Phosphorylation of Stathmin and Other Proteins Related to Nerve Growth Factor-induced Regulation of PC12 Cells*

We previously identified a set of soluble proteins whose phosphorylation could be originally related to the multihormonal regulations of anterior pituitary cells. Among these proteins, stathmin (proteins 7 and 8) was found to be ubiquitous and mostly abundant in neurons. Interestingly, stathmin and some other phosphoproteins of the same set could be identified also in PC12 cells in culture. Their phosphorylation was stimulated in these cells by nerve growth factor (NGF) in a way associated with its short term actions, probably corresponding to the early steps of its neuronal differentiating activity. In addition, the same proteins had their phosphorylation stimulated in the presence of fibroblast growth factor, known to stimulate PC12 cell differentiation in a way similar to NGF. A pharmacological analysis allowed us to distinguish three characteristic subsets of phosphoproteins, respectively, affected by CAMP-dependent agents, by CAMP-independent ones, or by both types of agents. Moreover, phosphorylation of stathmin and some other proteins was additive in the presence of NGF and of the CAMPpromoting agent forskolin. Altogether, the present results unravel some intracellular mechanisms related to the regulation of PC12 cells by extracellular effecters. They extend to the regulation of cell differentiation in our recent model for stathmin (Sobel, A., Boutterin, M-C., Beretta, L., Chneiweiss, H., Doye, V., and PeyroSaint-Paul, H. (1989) J. Sol. Chem. 264,3765-3772) as an ubiquitous intracellular relay possibly integrating the actions of diverse second messenger pathways involved in cell regulations.


Phosphorylation
of Stathmin and Other Proteins Related to Nerve Growth Factor-induced Regulation of PC12 Cells* (Received for publication, December 11, 1989) Valerie Doye, Marie-Claude Boutterin, and Andre Sobel From  We previously identified a set of soluble proteins whose phosphorylation could be originally related to the multihormonal regulations of anterior pituitary cells. Among these proteins, stathmin (proteins 7 and 8) was found to be ubiquitous and mostly abundant in neurons. Interestingly, stathmin and some other phosphoproteins of the same set could be identified also in PC12 cells in culture. Their phosphorylation was stimulated in these cells by nerve growth factor (NGF) in a way associated with its short term actions, probably corresponding to the early steps of its neuronal differentiating activity. In addition, the same proteins had their phosphorylation stimulated in the presence of fibroblast growth factor, known to stimulate PC12 cell differentiation in a way similar to NGF. A pharmacological analysis allowed us to distinguish three characteristic subsets of phosphoproteins, respectively, affected by CAMP-dependent agents, by CAMP-independent ones, or by both types of agents. Moreover, phosphorylation of stathmin and some other proteins was additive in the presence of NGF and of the CAMPpromoting agent forskolin. Altogether, the present results unravel some intracellular mechanisms related to the regulation of PC12 cells by extracellular effecters. They extend to the regulation of cell differentiation in our recent model for stathmin (Sobel,  Many extracellular agents regulate cell differentiation and functions through their binding to specific receptors at the cell surface. In the subsequent intracellular transduction of these signals, phosphorylation-dephosphorylation of proteins is known to be a major regulatory mechanism in all biological systems (l-5).
We previously identified, by two-dimensional polyacrylamide gel electrophoresis (BD-PAGE),' a set of cytoplasmic * This work was supported by funds from the Institut National de la Sante et de la Recherche M&licale, the Centre National de la Recherche Scientifique, the Ministire de la Recherche et de la Technologie, and the Association Francaise contre les Myopathies. Part of this work was published previously in abstract form (58). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solelv to indicate this fact.
One 19-kDa phosphoprotein, corresponding to the proteins that we originally designated as 7 and 8 and that we recently proposed to name "stathmin," appears actually to be ubiquitous (11) and identical to proteins P19 (12), pp17 or prosolin (13), and p18 (14). We proposed that it might play a general role as an intracellular relay integrating the various second messenger pathways activated by diverse extracellular signals (11). Two distinct isoforms LY and /3 of stathmin have been identified (15), differing by post-translational modifications (16). Their common cDNA was recently cloned from PC12 cell (16) and rat testis (17) cDNA libraries. However, stathmin is most abundant in neurons and displays a peak of expression during ontogenesis of the central nervous system around birth (12,16,18,19).
The rat pheochromocytoma cell line PC12 (20) has proved to be a very good model for studying the mechanisms and regulations of neuronal differentiation. Indeed, when treated for several days with nerve growth factor (NGF), the chromaffin-resembling cells cease proliferating and acquire a sympathetic neuron-like phenotype with the outgrowth of neurites (reviewed in Ref. 21). This long term action of NGF is preceded by a number of short term responses such as membrane ruffling, activation of ornithine decarboxylase, and protein phosphorylation (reviewed in Ref. 21). PC12 cells also respond to other regulatory agents such as fibroblast growth factor (FGF), which has been shown to reproduce most if not all the biological actions of NGF (22). Other agents trigger only some of the actions of NGF such as epidermal growth factor (23,24), tumor promoters (25), adenosine and analogues (26), or CAMP analogues which induce some neurite outgrowth but with different characteristics than with NGF (27).
NGF has been shown to activate several protein kinases (31,36,37) including a specific NGF-activated protein kinase (38). Stimulation of these kinases results in the phosphorylation of a number of intracellular proteins, some of which have been characterized by their BD-PAGE migration properties (39)(40)(41) to 100 mM NaCl, treated at 100 'C for 2 min, and centrifuged again as above (11). In these conditions, stathmin remained in the resulting "S3" supernatant which was further prepared for electrophoresis as described above. Two-dimensional Polyacrylamide Gel Electrophoresis BD-PAGE-ZD-PAGE was performed according to Garrels (44) and as previously described (6). The isoelectric focusing gels contained 2% total ampholines, pH 5-7 and 3-10, in the proportions 4:l. The second dimension was run on 12% acrylamide gels. The fixed gels were either first stained or dried directly and exposed for autoradiography (3-

AND DISCUSSION
Cells were labeled with "PO:-and treated with the appropriate pharmacological agents, and the phosphorylated proteins were analyzed by PD-PAGE autoradiography. This approach led previously to the characterization, in anterior pituitary cells, of the set of cytoplasmic phosphoproteins designated as proteins 1-16 (6,8,9). More recently, it has been shown that the radioactive spots numbered 7 and 8 (Mr = 19,000) correspond to the phosphorylated forms Pl and P2 of an ubiquitous protein that we proposed to name stathmin (11). Two isoforms, (Y and ,f3, of this protein were identified (15), whose respective nonphosphorylated (cu0, PO) and phosphorylated forms (al-cu3, pl-p3) yield corresponding spots of decreasing p1 (6.2-5.5) on BD-PAGE gels: Nl (aO), N2 (/30), Pl (cul), P2 (/?l, LYE), and P3 (@2, a3, p3). GH4Cl proteins is further stressed by a comparable regulation of their phosphorylation by various extracellular effecters (see below). In addition, the PC12 cell proteins which migrated at the positions of the unphosphorylated (Nl and N2) and phosphorylated (Pl, P2, and P3) spots of stathmin shared the characteristic heat resistance property (11) of this protein (see Fig. 4) and were all recognized by an antibody against brain stathmin (not shown). We also recently cloned a cDNA for stathmin from PC12 cells, coding for amino acid sequences identical to sequences determined for rat brain stathmin (16). Together, these observations indicate that the comigrating proteins are at least homologous, if not identical.
We therefore use here the same designations (stathmin and proteins 9-10 and 16) for the proteins in PC12 cells as for the corresponding phosphoproteins in other biological systems.
Phosphoproteins characteristic of PC12 cells were also identified on the PD-PAGE autoradiograms (Fig. lA). The different forms of tyrosine hydroxylase, first detected by reference to Nose et al. (39), were further identified on immunoblots with a specific antiserum (not shown). Chartin microtubule-associated proteins were also identified by analogy with the 2D-PAGE patterns presented by Black et al, (40) and by the observation of some of their previously described properties (47) such as long-term increased phosphorylation induced by NGF and inhibition of this effect by lithium (not shown). For comigration (middle panei), equal amounts of trichloroacetic acid-precipitable radioactive material from both treated cell types were coelectrophoresed. Note that only one radioactive spot is detectable on the corn&ration gel at the location of each of the GH4Cl proteins (spot 16 was more clearly detected on the original autoradiograms), indicating that the corresponding PC12 proteins exactly comigrate with their respective GH4Cl homologs.
Since PC12 cells can be converted by NGF to sympathetic neuron-like cells (21), we compared their phosphorylation pattern with that of SCG neurons in primary culture (Fig.  L4). As in PC12 cells, phosphoproteins of the set 7-16, including stathmin, were present in these peripheral neurons from nontumoral origin (some of these proteins, which appear faintly on the figure, were clearly detectable on the original autoradiograms).

Regulation of PC12 Cell Protein Phosphorylation by NGF
2D-PAGE Phosphoprotein Pattern Changes Induced by NGF-NGF has been shown to induce the phosphorylation of various well identified intracellular substrates like tyrosine hydroxylase (28), ribosomal protein S6 (37), vinculin (48), synapsin (49), histones (28), and other nuclear proteins (50,51). When PC12 cells were prelabeled with [32P]orthophosphate and treated with NGF (200 rig/ml) for 30 min, the phosphorylation of several proteins detected on SD-PAGE autoradiograms, including tyrosine hyclroxylase, was stimulated. This latter effect was thus used in all experiments as an internal control for effective NGF stimulation.
Phosphorylation of stathmin, protein 16 (Figs. 2-6), and of the additional protein 17 (Fig. 6) was clearly and consistently stimulated by NGF. In parallel, a decrease in the intensity of spot 9 was accompanied by a concomitant increase of the slightly more acidic spot 10 (Figs. 3 and 5), appearing, like in pituitary cells (6,8,9), as a possible conversion of phospho- in proteins 9-10, 16, and stathmin. 32PO:--prelabeled PC12 cells were treated for increasing times with 2.5 S NGF (200 rig/ml). Details of the 2D-PAGE autoradiograms in the region of proteins 9-10, 16, and stathmin (spots Pl, P2, and P3) are presented. NGF induced an apparent conversion of spot 9 to spot 10 and a shift of stathmin to its more acidic spots (Pa). Spot 16, not detected in untreated cells, appears clearly after 2 min of NGF treatment. protein 9 to 10, due to phosphorylation or to another molecular modification. Stimulation of PC12 cells by NGF simultaneously induced a conversion of both a! and @ isoforms of stathmin from their unphosphorylated to their phosphorylated forms, as reflected on silver-stained gels by a shift from spots Nl ((~0) and N2 (PO) toward Pl (cul) and P2 (Pl, (r2). Spot P3 (LIZ, ru3,/33) was not clearly detected by silver staining in our study (Fig. 4). This result indicates that the NGF-stimulated 32P incorporation reflects not only an activated phosphate turnover but an actual increase in the degree of phosphorylation of stathmin. Together with the observation that forskolin and adenosine induced the phosphorylation of the 01 and cr2 forms of stathmin migrating as P2 (Figs. 5 and 6), it also demonstrates that, as in GH cell regulations (9), both the cy and p isoforms Dose Response and Kinetics of NGF Effects-Phosphorylation of tyrosine hydroxylase, stathmin, and proteins 16 (Fig.  2) and 17 and the apparent conversion of protein 9 to 10 (not shown) were stimulated in the concentration range where NGF elicits its biological actions, with a maximum effect at 20 rig/ml, stable up to 200 rig/ml.
In the presence of 200 rig/ml 2.5 S NGF, stimulation of the phosphorylation of all the above described proteins occurred rapidly (Fig. 3). The apparent conversion of protein 9 to protein 10 was the earliest signal detected (1 min), followed by stimulation of stathmin (Pl-P3) and proteins 16 and 17 (2 min). Maximal effects were reached at 5 min for proteins 9 and 10 and at 1 h for the other proteins. In addition, the time course of the NGF-stimulated conversion of stathmin isoforms from Nl and N2 to Pl and P2 (Fig. 4) paralleled that of "'P incorporation into Pl-P3. After its maximal conversion at 1-2 h, stathmin was dephosphorylated, the distribution of its various forms returning to its basal state at 8 h (Fig. 4) and remaining in that state for up to 24 h in the continuous presence of NGF (not shown). In agreement with these data, the basal levels of '("P incorporation into stathmin and proteins 9-10, 16 (Fig. 5), and 17 (not shown) were similar in PC12 cells treated with NGF (200 rig/ml 7 S NGF) for 4 days and in untreated cells, and addition of fresh NGF induced no further stimulation (not shown). This relatively rapid dephosphorylation and subsequent "insensitivity" of stathmin thus reflects a "desensitization" of the stathmin phosphorylation pathway for the action of NGF, the cellular and molecular levels at which it takes place remaining to be determined.
Altogether, the relatively rapid stimulation and subsequent reversion of the phosphorylation of stathmin and proteins 9-10, 16, and 17 is thus most likely related to the acute biological actions of NGF, which themselves are likely to be the first steps of its long term neuronal differentiating activity. In addition to previous correlations with the regulations of cell proliferation and functions in diverse biological systems, this is the first example of a correlation between the regulation of cell differentiation and the phosphorylation of stathmin and proteins 9-10, 16, and 17.

Phosphorylation
Patterns Induced by Other Pharmacological Agents The same approach as with NGF was used to examine the effects of other pharmacological effecters whose biological, morphological, and/or biochemical actions on PC12 cells have been described. Since in NGF-pretreated cells phosphorylation of the above described proteins was no longer stimulated by NGF, we also determined if it could still be stimulated by other pharmacological effecters. Fibroblast Growth Factor FGF-Among various effecters regulating PC12 cell functions or differentiation, FGF induces the neuronal differentiation of PC12 cells in a way similar to NGF, whose wide spectrum of biological effects it most closely reproduces (22), including phosphorylation of several proteins like tyrosine hydroxylase (52) or a nuclear protein and NsplOO (22 Three sets of radioactive spots are indicated: 1) stathmin-related spots whose labeling was stimulated by both NGF and adenylate cyclase activators; 2) spots with arrcmheads whose intensity was enhanced only by NGF (like spots 16 and 17); and 3) spots pointed with arrows whose phosphorylation is modified by CAMP-stimulating agents (adenosine and forskolin).
In order to study the additivity of their actions, forskolin was added in the culture medium for the last 15 min of the l-h NGF treatment. Note the apparent shift of spots 16 and 17 to a more acidic spot (arrowhead) and the increased intensity of stathmin spot P3, whereas the intensities of some CAMP-related spots (unlabeled arrows) decreased as compared with the incubation with forskolin alone. protein 9 to 10, in the same way but to a lesser extent than NGF (Fig. 5A). As shown in Fig. 54 its effects were decreased in NGF-pretreated cells.
Thus, although the biological actions of FGF are mediated by specific receptors distinct from the NGF receptor (22,53), phosphorylation of the same proteins in response to FGF and NGF further suggests that the originally distinct intracellular pathways elicited by NGF and FGF converge in a common pathway leading to the same functional regulations. The original independence of the two pathways is, however, also underlined by the partial persistence of FGF-stimulated phosphorylations in NGF-treated cells. CAMP-dependent Phosphorylation Patterns-The second messenger(s) triggered by NGF is (are) unknown, and although the possible role of CAMP has been discussed (28,29), it seems that it is not mainly responsible for the numerous biological actions of NGF. However, CAMP and its analogues or promoting agents, as well as adenosine which activates adenylate cyclase through an A2 receptor (54), all induce some of the short term actions of NGF such as early stimulation of process outgrowth (55,56) or induction of ornithine decarboxylase (57). Interestingly, the adenylate cyclase activator forskolin (10 PM) and adenosine (100 PM) induced the apparent 9 to 10 conversion and the phosphorylation of stathmin in a way similar to the action of NGF (Figs. 5A and 6). Conversely, they did not stimulate the phosphorylation of some proteins affected by the growth factors, such as proteins 16 and 17, and they also induced phosphorylations not detected with NGF, such as proteins A and B (Fig. 6). These observations extend previous ones by Halegoua and Patrick (28) of a partial overlap of the spectrum of proteins phosphorylated by NGF and CAMP.
The lack of stimulation of proteins 16 and 17 (Fig. 6) by agents acting through CAMP indicated, however, that the spectrum of protein phosphorylation stimulated by NGF or FGF was partially distinct from that induced by pharmacological effecters enhancing intracellular CAMP. We thus completed this approach by studying the additivity of phosphorylation patterns induced by saturating concentrations of 2.5 S NGF (200 rig/ml) and forskolin (10 pM) (Fig. 6).
Although they are not quantitative, our data indicate that the effects of NGF and forskolin were at least partially additive; phosphorylation of stathmin was higher than with either agent alone, as attested by the strong intensity of its most phosphorylated spot, P3. In addition, forskolin and NGF mutually potentiated or inhibited some of their respective phosphorylation effects (Fig. 6). These results, together with the fact that long term NGF pretreatment did not affect forskolin (Fig. 5) or adenosine (not shown) responses, are also in agreement with the fact that NGF and CAMP trigger distinct regulatory pathways, which only partially overlap in their downstream effects.
TPA-Ca" (30)(31)(32)(33)36) and diacylglycerols (34) were also proposed to be involved in the biological actions of NGF. Since both agents are known to regulate the Ca'+/phospholipid-dependent protein kinase (protein kinase C), we also examined the effects on protein phosphorylation of the tumor promoter TPA, a direct activator of this enzyme which mimics some of the actions of NGF (25). TPA (100 rig/ml) had only a small effect on undifferentiated PC12 cells, significantly enhanced after the 4-day differentiating treatment by NGF. It induced the apparent conversion from spot 9 to 10 and the phosphorylation of spots Pl, P2, 16 (Fig. 5), and 17 (not shown).
Interestingly, phosphorylation of stathmin (spots Pl and P2) and proteins 16 and 17 was shown in NGF-pretreated cells to be stimulated also by K' depolarization (39), most likely through the resulting increase of the intracellular Ca'+ concentration.
Phosphorylation of these proteins is thus related to the activation of diverse second messenger pathways, the Ca" and/or phospholipid one(s) being more effective in differentiated than in untreated PC12 cells for reasons still unclear at the present stage.

Characteristic Features of Phosphoproteins
Related to NGFand CAMP-triggered Intracellular Regulatory Pathways Proteins 9-1 O-The apparent conversion of phosphoprotein 9 to 10, although possibly occurring through a modification other than phosphorylation, is a general and ubiquitous phenomenon observed in diverse biological systems (6,8,9). It was related in each case to activations of cell responses by various extracellular agents and, as for stathmin, in a way not specific to any particular second messenger pathway. Proteins A and B-On the other hand, proteins labeled A and B on Fig. 6 were stimulated only by CAMP-related effectors. Interestingly, they most likely correspond to protein 11 previously identified in pituitary cells as a specific CAMPresponsive protein (6,8,9). Unlike stathmin, they appear thus more generally as ubiquitous markers of the activation of a distinct and specific second messenger pathway. Proteins  However, the decrease of the intensities of spots 16 and 17, after the simultaneous addition of both forskolin and NGF, together with the increase of slightly more acidic spots at the same M, level (Fig. 6) suggests that these latter spots derive, respectively, from spots 16 and 17, possibly by further phosphorylation.
If this model is correct, the actions of the two types of effecters are also additive on these proteins in a sequential fashion since the phosphorylation induced by NGF has to take place to allow the CAMP-induced phosphorylation.

Similar observations
were made with proteins 16 and 17 in other biological systems, such as brain neurons in culture,' indicating that they might be, like stathmin, ubiquitous phosphoproteins related to the biological actions of various second messenger pathways activated by extracellular regulatory agents. Preliminary observations indicate that proteins 16 and 17 might actually be very closely related to stathmin at the molecular level.
Stuthmin-Among proteins 7-11, 16, and 17, stathmin is the best characterized on both the biological and biochemical levels. It was detected in many different biological systems (11)(12)(13)19) where its phosphorylation could be related to the regulations of cells by various extracellular agents (reviewed in Ref. 11). In anterior pituitary cells, its phosphorylation could be related to regulations of the cell's differentiated functions (6,8). In muscle cells, phosphorylation of stathmin was related to regulations of cell proliferation and functions in a way depending on their state of differentiation (10). Our present results with PC12 cells indicate that stathmin is also phosphorylated in response to NGF and other extracellular agents regulating PC12 cell differentiation.
They thus extend to cell differentiation the role proposed for stathmin (11) of an ubiquitous intracellular relay possibly integrating the diverse extracellular signals regulating cellular responses. As mentioned above, stathmin was phosphorylated in response to activation of both CAMP-dependent and -independent pathways in PC12 cells. Furthermore, the additivity of NGF and forskolin might reflect the fact that the distinct transduction pathways induce sequential phosphorylations on different sites and possibly even a synergism between the sites phosphorylated.
Altogether, these observations suggest that stathmin may reflect more generally the state of regulation of the cell by integrating the levels of activation of the various intracellular messenger pathways.