Stimulation of mitogenic pathways through kinase-impaired mutants of the epidermal growth factor receptor
J A Ewald1, K J Coker, J O Price
1Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.
Abstract:
Two residues have been shown to be critical for the kinase activity of the receptor for epidermal growth factor (EGF): lysine-721, which functions in the binding of ATP by correctly positioning the gamma-phosphate for phosphoryl transfer, and aspartate-813, which functions as the catalytic base of the kinase. Mutation of either of these two residues has been shown to disrupt kinase activity of the receptor. However, studies performed in different laboratories had suggested that while EGF receptors mutated at lysine-721 are unable to stimulate significant increases of [(3)H]thymidine incorporation into DNA in response to EGF treatment, cells expressing EGF receptors mutated at aspartate-813 do stimulate significant incorporation of [(3)H]thymidine into DNA in response to EGF. In the present study, EGF receptors mutated at lysine-721 or aspartate-813 (K721R and D813A, respectively), as well as wild-type EGF receptors, were expressed in the same cellular background, Chinese hamster ovary cells, and side-by-side experiments were performed to investigate possible signaling-related differences. Our results indicate that while there are measurable differences in the abilities of the two mutant receptors to stimulate [(3)H]thymidine incorporation between 20 and 24 h after addition of EGF, these differences cannot be correlated with significant differences in EGF-stimulated tyrosine phosphorylation of mutant EGF receptor and endogenous ErbB2, the extent of receptor internalization, EGF-stimulated ion uptake, stimulation of SHC activity, or receptor association with Grb2. Flow cytometric data suggest that populations of cells expressing either kinase-impaired mutant EGF receptor progress similarly into S phase in response to addition of EGF. These observations suggest that D813A and K721R retain similar ability to stimulate mitogenic signaling events through transactivation of ErbB2 with only subtle temporal differences, and they emphasize the importance of expressing mutant receptors in an identical cellular context to make valid comparisons of functions.
Insights
Mutations in epidermal growth factor (EGF) receptor kinase activity were studied. Both lysine-721 and aspartate-813 mutations impact EGF receptor function, but exhibit similar mitogenic signaling capabilities.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- The epidermal growth factor (EGF) receptor is crucial for cell growth and proliferation.
- Key residues, lysine-721 (ATP binding) and aspartate-813 (catalytic base), are essential for EGF receptor kinase activity.
- Previous studies showed conflicting results regarding the mitogenic potential of EGF receptors mutated at these sites.
Purpose of the Study:
- To directly compare the signaling functions of EGF receptors mutated at lysine-721 (K721R) and aspartate-813 (D813A) within an identical cellular context.
- To investigate differences in mitogenic signaling, receptor activity, and downstream signaling pathways between K721R and D813A mutants.
- To clarify the roles of K721 and D813 in EGF receptor-mediated cellular responses.
Main Methods:
- Expression of wild-type, K721R, and D813A EGF receptors in Chinese hamster ovary (CHO) cells.
- Measurement of [(3)H]thymidine incorporation into DNA following EGF stimulation.
- Assessment of EGF-stimulated tyrosine phosphorylation of EGF receptor and ErbB2.
- Analysis of receptor internalization, ion uptake, SHC activity, and Grb2 association.
- Flow cytometry to evaluate cell cycle progression (S phase entry).
Main Results:
- Both K721R and D813A mutants showed measurable differences in [(3)H]thymidine incorporation at later time points (20-24h) post-EGF addition.
- No significant differences were observed between the mutants in EGF-stimulated tyrosine phosphorylation, receptor internalization, ion uptake, SHC activity, or Grb2 association.
- Flow cytometry indicated similar progression into S phase for cells expressing either kinase-impaired mutant EGF receptor in response to EGF.
- Subtle temporal differences in mitogenic signaling were noted, but overall similar abilities to stimulate downstream events via ErbB2 transactivation were retained.
Conclusions:
- The K721R and D813A mutations in the EGF receptor result in largely similar abilities to stimulate mitogenic signaling through ErbB2 transactivation, with only minor temporal variations.
- These findings highlight the importance of using an identical cellular background for expressing mutant receptors to ensure valid functional comparisons.
- Despite disruptions in kinase activity, both mutants retain significant capacity to drive cellular proliferation in response to EGF.
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