Phosphorylation of the epidermal growth factor receptor at threonine 654 inhibits ligand-induced internalization and down-regulation

Lund, Kim; Lazar, Cheri S.; Chen, W S; Walsh, Bradley J.; Welsh, J B; Herbst, John J.; Walton, Gordon M.; Rosenfeld, M G; Gill, G N; Wiley, H. Steven

doi:10.1016/s0021-9258(17)30533-1

Cited by 120 publications

(15 citation statements)

References 72 publications

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“…The EGFR can also be phosphorylated by regulatory serine\threonine-protein kinases, such as protein kinase C [6][7][8][9][10] and CaM-dependent protein kinase II [11][12][13]. Moreover, we have shown previously that the binding of CaM to a fusion protein containing the juxtamembrane region (residues 645-660) of the human EGFR was prevented by the phosphorylation of Thr'&% by protein kinase C [16].…”

Section: Figure 6 Binding Of Biotinylated Cam To the Human Egfr Was Not Affected By Egf Or Alkaline Phosphatase Treatmentmentioning

confidence: 99%

“…Signalling by the EGFR is accompanied in many cell types by a transient increase in the cytosolic concentration of free Ca# + (for review, see [5]). This early calcium signal mediates the retroinhibition of the intrinsic tyrosine kinase activity of the EGFR using regulatory Ca# + -dependent serine\threonine-protein kinases that phosphorylate the receptor, such as protein kinase C [6][7][8][9][10] and calmodulin (CaM)-dependent protein kinase II [11][12][13]. In addition, the Ca# + -CaM complex also regulates the tyrosine kinase activity of the EGFR acting on the receptor itself [5,[14][15][16].…”

Section: Introductionmentioning

confidence: 99%

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Evidence for the direct interaction between calmodulin and the human epidermal growth factor receptor

Villalobo

2002

Biochemical Journal

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Previous work from our laboratory has demonstrated that the Ca2+—calmodulin complex inhibits the intrinsic tyrosine kinase activity of the epidermal growth factor receptor (EGFR), and that the receptor can be isolated by Ca2+-dependent calmodulin-affinity chromatography [San José, Benguría, Geller and Villalobo (1992) J. Biol. Chem. 267, 15237–15245]. Moreover, we have demonstrated that the cytosolic juxtamembrane region of the human receptor (residues 645–660) binds calmodulin in a Ca2+-dependent manner when this segment forms part of a recombinant fusion protein [Martín-Nieto and Villalobo (1998) Biochemistry 37, 227–236]. However, demonstration of the direct interaction between calmodulin and the whole receptor has remained elusive. In this work, we show that calmodulin, in the presence of Ca2+, forms part of a high-molecular-mass complex built upon covalent cross-linkage of the human EGFR immunoprecipitated from two cell lines overexpressing this receptor. Although several calmodulin-binding proteins co-immunoprecipitated with the EGFR, suggesting that they interact with the receptor, we demonstrated using overlay techniques that biotinylated calmodulin binds directly to the receptor in a Ca2+-dependent manner without the mediation of any adaptor calmodulin-binding protein. Calmodulin binds to the EGFR with an apparent dissociation constant (K′d) of approx. 0.2–0.3μM. Treatment of cells with epidermal growth factor, or with inhibitors of protein kinase C and calmodulin-dependent protein kinase II, or treatment of the immunoprecipitated receptor with alkaline phosphatase, does not significantly affect the binding of biotinylated calmodulin to the receptor.

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Section: Figure 6 Binding Of Biotinylated Cam To the Human Egfr Was Not Affected By Egf Or Alkaline Phosphatase Treatmentmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Evidence for the direct interaction between calmodulin and the human epidermal growth factor receptor

Villalobo

2002

Biochemical Journal

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“…The antiparallel helix dimer of JM is thought to facilitate asymmetric interaction between the kinase domains of EGFR, and hence its activation, in order to phosphorylate tyrosine residues in the C-tail (11, 34). This tyrosine phosphorylation results in the recruitment of PKC and other threonine kinases from the cytoplasm to the EGFR molecules for the phosphorylation of Thr654, which is known to negatively regulate EGFR signaling (26, 35). Our previous results suggested that the mechanism underlying this negative effect of pT654 is the dissociation of JM dimers in the presence of acidic lipids (20).…”

Section: Discussionmentioning

confidence: 99%

“…Another important factor in the regulation of EGFR through the TM-JM is the phosphorylation of Thr654 at the JM-A domain. Although Thr phosphorylation is known to be involved in EGFR deactivation, the precise mechanism of this is still elusive (25).…”

Section: Introductionmentioning

confidence: 99%

Threonine phosphorylation regulates the molecular assembly and signaling of EGFR in cooperation with membrane lipids

Maeda

Tamagaki-Asahina

Sato

et al. 2021

Preprint

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The cytoplasmic domain of the receptor tyrosine kinases (RTKs) plays roles as a phosphorylation enzyme and a protein scaffold but the regulation of these two functions is not fully understood. We here analyzed assembly of the transmembrane (TM)-juxtamembrane (JM) region of EGFR, one of the best studied species of RTKs, by combining single-pair FRET imaging and a nanodisc technique. The JM domain of EGFR contains a threonine residue that is phosphorylated after ligand association. We observed that the TM-JM peptides of EGFR form anionic lipid-induced dimers and cholesterol-induced oligomers. The two forms involve distinct molecular interactions, with a bias towards oligomer formation upon threonine phosphorylation. We further analyzed the functions of whole EGFR molecules, with or without a threonine to alanine substitution in the JM domain, in living cells. The results suggested an autoregulatory mechanism in which threonine phosphorylation of the JM domain causes a switch from kinase activation dimers to scaffolding oligomers.

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“…It is however, highly similar between RTK family members and shown a regulatory target in modulating RTK activity. The JM region of EGFR for instance, is phosphorylated on threonine 654 (Thr654) residue by PKC during 'receptor transmodulation' 222,223 . Receptor transmodulation is a negative feedback mechanism which modulates the intensity and duration of RTK signaling, as in the case of EGFR following GPCR transactivation 219,223 .…”

Section: -Rtk Structure: Three Prototypic Domainsmentioning

confidence: 99%

The effects of Mitogenic and Metabolic cues on clathrin-mediated endocytosis

Santos¹

2021

Preprint

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The cell ‘surfaceome’ collectively describes proteins found on the plasma membrane (PM), which functions in fundamental cellular processes including growth and proliferation. The surfaceome undergoes dynamic remodeling via the addition/removal of surface proteins in response to changing environmental conditions. In mammalian cells, surfaceome remodeling is predominantly facilitated by clathrin-mediated endocytosis (CME) which involves the invagination and internalization of PM regions via clathrin-coated structures—removing proteins from the cell surface. As a major regulator of the surfaceome, it is important to understand the underlying mechanisms governing CME, given that its dysregulation has been implicated in human pathologies including neurologic and oncogenic disorders. Cellular cues including mitogenic (e.g. growth factors) and metabolic signals (e.g. cellular energy levels) induce diverse cellular processes (e.g. growth and proliferation) requiring surfaceome/PM remodeling. Precisely how mitogenic and metabolic signals may induce surfaceome remodeling however, is under-examined. As a major regulator of the surfaceome, CME is a likely mechanism through which cellular cues may remodel the PM. Poorly understood, I thus sought to investigate how mitogenic and metabolic signals may regulate CME. Mitogenic signaling by the epidermal growth factor receptor (EGFR) triggers PLCγ1-calcium signals, which I found a requirement for CME of EGFR—likely via calcium control of the Sjn1 protein. Consistently, using TIRF-M imaging coupled to automated software analysis, I demonstrate that inhibition of PLCγ1-calcium signals impairs the formation/assembly of GFR-containing clathrin structures. In addition, I hypothesize that PLCγ1-calcium signals also regulates the CME of other surface proteins given its robust control of Sjn1—which localizes broadly amongst clathrin-coated structures. AMPK is a cellular energy sensor activated by metabolic stress (e.g. starvation). Using TIRF-M imaging coupled to automated software analysis, I found that AMPK activation broadly reduces the formation/assembly of bona fide clathrin-coated pits, without impairing the internalization rates of CME cargoes (e.g. EGFR, TfR and β1-integrin). Furthermore, I found that AMPK may regulate CME throughcontrol of the Arf6 protein. Collectively, my findings uncover and provide novel mechanisms by which mitogenic (via EGFR-induced calcium signals) and metabolic signals (via AMPK control of Arf6) may induce regulation of CME—in eliciting global reorganization of the plasma membrane. 1,2,11–13,3–10

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Phosphorylation of the epidermal growth factor receptor at threonine 654 inhibits ligand-induced internalization and down-regulation

Cited by 120 publications

References 72 publications

Evidence for the direct interaction between calmodulin and the human epidermal growth factor receptor

Evidence for the direct interaction between calmodulin and the human epidermal growth factor receptor

Threonine phosphorylation regulates the molecular assembly and signaling of EGFR in cooperation with membrane lipids

The effects of Mitogenic and Metabolic cues on clathrin-mediated endocytosis

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