Abstract
The major site of phosphorylation of the epidermal growth factor (EGF) receptor after treatment of cells with EGF is threonine 669. Phosphorylation of this site is also associated with the transmodulation of the EGF receptor caused by platelet-derived growth factor and phorbol ester. A distinctive feature of the primary sequence surrounding threonine 669 is the proximity of 2 proline residues (-Pro-Leu-Thr669-Pro-). This site is not a substrate for phosphorylation by protein kinase C. To investigate the mechanism of the increased phosphorylation of the EGF receptor at threonine 669, in vitro assays were used to measure protein kinase and protein phosphatase activities present in homogenates prepared from cells treated with and without EGF. No evidence for the regulation of protein phosphatase activity was obtained in experiments using the [32P]phosphate-labeled EGF receptor as a substrate. A synthetic peptide corresponding to residues 663-681 of the EGF receptor was used as a substrate for protein kinase assays. Incubation of murine 3T3 L1 pre-adipocytes and human WI-38 fibroblasts with EGF caused a rapid increase (3-10-fold) in the level of threonine protein kinase activity detected in cell homogenates. Similar results were obtained after EGF treatment of Chinese hamster ovary cells expressing wild-type (Thr669) and mutated (Ala669) human EGF receptors. Activation of the threonine protein kinase activity was also observed in cells treated with platelet-derived growth factor, serum, and phorbol ester. Insulin-like growth factor-1 caused no significant change in protein kinase activity. Together these data indicate a role for the regulation of the activity of a threonine protein kinase in the control of the phosphorylation state of the EGF receptor at threonine 669. The significance of the identification of a growth factor-stimulated threonine protein kinase to the mechanism of signal transduction is discussed.
Highlights
The major site of phosphorylation of the epidermal an increase in the tyrosine protein kinase activity of the growth factor (EGF) receptor after treatment of cells receptor cytoplasmic domain [1].Treatment of cells with with EGF is threonine 669
Incubation of murine 3T3 L1 pre-adipocytes These data indicate that the phosphorylation of threonine and human WI-38 fibroblasts withEGF caused a rapid 654 by protein kinase C is not required for the inhibition of increase (3-10-fold) in the level of threonine protein EGF binding caused by PMA and that other mechanisms kinase activity detected in cell homogenates
These threonine residues are located at positions 669, 759, and 969 in the predicted primary sequence of the EGF receptor [26]
Summary
The major site of phosphorylation of the epidermal an increase in the tyrosine protein kinase activity of the growth factor (EGF) receptor after treatment of cells receptor cytoplasmic domain [1].Treatment of cells with with EGF is threonine 669. The peptide containing the major site of EGF receptor threonine phosphorylation eluted from the column a t 27%acetonitrile and has been described previously [4,5,16].
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
