HER-2/c-erbB2 is phosphorylated by calmodulin-dependent protein kinase II on a single site in the cytoplasmic tail at threonine-1172

Oncogene. 1996 Jun 20;12(12):2725-30.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2
  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism*
  • Cytoplasm / chemistry*
  • Epidermal Growth Factor / pharmacology
  • ErbB Receptors / chemistry
  • ErbB Receptors / metabolism
  • Fibroblasts / drug effects
  • Humans
  • Molecular Sequence Data
  • Phosphopeptides / biosynthesis
  • Phosphopeptides / genetics
  • Phosphorylation
  • Rats
  • Receptor, ErbB-2 / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Threonine / metabolism*

Substances

  • Phosphopeptides
  • Recombinant Fusion Proteins
  • Threonine
  • Epidermal Growth Factor
  • ErbB Receptors
  • Receptor, ErbB-2
  • CAMK2A protein, human
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Camk2a protein, rat