EGF-dependent phosphorylation of the EGF receptor in plasma membranes isolated from young and senescent WI-38 cells.
Tyrosine-specific phosphorylation of the receptor for epidermal growth factor (EGF) in plasma membranes isolated from WI-38 cells is EGF-dependent and occurs to an equivalent extent and on identical tryptic peptides in preparations from cells of various in vitro ages. There is a marked reduction, however, in phosphorylation of receptor molecules from senescent as compared with young WI-38 cells, if enzyme activity is assayed in an immune complex following solubilization of plasma membranes with Nonidet P-40 (NP-40). Differences in the level of receptor phosphorylation in young vs. senescent NP-40 extracts are not resolved by changing the temperature at which the assay is performed, or the length of incubation. Moreover, addition of NP-40 or chloroform-methanol extracts of young cells to assays measuring receptor phosphorylation in senescent cell NP-40 preparations does not augment the senescent enzyme activity. The immunopurified senescent receptor is, however, capable of catalyzing phosphorylation of exogenous substrates. These results indicate that the loss of receptor autophosphorylation in solubilized preparations may result from a differential sensitivity of the senescent cell receptor to the detergent. This finding provides a marker for senescence and suggests subtle changes in protein structure, conformation, or regulation of the EGF receptor in senescent cells.
Brooks
K M
KM
Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104.
Phillips
P D
PD
Carlin
C R
CR
Knowles
B B
BB
Cristofalo
V J
VJ
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002470:Cell Survival; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007158:Immunologic Techniques; D008055:Lipids / Q000494:pharmacology; D010766:Phosphorylation; D013696:Temperature; D013997:Time Factors
D008055:Lipids; D004815:Epidermal Growth Factor; D066246:ErbB Receptors