Rl. Feinmesser et al., 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), 1996, pp. 2725-2730
Calmodulin-dependent protein kinase II (Cam kinase II) is known to des
ensitise epidermal growth factor receptor (HER-1) tyrosine kinase acti
vity by a process involving phosphorylation at serines 1046/47 in the
cytoplasmic tail, We have developed an experimental system to investig
ate phosphorylation of the related HER-2/cerbB2 proto-oncogene utilisi
ng purified Cam kinase II and recombinant glutathione-S-transferase (G
ST) fusion proteins, The cDNA for rat Cam kinase II-alpha was transfec
ted into human embryonic kidney (HEK) 293 fibroblasts and the expresse
d protein purified to homogeneity by calmodulin-agarose affinity chrom
atography, A GST fusion protein comprising residues 1126-1255 of HER-2
was phosphorylated by purified Cam kinase II, in contrast to a GST pr
otein comprising residues 1005-1125, Phosphoamino-acid analysis and si
te-directed mutagenesis indicated that HER-2 was phosphorylated on a s
ingle site at threonine-1172 which resides within a consensus Cam kina
se II phosphorylation site (RAKT), HER-2 (threonine-1172-alanine), in
the form of a ligand-inducible chimaera HER-1/2, was co-transfected in
to HEK-293 fibroblasts with a constitutively active form of Cam kinase
II, followed by in vivo labelling of these cells with P-32-orthophosp
hate, Immunoprecipitation of ligand-activated receptors followed by tw
o-dimensional phosphopeptide mapping indicated that threonine-1172 in
HER-2 is a newly identified in vivo site which can be hyper-phosphoryl
ated by constitutively active Cam kinase II, In addition, when over-ex
pressed in HEK 293 fibroblasts, HER-1/2 (threonine-1172-alanine) showe
d a defect in desensitisation and underwent a more sustained EGF-induc
ed receptor autophosphorylation compared to wild-type HER-1/2.