The human epidermal growth factor receptor contains a juxtamembrane calmodulin-binding site.
A ligand-insensitive form of the human epidermal growth factor receptor (EGFR) was enriched by Ca2+-dependent calmodulin-affinity chromatography purification. The basic amphiphilic segment Arg645-Arg-Arg-His-Ile-Val-Arg-Lys-Arg-Thr654-Leu-Arg-Arg-Le u-Leu-Gln 660, located within the cytoplasmic juxtamembrane domain of this receptor, was purified as a fusion protein with glutathione S-transferase and shown to bind calmodulin in a Ca2+-dependent manner. An apparent dissociation constant of 0.4 microM calmodulin (Kd'(CaM)) and an apparent affinity constant of 0.5 microM free Ca2+ (Ka'(Ca)) were measured for this binding process. Binding of calmodulin at the juxtamembrane site prevented the phosphorylation of residue Thr-654 by protein kinase C, and an apparent inhibition constant of 0.5-1 microM calmodulin (Ki'(CaM)) was determined. Conversely, phosphorylation of this site by protein kinase C prevented its subsequent interaction with calmodulin. We therefore propose that cross talk between signaling pathways mediated by calmodulin and protein kinase C occurs at the juxtamembrane domain of the EGFR. This calmodulin-binding sequence is highly conserved among protein tyrosine kinases of the vertebrate EGFR family.
Martín-Nieto
J
J
Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Villalobo
A
A
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002846:Chromatography, Affinity; D003593:Cytoplasm / Q000378:metabolism; D066246:ErbB Receptors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D013912:Threonine; D066246:ErbB Receptors; D011493:Protein Kinase C