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

Publication ,  Journal Article
Feinmesser, RL; Gray, K; Means, AR; Chantry, A
Published in: Oncogene
June 20, 1996

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.

Duke Scholars

Published In

Oncogene

ISSN

0950-9232

Publication Date

June 20, 1996

Volume

12

Issue

12

Start / End Page

2725 / 2730

Location

England

Related Subject Headings

  • Threonine
  • Substrate Specificity
  • Sequence Homology, Amino Acid
  • Recombinant Fusion Proteins
  • Receptor, erbB-2
  • Receptor, ErbB-2
  • Rats
  • Proto-Oncogene Mas
  • Phosphorylation
  • Phosphopeptides
 

Citation

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ICMJE
MLA
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Feinmesser, R. L., Gray, K., Means, A. R., & Chantry, A. (1996). HER-2/c-erbB2 is phosphorylated by calmodulin-dependent protein kinase II on a single site in the cytoplasmic tail at threonine-1172. Oncogene, 12(12), 2725–2730.
Feinmesser, R. L., K. Gray, A. R. Means, and A. Chantry. “HER-2/c-erbB2 is phosphorylated by calmodulin-dependent protein kinase II on a single site in the cytoplasmic tail at threonine-1172.Oncogene 12, no. 12 (June 20, 1996): 2725–30.

Published In

Oncogene

ISSN

0950-9232

Publication Date

June 20, 1996

Volume

12

Issue

12

Start / End Page

2725 / 2730

Location

England

Related Subject Headings

  • Threonine
  • Substrate Specificity
  • Sequence Homology, Amino Acid
  • Recombinant Fusion Proteins
  • Receptor, erbB-2
  • Receptor, ErbB-2
  • Rats
  • Proto-Oncogene Mas
  • Phosphorylation
  • Phosphopeptides