HER-2/c-erbB2 is phosphorylated by calmodulin-dependent protein kinase II on a single site in the cytoplasmic tail at threonine-1172.
Calmodulin-dependent protein kinase II (Cam kinase II) is known to desensitise epidermal growth factor receptor (HER-1) tyrosine kinase activity by a process involving phosphorylation at serines 1046/47 in the cytoplasmic tail. We have developed an experimental system to investigate phosphorylation of the related HER-2/c-erbB2 proto-oncogene utilising purified Cam kinase II and recombinant glutathione-S-transferase (GST) fusion proteins. The cDNA for rat Cam kinase II-alpha was transfected into human embryonic kidney (HEK) 293 fibroblasts and the expressed protein purified to homogeneity by calmodulin-agarose affinity chromatography. A GST fusion protein comprising residues 1126-1255 of HER-2 was phosphorylated by purified Cam kinase II, in contrast to a GST protein comprising residues 1005-1125. Phosphoamino-acid analysis and site-directed mutagenesis indicated that HER-2 was phosphorylated on a single site at threonine-1172 which resides within a consensus Cam kinase II phosphorylation site (RAKT). HER-2 (threonine-1172-alanine), in the form of a ligand-inducible chimaera HER-1/2, was co-transfected into HEK-293 fibroblasts with a constitutively active form of Cam kinase II, followed by in vivo labelling of these cells with 32 P-orthophosphate. Immunoprecipitation of ligand-activated receptors followed by two-dimensional phosphopeptide mapping indicated that threonine-1172 in HER-2 is a newly identified in vivo site which can be hyper-phosphorylated by constitutively active Cam kinase II. In addition, when over-expressed in HEK-293 fibroblasts, HER-1/2 (threonine-1172-alanine) showed a defect in desensitisation and underwent a more sustained EGF-induced receptor autophosphorylation compared to wild-type HER-1/2.
Feinmesser
R L
RL
Department of Biochemistry, Charing Cross and Westminster Medical School, London, UK.
Gray
K
K
Means
A R
AR
Chantry
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D003593:Cytoplasm / Q000737:chemistry*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000096:biosynthesis / Q000235:genetics; D010766:Phosphorylation; D000090063:Proto-Oncogene Mas; D051381:Rats; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism*
D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D010748:Phosphopeptides; D000090063:Proto-Oncogene Mas; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D013912:Threonine; C000715470:MAS1 protein, human; C517657:CAMK2A protein, human; C517675:Camk2a protein, rat