Human Calmodulin Methyltransferase: Expression, Activity on Calmodulin, and Hsp90 Dependence
Figure 5
CaM KMT binds to the middle domain of Hsp90.
(A) Schematic representation of the structural domains of human Hsp90 used in these experiments (numbers refer to the sequence of human Hsp90α). (B) Binding of CaM KMT and the middle domain of the Hsp90. GST fused Hsp90 fragments (10 µg protein) used in the pull-down experiments: GST- Hsp90N - N-terminal (9–236aa), GST- Hsp90M - middle domain (273–617aa), GST- Hsp90C- C-terminal (629–732aa), were expressed in E.Coli, purified and immobilized to glutathione agarose beads. After overnight incubation with Myc-CaM KMT transfected HEK293 cells’ lysates, the beads were washed and the bound proteins were subjected to SDS-PAGE. The membrane was stained with ponceau red before immunoblotting to demonstrate similar quantities of GST-Hsp90. Immunoblotting with anti-Myc antibody revealed that CaM KMT binds only the middle domain. Representative results from six independent experiments are shown.