr(p+1loop) residue in RTK autophosphorylation and constitutive activation of STAT3 in metastatic cancer cells.     

Focal adhesion kinase (FAK) binds RET kinase via its FERM domain, priming a direct and reciprocal RET-FAK transactivation mechanism

Whether RET is able to directly phosphorylate and activate downstream targets independently of the binding of proteins that contain Src homology 2 or phosphotyrosine binding domains and whether mechanisms in trans by cytoplasmic kinases can modulate RET function and signaling remain largely unexplored. In this study, oligopeptide arrays were used to screen substrates directly phosphorylated by purified recombinant wild-type and oncogenic RET kinase domain in the presence or absence of small molecule inhibitors. The results of the peptide array were validated by enzyme kinetics, in vitro kinase, and cell-based experiments. The identification of focal adhesion kinase (FAK) as a direct substrate for RET kinase revealed (i) a RET-FAK transactivation mechanism consisting of direct phosphorylation of FAK Tyr-576/577 by RET and a reciprocal phosphorylation of RET by FAK, which crucially is able to rescue the kinase-impaired RET K758M mutant and (ii) that FAK binds RET via its FERM domain. Interestingly, this interaction is abolished upon RET phosphorylation, indicating that RET binding to the FERM domain of FAK is a priming step for RET-FAK transactivation. Finally, our data indicate that FAK inhibitors could be used as potential therapeutic agents for patients with multiple endocrine neoplasia type 2 tumors because both, treatment with the FAK kinase inhibitor NVP-TAE226 and FAK down-regulation by siRNA reduced RET phosphorylation and signaling as well as the proliferation and survival of tumor and transfected cell lines expressing oncogenic RET.     

Brain derived neurotrophic factor is involved in the regulation of glycogen synthase kinase 3β (GSK3β) signalling

Glycogen synthase kinase 3β (GSK3β) is involved in several biochemical processes in neurons regulating cellular survival, gene expression, cell fate determination, metabolism and proliferation. GSK3β activity is inhibited through the phosphorylation of its Ser-9 residue. In this study we sought to investigate the role of BDNF/TrkB signalling in the modulation of GSK3β activity. BDNF/TrkB signalling regulates the GSK3β activity both in vivo in the retinal tissue as well as in the neuronal cells under culture conditions. We report here for the first time that BDNF can also regulate GSK3β activity independent of its effects through the TrkB receptor signalling. Knockdown of BDNF lead to a decline in GSK3β phosphorylation without having a detectable effect on the TrkB activity or its downstream effectors Akt and Erk1/2. Treatment with TrkB receptor agonist had a stimulating effect on the GSK3β phosphorylation, but the effect was significantly less pronounced in the cells in which BDNF was knocked down. The use of TrkB receptor antagonist similarly, manifested itself in the form of downregulation of GSK3β phosphorylation, but a combined TrkB inhibition and BDNF knockdown exhibited a much stronger negative effect. In vivo, we observed reduced levels of GSK3β phosphorylation in the retinal tissues of the BDNF^+/− animals implicating critical role of BDNF in the regulation of the GSK3β activity. Concluding, BDNF/TrkB axis strongly regulates the GSK3β activity and BDNF also exhibits GSK3β regulatory effect independent of its actions through the TrkB receptor signalling.     

Novel mechanism of regulation of Rac activity and lamellipodia formation by RET tyrosine kinase

Rac activation in neuronal cells plays an important role in lamellipodia formation that is a critical event for neuritogenesis. It is well known that the Rac activity is regulated via activation of phosphatidylinositol 3-kinase (PI3K) by a variety of receptor tyrosine kinases. Here we show that increased serine phosphorylation on RET receptor tyrosine kinase following cAMP elevation promotes lamellipodia formation of neuronal cells induced by glial cell line-derived neurotrophic factor (GDNF). We identified serine 696 in RET as a putative phosphorylation site by protein kinase A and found that mutation of this serine almost completely inhibited lamellipodia formation by GDNF without affecting activation of the PI3K/AKT signaling pathway. Mutation of tyrosine 1062 in RET, whose phosphorylation is crucial for activation of PI3K, also inhibited lamellipodia formation by GDNF. Inhibition of lamellipodia formation by mutation of either serine 696 or tyrosine 1062 was associated with decrease of the Rac1-guanine nucleotide exchange factor (GEF) activity, suggesting that this activity is regulated by two different signaling pathways via serine 696 and tyrosine 1062 in RET. Moreover, in the presence of serine 696 mutation, lamellipodia formation was rescued by replacing tyrosine 687 with phenylalanine. These findings propose a novel mechanism that receptor tyrosine kinase modulates actin dynamics in neuronal cells via its cAMP-dependent phosphorylation.     

Structure and chemical inhibition of the RET tyrosine kinase domain

The RET proto-oncogene encodes a receptor tyrosine kinase for the glial cell line-derived neurotrophic factor family of ligands. Loss-of-function mutations in RET are implicated in Hirschsprung disease, whereas activating mutations in RET are found in human cancers, including familial medullar thyroid carcinoma and multiple endocrine neoplasias 2A and 2B. We report here the biochemical characterization of the human RET tyrosine kinase domain and the structure determination of the non-phosphorylated and phosphorylated forms. Both structures adopt the same active kinase conformation competent to bind ATP and substrate and have a pre-organized activation loop conformation that is independent of phosphorylation status. In agreement with the structural data, enzyme kinetic data show that autophosphorylation produces only a modest increase in activity. Longer forms of RET containing the juxtamembrane domain and C-terminal tail exhibited similar kinetic behavior, implying that there is no cis-inhibitory mechanism within the RET intracellular domain. Our results suggest the existence of alternative inhibitory mechanisms, possibly in trans, for the autoregulation of RET kinase activity. We also present the structures of the RET tyrosine kinase domain bound to two inhibitors, the pyrazolopyrimidine PP1 and the clinically relevant 4-anilinoquinazoline ZD6474. These structures explain why certain multiple endocrine neoplasia 2-associated RET mutants found in patients are resistant to inhibition and form the basis for design of more effective inhibitors.     

Sorafenib functions to potently suppress RET tyrosine kinase activity by direct enzymatic inhibition and promoting RET lysosomal degradation independent of proteasomal targeting

Germ line missense mutations in the RET (rearranged during transfection) oncogene are the cause of multiple endocrine neoplasia, type 2 (MEN2), but at present surgery is the only treatment available for MEN2 patients. In this study, the ability of Sorafenib (BAY 43-9006) to act as a RET inhibitor was investigated. Sorafenib inhibited the activity of purified recombinant kinase domain of wild type RET and RET(V804M) with IC(50) values of 5.9 and 7.9 nm, respectively. Interestingly, these values were 6-7-fold lower than the IC(50) for the inhibition of B-RAF(V600E). In cell-based assays, Sorafenib inhibited the kinase activity and signaling of wild type and oncogenic RET in MEN2 tumor and established cell lines at a concentration between 15 and 150 nm. In contrast, inhibition of oncogenic B-RAF- or epidermal growth factor-induced ERK1/2 phosphorylation required micromolar concentrations of Sorafenib demonstrating the high specificity of this drug in targeting RET. Moreover, prolonged exposure to Sorafenib resulted in inhibition of cell proliferation and RET protein degradation. Using lysosomal and proteasomal inhibitors, we demonstrate that Sorafenib induces RET lysosomal degradation independent of proteasomal targeting. Furthermore, we provide a structural model of the Sorafenib.RET complex in which Sorafenib binds to and induces the DFG(out) conformation of the RET kinase domain. These results strengthen the argument that Sorafenib may be effective in the treatment of MEN2 patients. In addition, because inhibition of RET is not impaired by mutation of the Val(804) gatekeeper residue, MEN2 tumors may be less susceptible to acquired Sorafenib resistance.     

The structure of GFRalpha1 domain 3 reveals new insights into GDNF binding and RET activation

Glial cell line-derived neurotrophic factor (GDNF) binds to the GDNF family co-receptor alpha1 (GFRalpha1) and activates RET receptor tyrosine kinase. GFRalpha1 has a putative domain structure of three homologous cysteine-rich domains, where domains 2 and 3 make up a central domain responsible for GDNF binding. We report here the 1.8 A crystal structure of GFRalpha1 domain 3 showing a new protein fold. It is an all-alpha five-helix bundle with five disulfide bridges. The structure was used to model the homologous domain 2, the other half of the GDNF-binding fragment, and to construct the first structural model of the GDNF-GFRalpha1 interaction. Using site-directed mutagenesis, we identified closely spaced residues, Phe213, Arg224, Arg225 and Ile229, comprising a putative GDNF-binding surface. Mutating each one of them had slightly different effects on GDNF binding and RET phosphorylation. In addition, the R217E mutant bound GDNF equally well in the presence and absence of RET. Arg217 may thus be involved in the allosteric properties of GFRalpha1 or in binding RET.     

A cytoplasmic PPPSP motif determines megalin's phosphorylation and regulates receptor's recycling and surface expression

Megalin is a large endocytic receptor expressed at the apical surface of several absorptive epithelia. It binds multiple ligands including apolipoproteins, vitamin and hormone carrier proteins and signaling molecules such as parathyroid hormone and the morphogen sonic hedgehog. An important characteristic of megalin is its high endocytic activity, which is mediated by tyrosine-based endocytic motifs within the receptor's cytoplasmic tail. This domain also harbors several putative consensus phosphorylation motifs for protein kinase (PK) C and casein kinase-II and one consensus motif for PKA and glycogen synthase kinase-3 (GSK3). Here we report that the cytoplasmic domain of megalin is constitutively phosphorylated depending on the integrity of a PPPSP motif, a putative GSK3 site, with a minor participation of the other phosphorylation motifs. Mutation of the serine residue within the PPPSP motif as well as blocking GSK3 activity, with two different inhibitors, significantly decreased the phosphorylation levels of the receptor. Both the megalin PPPAP mutant and the underphosphorylated wild-type receptor, by inhibition of GSK3 activity, were more expressed at the cell surface and more efficiently recycled, but they were not inhibited in their initial endocytosis rates. Altogether, these results show that the PPPSP motif and the GSK3 activity are critical to allow megalin phosphorylation and also negatively regulate the receptor's recycling.     

Dominant effects of RET receptor misexpression and ligand-independent RET signaling on ureteric bud development

During kidney development, factors from the metanephric mesenchyme induce the growth and repeated branching of the ureteric bud, which gives rise to the collecting duct system and also induces nephrogenesis. One signaling pathway known to be required for this process includes the receptor tyrosine kinase RET and co-receptor GFR(&agr;)-1, which are expressed in the ureteric bud, and the secreted ligand GDNF produced in the mesenchyme. To examine the role of RET signaling in ureteric bud morphogenesis, we produced transgenic mice in which the pattern of RET expression was altered, or in which a ligand-independent form of RET kinase was expressed. The Hoxb7 promoter was used to express RET throughout the ureteric bud branches, in contrast to its normal expression only at the bud tips. This caused a variable inhibition of ureteric bud growth and branching reminiscent of, but less severe than, the RET knockout phenotype. Manipulation of the level of GDNF, in vitro or in vivo, suggested that this defect was due to insufficient rather than excessive RET signaling. We propose that RET receptors expressed ectopically on ureteric bud trunk cells sequester GDNF, reducing its availability to the normal target cells at the bud tips. When crossed to RET knockout mice, the Hoxb7/RET transgene, which encoded the RET9 isoform, supported normal kidney development in some RET-/- animals, indicating that the other major isoform, RET51, is not required in this organ. Expression of a Hoxb7/RET-PTC2 transgene, encoding a ligand-independent form of RET kinase, caused the development of abnormal nodules, outside the kidney or at its periphery, containing branched epithelial tubules apparently formed by deregulated growth of the ureteric bud. This suggests that RET signaling is not only necessary but is sufficient to induce ureteric bud growth, and that the orderly, centripetal growth of the bud tips is controlled by the spatially and temporally regulated expression of GDNF and RET.     

Alterations at dispersed sites cause phosphorylation and activation of SNF1 protein kinase during growth on high glucose

The SNF1/AMP-activated protein kinases are central energy regulators in eukaryotes. SNF1 of Saccharomyces cerevisiae is inhibited during growth on high levels of glucose and is activated in response to glucose depletion and other stresses. Activation entails phosphorylation of Thr(210) on the activation loop of the catalytic subunit Snf1 by Snf1-activating kinases. We have used mutational analysis to identify Snf1 residues that are important for regulation. Alteration of Tyr(106) in the αC helix or Leu(198) adjacent to the Asp-Phe-Gly motif on the activation loop relieved glucose inhibition of phosphorylation, resulting in phosphorylation of Thr(210) during growth on high levels of glucose. Substitution of Arg for Gly(53), at the N terminus of the kinase domain, increased activation on both high and low glucose. Alteration of the ubiquitin-associated domain revealed a modest autoinhibitory effect. Previous studies identified alterations of the Gal83 (β) and Snf4 (γ) subunits that relieve glucose inhibition, and we have here identified a distinct set of Gal83 residues that are required. Together, these results indicate that alterations at dispersed sites within each subunit of SNF1 cause phosphorylation of the kinase during growth on high levels of glucose. These findings suggest that the conformation of the SNF1 complex is crucial to maintenance of the inactive state during growth on high glucose and that the default state for SNF1 is one in which Thr(210) is phosphorylated and the kinase is active.     

Ureteric bud outgrowth in response to RET activation is mediated by phosphatidylinositol 3-kinase

The c-ret gene encodes a receptor tyrosine kinase (RET) essential for the development of the kidney and enteric nervous system. Activation of RET requires the secreted neurotrophin GDNF (glial cell line-derived neurotrophic factor) and its high affinity receptor, a glycosyl phosphatidylinositol-linked cell surface protein GFRalpha1. In the developing kidney, RET, GDNF, and GFRalpha1 are all required for directed outgrowth and branching morphogenesis of the ureteric bud epithelium. Using MDCK renal epithelial cells as a model system, activation of RET induces cell migration, scattering, and formation of filopodia and lamellipodia. RET-expressing MDCK cells are able to migrate toward a localized source of GDNF. In this report, the intracellular signaling mechanisms regulating RET-dependent migration and chemotaxis are examined. Activation of RET resulted in increased levels of phosphatidylinositol 3-kinase (PI3K) activity and Akt/PKB phosphorylation. This increase in PI3K activity is essential for regulating the GDNF response, since the specific inhibitor, LY294002, blocks migration and chemotaxis of MDCK cells. Using an in vitro organ culture assay, inhibition of PI3K completely blocks the GDNF-dependent outgrowth of ectopic ureter buds. PI3K is also essential for branching morphogenesis once the ureteric bud has invaded the kidney mesenchyme. The data suggest that activation of RET in the ureteric bud epithelium signals through PI3K to control outgrowth and branching morphogenesis.     

Activation of RET tyrosine kinase regulates interleukin-8 production by multiple signaling pathways

Interleukin-8 (IL-8) is known to contribute to human cancer progression through its potential function as a mitogenic, angiogenic, or motogenic factor. We found a high level of IL-8 production in SK-N-MC human primitive neuroectodermal tumor cells transfected with the human RET gene (SK-N-MC (RET) cells) in response to glial cell line-derived neurotrophic factor (GDNF) stimulation. IL-8 was also produced at high levels in TT human medullary thyroid carcinoma and TPC-1 human papillary thyroid carcinoma cell lines both of which express activated RET tyrosine kinase. To investigate which signaling pathways are responsible for IL-8 expression, we treated SK-N-MC (RET) cells with several kinase inhibitors before GDNF stimulation. The results showed that a MEK1 inhibitor, PD98059, a p38MAPK inhibitor, SB202190, and a protein kinase C (PKC) inhibitor, Calphostin C, markedly decreased the IL-8 secretion from SK-N-MC (RET) cells at 24 h after GDNF stimulation. In contrast, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, LY294002, increased its secretion. These results thus suggested that IL-8 production by RET tyrosine kinase is regulated by multiple signaling pathways.     

Phosphorylation of Thr654 but not Thr669 within the juxtamembrane domain of the EGF receptor inhibits calmodulin binding

Calcium-calmodulin (CaM) binding to the epidermal growth factor receptor (EGFR) has been shown to both inhibit and stimulate receptor activity. CaM binds to the intracellular juxtamembrane (JM) domain (Met645-Phe688) of EGFR. Protein kinase C (PKC) mediated phosphorylation of Thr654 occurs within this domain. CaM binding to the JM domain inhibits PKC phosphorylation and conversely PKC mediated phosphorylation of Thr654 or Glu substitution of Thr654 inhibits CaM binding. A second threonine residue (Thr669) within the JM domain is phosphorylated by the mitogen-activated protein kinase (MAPK). Previous results have shown that CaM interferes with EGFR-induced MAPK activation. If and how phosphorylation of Thr669 affects CaM-EGFR interaction is however not known.In the present study we have used surface plasmon resonance (BIAcore) to study the influence of Thr669 phosphorylation on real time interactions between the intracellular juxtamembrane (JM) domain of EGFR and CaM. The EGFR-JM was expressed as GST fusion proteins in Escherichia coli and phosphorylation was mimicked by generating Glu substitutions of either Thr654 or Thr669. Purified proteins were coupled to immobilized anti-GST antibodies at the sensor surface and increasing concentration of CaM was applied. When mutating Thr654 to Glu654 no specific CaM binding could be detected. However, neither single substitutions of Thr669 (Gly669 or Glu669) nor double mutants Gly654/Gly669 or Gly654/Glu669 influenced the binding of CaM to the EGFR-JM. This clearly shows that PKC may regulate EGF-mediated CaM signalling through phosphorylation of Thr654 whereas phosphorylation of Thr669 seems to play a CaM independent regulatory role. The role of both residues in the EGFR-calmodulin interaction was also studied in silico. Our modelling work supports a scenario where Thr654 from the JM domain interacts with Glu120 in the calmodulin molecule. Phosphorylation of Thr654 or Glu654 substitution creates a repulsive electrostatic force that would diminish CaM binding to the JM domain. These results are in line with the Biacore experiments showing a weak binding of the CaM to the JM domain with Thr654 mutated to Glu. Furthermore, these results provide a hypothesis to how CaM binding to EGFR might both positively and negatively interfere with EGFR-activity.