Nerve growth factor-induced changes in the intracellular localization of the protein kinase C substrate B-50 in pheochromocytoma PC12 cells

High levels of the neuron-specific protein kinase C substrate, B-50 (= GAP43), are present in neurites and growth cones during neuronal development and regeneration. This suggests a hitherto nonelucidated role of this protein in neurite outgrowth. Comparable high levels of B-50 arise in the pheochromocytoma PC12 cell line during neurite formation. To get insight in the putative growth-associated function of B-50, we compared its ultrastructural localization in naive PC12 cells with its distribution in nerve growth factor (NGF)- or dibutyryl cyclic AMP (dbcAMP)-treated PC12 cells. B-50 immunogold labeling of cryosections of untreated PC12 cells is mainly associated with lysosomal structures, including multivesicular bodies, secondary lysosomes, and Golgi apparatus. The plasma membrane is virtually devoid of label. However, after 48-h NGF treatment of the cells, B-50 immunoreactivity is most pronounced on the plasma membrane. Highest B-50 immunoreactivity is observed on plasma membranes surrounding sprouting microvilli, lamellipodia, and filopodia. Outgrowing neurites are scattered with B-50 labeling, which is partially associated with chromaffin granules. In NGF-differentiated PC12 cells, B-50 immunoreactivity is, as in untreated cells, also associated with organelles of the lysosomal family and Golgi stacks. B-50 distribution in dbcAMP-differentiated cells closely resembles that in NGF-treated cells. The altered distribution of B-50 immunoreactivity induced by differentiating agents indicates a shift of the B-50 protein towards the plasma membrane. This translocation accompanies the acquisition of neuronal features of PC12 cells and points to a neurite growth-associated role for B-50, performed at the plasma membrane at the site of protrusion.     

Nerve growth factor-induced increase in the cell-free phosphorylation of a nuclear protein in PC12 cells

In previous studies from this laboratory (Yu, M.W., Tolson, N. W., and Guroff, G. (1980) J. Biol. Chem. 255, 10481-10492) nerve growth factor treatment of PC12 cells was shown to increase the phosphorylation of a specific nonhistone nuclear protein. In the present work these whole-cell observations have been pursued and a cell-free system developed, based on the detergent treatment devised by Lenk et al. (Lenk, R., Ransom, L., Kaufmann, Y., and Penman, S. (1977) Cell 10, 67-78), in order to explore the nerve growth factor-sensitive phosphorylation system in biochemical detail. Using this preparation it has been shown that treatment of the whole cells with nerve growth factor for 30 min or more leads to a marked increase in the subsequent cell-free phosphorylation of the same nonhistone nuclear protein. A characterization of this phosphorylation indicates that it is quite labile to heat and to structural disruption, that it prefers ATP as phosphate donor, and that it requires Mg2+, but is inhibited by high Mg2+ levels as well as by certain other divalent cations. The site of phosphorylation appears to be on serine residues of the protein, as was the phosphorylation observed previously in whole cells. The use of various inhibitors and stimulators suggests that the kinase catalyzing this phosphorylation is not cAMP-dependent, nor is it similar to protein kinase C or casein kinase. The increased phosphorylation produced by nerve growth factor is not transient, the stimulation being constant for at least 3 days in the continuous presence of nerve growth factor. Increases in the phosphorylation of the same nuclear protein can be seen upon treatment of the cells with other effectors such as epidermal growth factor and dibutyryl cyclic AMP, the latter in spite of the fact that cAMP-dependence could not be established in the cell-free system. Finally, a similar system, with a similar stimulation of phosphorylation due to nerve growth factor treatment, can be prepared from sympathetic ganglia from neonatal animals.     

Nerve growth factor-induced decrease in the cell-free phosphorylation of a soluble protein in PC12 cells

Incubation of cell-free extracts from PC12 cells with [32P]ATP leads to the phosphorylation of a 100,000-dalton protein. In extracts from cells treated with nerve growth factor, the labeling of the 100,000-dalton protein is substantially and selectively reduced. Direct quantitation indicates that the reduction is a minimum of 30-50% in the various experiments. The decrease is evident after as little as 15 min of nerve growth factor treatment, and disappears within 2 h after the removal of nerve growth factor. The decrease is dose dependent; a complete response is seen after treatment with 10 ng of nerve growth factor/ml. Some decrease in phosphorylation is also seen after treatment of the cells with epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, or 5'-N-ethylcarboxamideadenosine, a potent adenosine receptor agonist, but not after treatment with insulin. The phosphorylation of the 100,000-dalton protein, in extracts from either control or nerve growth factor-treated cells, leads almost exclusively to the formation of phosphothreonine. The addition of equal amounts of extract from untreated cells and extract from nerve growth factor-treated cells produces a level of phosphorylation exactly intermediate between those of the two extracts used separately, indicating the absence of a soluble kinase inhibitor. The data suggest that nerve growth factor treatment produces either a covalent inhibition or a physical removal of the kinase for the 100,000-dalton protein.     

Nerve growth factor-induced alteration in the response of PC12 pheochromocytoma cells to epidermal growth factor

PC12 cells, which differentiate morphologically and biochemically into sympathetic neruonlike cells in response to nerve growth fact, also respond to epidermal growth factor. The response to epidermal growth factor is similar in certain respects to the response to nerve growth fact. Both peptides produce rapid increases in cellular adhesion and 2-deoxyglucose uptake and both induce ornithine decarboxylase. But nerve growth factor causes a decreased cell proliferation and a marked hypertrophy of the cells. In contrast, epidermal growth factor enhances cell proliferation and does not cause hypertrophy. Nerve growth factor induces the formation of neuritis; epidermal growth factor does not. When both factors are presented simultaneously, the cells form neurites. Furthermore, the biological response to epidermal growth fact, as exemplified by the induction of ornithine decarboxylase, is attenuated by prior treatment of the cells with nerve growth factor. PC12 cells have epidermal growth factor receptors. The binding of epidermal growth factor to these receptors is rapid and specific, and exhibits an equilibrium constant of 1.9 x 10(-9) M. Approximately 80,000 receptors are present per cell, and this number is independent of cell density. Treatment of the cells with nerve growth factor reduces the amount of epidermal growth factor binding by at least 80 percent. The decrease in receptor binding begins after approximately 12-18 h of nerve growth factor treatment and is complete within 3 d. Scratchard plots indicate that the number of binding sites decreases, not the affinity of the binding sites for epidermal growth factor.     

Nerve growth factor stimulates the tyrosine phosphorylation of MAP2 kinase in PC12 cells.

NGF treatment of PC12 cells results in the rapid activation of MAP2 kinase. We report here that the induction of enzyme activity was correlated with the phosphorylation of MAP2 kinase, detected by metabolic labeling of the enzyme and with anti-phosphotyrosine antibodies. NGF stimulated the phosphorylation of MAP2 kinase on tyrosine, as well as serine and threonine residues. Western blot analysis using a polyclonal anti-phosphotyrosine antibody demonstrated that the tyrosine phosphorylation of MAP2 kinase was maximal within 2 min following NGF exposure and preceded the induction of MAP2 kinase activity. The NGF-stimulated tyrosine phosphorylation of an identified substrate provides direct evidence for the participation of a tyrosine kinase in the mechanism of action of NGF.     

Regulation of protein kinase activities in PC12 pheochromocytoma cells.

Stimulation of serine protein kinase activity (referred to as S6 kinase) occurs within minutes of addition of nerve growth factor (NGF) to PC12 rat pheochromocytoma cells. This enzyme activity is not related to the cAMP-dependent protein kinase (protein kinase A) or the Ca2+- and phospholipid-dependent protein kinase (protein kinase C), two other protein kinases potentially involved in signal transduction. Two peaks of NGF-stimulated S6 phosphotransferase activity are observed upon ion exchange chromatography; one that comigrates with the serine kinase previously described in chicken embryo fibroblasts and another with distinct elution properties. Several other factors are also found to regulate S6 phosphotransferase activity in PC12 cells including epidermal growth factor, insulin, and phorbol myristate acetate. Dibutyryl cAMP stimulates S6 phosphotransferase activity; however, this activity is strongly inhibited by the protein kinase A heat stable inhibitor. At least two mechanisms exist through which the NGF-stimulated S6 kinase activity can be regulated, one that apparently can use protein kinase C whereas the other(s) does not. The potential roles of these protein kinase activities in signal transduction and regulation of cell growth and differentiation is discussed.     

Nerve growth factor-induced decrease in the calpain activity of PC12 cells

PC12 cells are a nerve growth factor-responsive clone derived from a rat pheochromocytoma. Treatment with nerve growth factor causes the cells to differentiate. One of the hallmarks of this differentiation is the generation of neurites. PC12 cells contain both calpain I and calpain II; about 90% of the total calpain activity is due to calpain II. Treatment of the cells with nerve growth factor causes a time-dependent decrease in calpain activity, more than 50% being lost over a 5-day period. Both the decrease in calpain activity and the growth of neurites are reversible upon the removal of nerve growth factor from the cultures. Agents other than nerve growth factor that cause neurite outgrowth, such as fibroblast growth factor and dibutyryl cyclic AMP, also cause a decrease in calpain activity. Calpain levels, as detected with immunoblotting or immunohistochemistry, show no decrease. Removal of calpastatin, the endogenous inhibitor of the calpains, by phenyl-Sepharose chromatography increases the calpain activity of extracts from both control and nerve growth factor-treated cells and brings the activity in the extracts from treated cells up to the activity in those from controls. Calpastatin-containing fractions from extracts of nerve growth factor-treated cells inhibit more calpain activity than do comparable fractions from control cells. These studies suggest that nerve growth factor causes a decrease in the activity of calpain in morphologically differentiating PC12 cells by causing an increase in the activity of calpastatin.     

Selective translocation of protein kinase C-delta in PC12 cells during nerve growth factor-induced neuritogenesis.

The specific intracellular signals initiated by nerve growth factor (NGF) that lead to neurite formation in PC12 rat pheochromocytoma cells are as of yet unclear. Protein kinase C-delta (PKC delta) is translocated from the soluble to the particulate subcellular fraction during NGF-induced-neuritogenesis; however, this does not occur after treatment with the epidermal growth factor, which is mitogenic but does not induce neurite formation. PC12 cells also contain both Ca(2+)-sensitive and Ca(2+)-independent PKC enzymatic activities, and express mRNA and immunoreactive proteins corresponding to the PKC isoforms alpha, beta, delta, epsilon, and zeta. There are transient decreases in the levels of immunoreactive PKCs alpha, beta, and epsilon after 1-3 days of NGF treatment, and after 7 days there is a 2.5-fold increase in the level of PKC alpha, and a 1.8-fold increase in total cellular PKC activity. NGF-induced PC12 cell neuritogenesis is enhanced by 12-O-tetradecanoyl phorbol-13-acetate (TPA) in a TPA dose- and time-dependent manner, and this differentiation coincides with abrogation of the down-regulation of PKC delta and other PKC isoforms, when the cells are treated with TPA. Thus a selective activation of PKC delta may play a role in neuritogenic signals in PC12 cells.     

Dipeptide inhibitors of ubiquitin-mediated protein turnover prevent growth factor-induced neurite outgrowth in rat pheochromocytoma PC12 cells.

Dipeptide inhibitors of the ubiquitin-dependent proteolysis pathway governed by N-terminal recognition (N-end rule) in reticulocyte lysates significantly suppress NGF- and bFGF-induced neurite outgrowth in rat pheochromocytoma PC12 cells, but do not cause retraction of already formed neurites. Peptides which do not inhibit proteolysis are also without effect on PC12 cell differentiation. Suppression of neurite outgrowth is readily reversible upon removal of the inhibitors. These data demonstrate a requirement for specific protein turnover in the process of neuron-like differentiation in PC12 cells and provide the first demonstration of a physiological role for the N-end rule.     

Nerve growth factor stimulates phosphorylation of phospholipase C-gamma in PC12 cells.

PC12 cells contain at least three immunologically distinct phospholipase C (PLC) isozymes, PLC-beta, PLC-gamma, and PLC-delta. Treatment of PC12 cells with nerve growth factor (NGF) leads to an increase in the phosphorylation of PLC-gamma, but not of PLC-beta or PLC-delta. This increase can be seen in as little as 1 minute. The increased phosphorylation occurs on both serine and tyrosine residues, with the major increase being in the former. This result suggests the possibility that the NGF-dependent increase in phosphoinositide hydrolysis in PC12 cells is due to selective phosphorylation of PLC-gamma by serine and tyrosine protein kinases associated with the NGF receptor.     

Nerve growth factor stimulates a protein kinase in PC-12 cells that phosphorylates microtubule-associated protein-2

Some of the effects of nerve growth factor (NGF) may be mediated by changes in protein phosphorylation. We have identified a protein kinase from PC-12 cells that catalyzes the phosphorylation of pig brain microtubule-associated protein (MAP)-2 in vitro. This activity is stimulated 2-4-fold in extracts from cells treated with NGF or epidermal growth factor (EGF). The partial purification and characterization of this MAP kinase indicate that it is distinct from previously described NGF-stimulated protein kinases. The NGF-stimulated kinase activity is unaffected by direct addition to the assay of the heat-stable cAMP-dependent kinase peptide inhibitor, staurosporine, or K-252A, is slightly stimulated by heparin and is inhibited by sodium fluoride and calcium ions. Treatment of cells with NGF increases the activity of the kinase within 2 min. The activity declines after 10 min, and a second phase of activation is observed at 20-30 min. Comparison of its behavior on gel permeation and sucrose density gradients indicates a molecular mass in the range of 40,000 daltons. The kinase activity is specific for ATP as substrate with a Km of 12 microM. Although the pathway of activation of MAP kinase by NGF is unknown, the stimulation can be reversed by treatment of the enzyme with alkaline phosphatase, suggesting that activation involves phosphorylation of the kinase itself. The properties and hormone sensitivity of the PC-12 MAP kinase suggest that it is similar to the previously identified, growth factor-sensitive MAP kinase from 3T3-L1 adipocytes.     

PC12 cells have caveolae that contain TrkA. Caveolae-disrupting drugs inhibit nerve growth factor-induced, but not epidermal growth factor-induced, MAPK phosphorylation

Nerve growth factor (NGF) induces survival and differentiation of the neural crest-derived PC12 cell line. Caveolae are cholesterol-enriched, caveolin-containing plasma membrane microdomains involved in vesicular transport and signal transduction. Here we demonstrate the presence of caveolae in PC12 cells and their involvement in NGF signaling. Our results showed the expression of caveolin-1 by Western blot and confocal immuno-microscopy. The presence of plasma membrane caveolae was directly shown by rapid-freeze deep-etching electron microscopy. Moreover, combined deep-etching and immunogold techniques revealed the presence of the NGF receptor TrkA in the caveolae of PC12 cells. These data together with the cofractionation of Shc, Ras, caveolin, and TrkA in the caveolae fraction supported a role for these plasma membrane microdomains in NGF signaling. To approach this hypothesis, caveolae were disrupted by treatment of PC12 cells with cholesterol binding drugs. Either filipin or cyclodextrin treatment increased basal levels of MAPK phosphorylation. In contrast, pretreatment of PC12 cells with these drugs inhibited the NGF- but not the epidermal growth factor-induced MAPK phosphorylation without affecting the TrkA autophosphorylation. Taken together, our results demonstrate the presence of caveolae in PC12 cells, which contain the high affinity NGF receptor TrkA, and the specific involvement of these cholesterol-enriched plasma membrane microdomains in the propagation of the NGF-induced signal.     

Nerve growth factor-induced phosphorylation of SNAP-25 in PC12 cells: a possible involvement in the regulation of SNAP-25 localization

Synaptosomal-associated protein of 25 kDa (SNAP-25), a t-SNARE protein essential for neurotransmitter release, is phosphorylated at Ser187 following activation of cellular protein kinase C by treatment with phorbol 12-myristate 13-acetate. However, it remains unclear whether neuronal activity or an endogenous ligand induces the phosphorylation of SNAP-25. Here we studied the phosphorylation of SNAP-25 in PC12 cells using a specific antibody for SNAP-25 phosphorylated at Ser187. A small fraction of SNAP-25 was phosphorylated when cells were grown in the absence of nerve growth factor (NGF). A brief treatment with NGF that was enough to activate the mitogen-activated protein kinase signal transduction pathway did not increase the phosphorylation of SNAP-25; however, phosphorylation was up-regulated after a prolonged incubation with NGF. Up-regulation was transitory, and maximum phosphorylation (a fourfold increase over basal phosphorylation) was achieved between 36 and 48 h after the addition of NGF. Immunofluorescent microscopy showed that SNAP-25 was localized primarily in the plasma membrane, although a significant population was also present in the cytoplasm. Quantitative microfluorometry revealed that prolonged treatment with NGF resulted in a preferential localization of SNAP-25 in the plasma membrane. A mutational study using a fusion protein with green fluorescent protein as a tag indicated that the point mutation of Ser187 to Ala abolished the NGF-dependent relocalization. A population of SNAP-25 in the plasma membrane was not increased by a point mutation at Ser187 to Glu; however, it was increased by prolonged treatment with NGF, indicating that the SNAP-25 phosphorylation is essential, but not sufficient, for the NGF-induced relocation to the plasma membrane. Our results suggest a close temporal relationship between the up-regulation of SNAP-25 phosphorylation and its relocation, and NGF-induced differentiation of PC12 cells.     

Nerve growth factor and fibroblast growth factor selectively activate a protein kinase that phosphorylates high molecular weight microtubule-associated proteins. Detection, partial purification, and characterization in PC12 cells

A cell-free assay has been developed to detect and characterize a nerve growth factor (NGF)-stimulated protein kinase activity in PC12 cells that phosphorylates high molecular weight microtubule-associated proteins (HMW-MAPs). The activity was partially purified and separated from other endogenous nonregulated HMW-MAP kinase activities by chromatography on heparin-Sepharose and Mono-Q resin. Characterization of the NGF-activated kinase (designated HMK) revealed the following features. 1) Both MAP1 and MAP2 are phosphorylated with approximately equal efficiencies. 2) Activation reaches a plateau within 3 min of NGF treatment and persists for approximately 60 min; subsequently, a substantial decline occurs by 5 h. 3) Maximal activation reaches 15-20-fold; activation is nearly as high with fibroblast growth factor, an agent that mimics NGF in promoting PC12 cell neuronal differentiation. 4) Epidermal growth factor and depolarizing levels of K+ stimulate HMK activity by only 2-4-fold; additional agents without PC12 cell differentiation activity (insulin, phorbol ester, and a permeant cAMP analogue) do not stimulate HMK activity. 5) The divalent cation requirement shows a preference for Mn2+ over Mg2+. 6) There is inhibition by 10 mM 2-aminopurine but not by 6-thioguanine, heparin, or NaF. 7) HMW-MAPs and myelin basic protein are effective substrates while histones IIIs and H1, dephospho-beta-casein, and S6 protein are not phosphorylated by HMK. These and other features appear to distinguish HMK from a variety of other well-characterized protein kinases as well as from other previously described NGF-activated kinases. The properties of HMK indicate that it could play a role in the signaling pathway for growth-factor-promoted neuronal differentiation.