Nerve growth factor- and epidermal growth factor-stimulated phosphorylation of a PC12 cytoskeletally associated protein in situ.

Abstract

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

K-252a: a specific inhibitor of the action of nerve growth factor on PC 12 cells

“…The details of this action are not clear as yet, but it appears increasingly likely that at least one means by which NGF acts is by altering the level of phosphorylation of a number of cellular proteins (Halegoua and Patrick, 1980;Yu et al, 1980;End et al, 1983;Landreth and Rieser, 1985;Lee et al, 1985;Matsuda et al, 1986). It is clear, from more recent studies, that the changes seen in the various phosphorylations in NGF-sensitive cells are due to the actions of more than one kinase.…”

Wheat germ agglutinin blocks the biological effects of nerve growth factor.

“…The experiments were performed at NGF concentrations of 50 ng/ml, a condition under which the level of NGF receptor occupancy on the PCI2 cells, even if reduced by 50%, would be more than sufficient to stimulate maximally each of the biological effects examined here. Both the NGFstimulated neurite outgrowth and the phosphorylation of the 250-kD cytoskeletal protein occur maximally at NGF concentrations below 1 ng/ml (18,19). The NGF-mediated alteration of cell surface morphology is also observed at low NGF concentrations (24).…”

“…Similar experiments were performed to test if WGA would alter the ability of epidermal growth factor to stimulate the phosphorylation of the 250-kD protein (19). WGA inhibited the epidermal growth factor-stimulated phosphorylation of the 250-kD protein, although not as much as NGF did (data not shown).…”

“…NGF treatment of PCI2 cells produces stable changes in the cells that persist upon extraction of the ceils in nonionic detergent and that can be detected by phosphorylation of a 250-kD cytoskeletal protein in situ (19). In this system, protein phosphorylation occurs between immobilized protein kinases and their protein substrates, both of which are associated with the detergent-insoluble cytoskeleton.…”

“…The phosphorylation of PCI2 cytoskeletal protein was performed as described by Landreth and Rieser (19). PC12 cells were removed from the tissue culture dishes by trituration in calcium-and magnesium-free phosphate-buffered saline (PBS), washed, and then resuspended at a final concentration of l0 s cells/ml in PBS containing l mg/ml bovine serum albumin and 1 mg/ml glucose.…”