Nerve growth factor (NGF) and epidermal growth factor (EGF) produce stable alterations in PC12 cells that persist in the detergent-insoluble cytoskeleton, resulting in the phosphorylation of a 250,000-mol-wt cytoskeletally associated protein in situ. Treatment of PC12 cells with NGF or EGF, followed by detergent lysis of the cells and incubation of the resulting cytoskeletons with 3,-32p-ATP, permitted detection of hormonally stimulated, energydependent events, which result in the enhanced phosphorylation of a cytoskeletally associated protein as an immediate consequence of receptor occupancy. These events were elicited only upon treatment of intact cells at physiological temperatures. The NGF-and EGFstimulated events occurred rapidly; however, they were a transient effect of hormone action. NGF and EGF were found to act through independent mechanisms to stimulate the in situ phosphorylation of the 250,000-mol-wt protein, as the effects of NGF, but not EGF, were blocked by methyltransferase inhibitors. The 250,000-mol-wt protein was phosphorylated on serine and threonine residues in response to both NGF and EGF although in somewhat different proportions. The data suggest that the hormone-stimulated labeling of the 250,000-mol-wt protein may be the result of either the direct activation of a protein kinase, the redistribution of the kinase relative to its substrates as a consequence of hormone action, or the coincident occurrence of these events.Although it is well known that the binding of peptide hormones to cell surface receptors results in various metabolic changes in the target cells, the mechanisms subserving these events are not well understood. Several hypotheses have been advanced to explain the myriad effects of peptide hormones, including the involvement of second messengers generated on hormone binding (1), internalization of the hormone and subsequent transport to the nucleus (2), and the activation of hormonally sensitive protein kinases (3). There is reasonably good evidence that at least some of the biological effects, including the stimulation of DNA synthesis, are the result of receptor aggregation within the plasma membrane (4-6).The role of protein kinases in hormone action is of particular interest, given the recent discovery that the membrane receptors for several peptide hormones possess an intrinsic protein kinase activity (7-12). The binding of hormone to its receptor immediately activates the protein kinase, resulting in autophosphorylation of the receptor itself as well as the phosphorylation of other cellular proteins (3,7,(12)(13)(14). It is

The epidermal growth factor receptor (EGF-R) on human epidermoid carcinoma cells, A431, was found to be predominantly associated with the detergent-insoluble cytoskeleton, where it retained both a functional ligand-binding domain and an intrinsic tyrosine kinase activity. The EGF-R was constitutively associated with the A431 cytoskeleton; this association was not a consequence of adventitious binding. The EGF-R was associated with cytoskeletal elements both at the cell surface, within intracellular vesicles mediating the internalization of the hormone-receptor complex, and within lysosomes. The EGF-R became more stably associated with cytoskeletal elements after its internalization. The cytoskeletal association of the EGF-R was partially disrupted on suspension of adherent cells, indicating that alteration of cellular morphology influences the structural association of the EGF-R, and that the EGF-R is not intrinsically insoluble.Cytoskeletons prepared from EGF-treated A431 cells, when incubated with ,1,-32p-ATP, demonstrated enhanced autophosphorylation of the EGF-R in situ as well as the phosphorylation of several high molecular weight proteins. In this system, phosphorylation occurs between immobilized kinase and substrate. The EGF-R and several high molecular weight cytoskeletal proteins were phosphorylated on tyrosine residues; two of the latter proteins were phosphorylated transiently as a consequence of EGF action, suggesting that EGF caused the active redistribution of the protein substrates relative to protein kinases. The ability of EGF to stimulate protein phosphorylation in situ required treatment of intact cells at physiological temperatures; addition of EGF directly to cytoskeletons had no effect. These data suggest that the structural association of the EGF-R may play a role in cellular processing of the hormone, as well as in regulation of the EGF-R kinase activity and in specifying its cellular substrates.

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