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 cAMP-promoting agent forskolin. Altogether, the present results unravel some intracellular mechanisms related to the regulation of PC12 cells by extracellular effectors. 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 Peyro-Saint-Paul, H. (1989) J. Biol. Chem. 264, 3765-3772) as an ubiquitous intracellular relay possibly integrating the actions of diverse second messenger pathways involved in cell regulations.     

Antileukemic agent alkyllysophospholipid regulates phosphorylation of distinct proteins in HL60 and K562 cells and differentiation of HL60 cells promoted by phorbol ester

The tumor-promoting 12-0-tetradecanoylphorbol-13-acetate (TPA) stimulated phosphorylation of several proteins in block I (including protein Ia) and protein 3 in HL60 cells. The antileukemic agent alkyllysophospholipid (ALP) inhibited the TPA-stimulated phosphorylation of these proteins and the TPA-induced differentiation of the cells. In comparison, TPA only stimulated phosphorylation of protein 3 in K562 cells which, in contrast, were not induced to differentiate by TPA and lacked protein Ia and had a very high basal phosphorylation of protein B. ALP inhibited phosphorylation of protein 3 as well as protein B in K562 cells. The data suggest that the presence of distinct phosphoproteins and regulation of their phosphorylation may be related to the selective susceptibility of the two leukemia cell lines to the maturating effect of TPA and cytotoxicity of ALP.     

Comparative analysis of phosphoprotein-enriched myocyte proteomes reveals widespread alterations during differentiation

The differentiation of skeletal muscle has been associated with altered phosphorylation status of individual proteins. However, a global analysis of protein phosphorylation during myogenesis has yet to be undertaken. Here, we report the identification of over 130 putative phosphoproteins from murine C2C12 muscle cells. Cell extracts were fractionated on phosphoprotein enrichment columns and the resulting proteins were detected by two-dimensional gel electrophoresis and silver stain, and identified by liquid chromatography coupled to electrospray tandem mass spectrometry. The early differentiation of C2C12 myoblasts was found to be accompanied by changes in the phosphorylation or expression of numerous proteins including cytoskeletal, heat shock and signaling proteins, the pp32 family of nuclear phosphoproteins, several disease-associated gene products and other characterized and uncharacterized proteins.     

Intracellular substrates for extracellular signaling. Characterization of a ubiquitous, neuron-enriched phosphoprotein (stathmin)

We previously identified (Sobel, A., and Tashjian, A. H., Jr. (1983) J. Biol. Chem. 258, 10312-10324) a group of cytoplasmic proteins whose phosphorylation could be related to the regulation by extracellular effectors of cells as different as pituitary and muscle cells. Among these phosphoproteins, proteins "7" and "8" (Mr approximately 19,000, pI approximately 5.8-6.0), that we now designate P1 and P2, are very abundant in rat brain. Partial purification of these proteins was therefore achieved after 100 degrees C precipitation of a rat brain-soluble fraction and further fractionation of the supernatant by ion exchange chromatography. Several related non-phosphorylated (N1, N2) and phosphorylated (P3) proteins were also identified in the heat-resistant supernatant. Antisera raised against P2 extracted from nitrocellulose blots of semipreparative two-dimensional gels recognized all the proteins N1, N2, P1, P2, and P3, confirming that they belong to the same protein family, and suggesting that they are likely various forms of a single protein core. The same protein could be detected biochemically and immunologically at various concentrations in all the tissues or cell types from diverse mammalian and nonmammalian species tested. Together with our previous data relating its phosphorylation to the regulation of the proliferation, differentiation, and/or the functions of the cells considered, this observation leads us to suggest that it might be an ubiquitous regulatory phosphoprotein playing the role of an intracellular "relay" for extracellular signals, after their binding to specific membrane receptors and the generation of second messengers. We propose to name this protein stathmin, from the greek "stathmos" (relay).     

Evidence on the participation of protein kinase C alpha in the proliferation of cultured myoblasts.

There is evidence involving protein kinase C (PKC) in the signal transduction pathways that regulate the differentiation of myoblasts into mature multinucleated muscle cells (myotubes). In order to obtain information on the possible role of individual PKC isozymes in myogenesis, in the present work we investigated the differential expression of PKC isoforms alpha, beta, delta, epsilon, and zeta during muscle cell development in vitro. Chick embryo myoblasts cultured from 1 to 6 days were used as experimental model. Morphological characterization and measurement of specific biochemical parameters in cultures, e.g., DNA synthesis, creatine kinase activity, and myosin levels, revealed a typical muscle cell developmental pattern consisting of an initial proliferation of myoblasts followed by their differentiation into myotubes. PKC activity was high at the proliferation stage, decreased as myoblasts elongated and fused, and increased again in differentiated myotubes. In proliferating myoblasts, the PKC inhibitors calphostin C and bisindolylmaleimide I decreased DNA synthesis whereas in myoblasts undergoing differentiation they exerted the opposite effect, suggesting that PKC plays a role at both stages of myogenesis. Western blot analysis of changes in the expression of PKC isoforms during muscle cell development showed high levels of PKC alpha in the proliferating phase which markedly decreased as myoblasts differentiated. Treatment with TPA of proliferative myoblasts inhibited DNA synthesis and selectively down-regulated PKC alpha, suggesting that this isozyme may have an important role in maintaining myoblast proliferation. On the other hand, an increase in the expression of PKC beta, delta, and epsilon was detected during myogenesis, suggesting that one or more of these isoforms may participate in the differentiation process of myoblasts.     

Phosphorylation of eukaryotic elongation factor 2 can be regulated by phosphoinositide 3-kinase in the early stages of myoblast differentiation

We have previously reported that phosphorylation of eukaryotic elongation factor 2 (eEF2) is related to the differentiation of chick embryonic muscle cells in culture. In the present study, we found that eEF2 phosphorylation declined shortly after induction of differentiation of L6 myoblasts, when the cells prepare for terminal differentiation by withdrawing from the cell cycle. This decrease in phosphorylation was prevented by inhibitors of phosphoinositide 3-kinase (PI3-kinase) that strongly inhibit myoblast differentiation. We hypothesized that PI3-kinase plays an important role in myoblast differentiation by regulating eEF2 phosphorylation in the early stages of differentiation. To test this hypothesis, myoblasts were synchronized at in G2/M and cultured in fresh differentiation medium (DM) or growth medium (GM). In DM the released cells accumulated in G0/G1 while in GM they progressed to S phase. In addition, cyclin D1 was more rapidly degraded in DM than in GM, and eEF2 phosphorylation decreased more. Inhibitors of PI3-kinase increased eEF2 phosphorylation, but PI3-kinase became more activated when eEF2 phosphorylation declined. These results suggest that the regulation of L6 myoblast differentiation by PI3-kinase is related to eEF2 phosphorylation.     

Comparative proteomes of the proliferating C(2)C(12) myoblasts and fully differentiated myotubes reveal the complexity of the skeletal muscle differentiation program

When cultured in low serum-containing growth medium, the mouse C(2)C(12) cells exit cell cycle and undergo a well-defined program of differentiation that culminates in the formation of myosin heavy chain-positive bona fide multinucleated muscle cells. To gain an understanding into this process, we compared total, membrane- and nuclear-enriched proteins, and phospho-proteins from the proliferating C(2)C(12) cells and the fully differentiated myotubes by the combined methods of two-dimensional PAGE, quantitative PDQuest image analysis, and MS. Quantification of more than 2,000 proteins from C(2)C(12) myoblasts and myotubes revealed that a vast majority of the abundant proteins appear to be relegated to the essential, housekeeping and structural functions, and their steady state levels remain relatively constant. In contrast, 75 proteins were highly regulated during the phenotypic conversion of rapidly dividing C(2)C(12) myoblasts into fully differentiated, multi-nucleated, post-mitotic myotubes. We found that differential accumulation of 26 phospho-proteins also occurred during conversion of C(2)C(12) myoblasts into myotubes. We identified the differentially expressed proteins by MALDI-TOF-MS and LC-ESI-quadrupole ion trap MS/MS. We demonstrate that more than 100 proteins, some shown to be associated with muscle differentiation for the first time, that regulate inter- and intracellular signaling, cell shape, proliferation, apoptosis, and gene expression impinge on the mechanism of skeletal muscle differentiation.     

'Early' mammalian myoblasts are resistant to phorbol ester-induced block of differentiation

Mesenchymal cells were isolated from somites and limbs of mouse embryos at different developmental stages. When grown in tissue culture, some of the cells underwent muscle differentiation as indicated by synthesis of sarcomeric myosin, acetylcholine receptor and, in the case of limb cells, fusion into multinucleated myotubes. When the tumour promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) was added to these cultures, it caused differential effects, depending upon the age of the embryo from which cells were isolated. In cultures of somites or limb bud from embryos up to 12 days post coitum, TPA did not interfere with the appearance of differentiated muscle cells. When TPA was added to cultures from older embryos, it inhibited muscle differentiation with an efficiency which increased with the age of the embryo, reaching about 90% inhibition at 15 days. After this period, a new population of myogenic cells appeared in the limb, which were able to differentiate in the presence of TPA and represented the great majority of myoblasts after day 18 of embryonic development. The simplest interpretation of these data can be based on the existence of three major classes of myogenic cell precursors, which appear sequentially during muscle histogenesis: 'early' myoblasts, which appear resistant to tumour promoters; 'late' myoblasts, whose differentiation is inhibited by tumour promoters and 'satellite' cells which, like early myoblasts, show no sensitivity to TPA.     

Control of differentiation in heterokaryons and hybrids involving differentiation-defective myoblast variants

Clones of differentiation-defective myoblasts were isolated by selecting clones of L6 rat myoblasts that did not form myotubes under differentiation-stimulating conditions. Rat skeletal myosin light chain synthesis was induced in heterokaryons formed by fusing these defective myoblasts to differentiated chick skeletal myocytes. This indicates that the structural gene for this muscle protein was still responsive to chick inducing factors and that the defective myoblasts were not producing large quantities of molecules that dominantly suppressed the expression of differentiated functions. The regulation of the decision to differentiate was then examined in hybrids between differentiation- defective myoblasts and differentiation-competent myoblasts. Staining with antimyosin antibodies showed that the defective myoblasts and homotypic hybrids formed by fusing defective myoblasts to themselves could in fact differentiate, but did so more than a thousand times less frequently than the 64% differentiation achieved by competent L6 myoblasts or homotypic competent X competent L6 hybrids. Heterotypic hybrids between differentiation-defective myoblasts and competent L6 cells exhibited an intermediate behavior of approximately 1% differentiation. A theoretical model for the regulation of the commitment to terminal differentiation is proposed that could explain these results by invoking the need to achieve threshold levels of secondary inducing molecules in response to differentiation-stimulating conditions. This model helps explain many of the stochastic aspects of cell differentiation.     

Rac1 inhibits myogenic differentiation by preventing the complete withdrawal of myoblasts from the cell cycle

The small GTPase protein Rac1 is involved in a wide range of biological processes, yet its role in cell differentiation is mostly unknown. Here we show that Rac1 activity is high in proliferating myoblasts and decreases during the differentiation process. To analyze the involvement of Rac1 in muscle differentiation, different forms of the protein were expressed in muscle cells. A constitutively activated form of Rac1 (Rac1Q61L) inhibited the activity of MyoD in promoting muscle differentiation, whereas a dominant negative form of Rac1 (Rac1T17N) induced the activity of MyoD in promoting muscle differentiation. Expression of Rac1T17N imposed myogenic differentiation on myoblasts growing under mitogenic conditions. In inquiring whether Rac1 affected the withdrawal of myoblasts from the cell cycle, we analyzed the expression of cyclin D1 and p21(WAF1) and the phosphorylation state of the retinoblastoma protein. According to these markers and bromodeoxyuridine incorporation, C2 myoblasts expressing Rac1T17N exited the cell cycle earlier than control C2 cells. Myoblasts expressing Rac1Q61L did not permanently withdraw from the cell cycle. An indication of the possible involvement of the mitogen-activated protein kinase (MAPK) pathway in Rac1-mediated myoblast proliferation was obtained by the use of MAPK kinase inhibitors U0126 and PD098059. These inhibitors arrested C2-Rac1Q61L cell cycling. Taken together, our results show that Rac1 activation interferes with myoblast exit from the cell cycle via or in concert with the MAPK pathway.     

Differences in the cyclic AMP-dependent phosphorylation of plasma membrane proteins of differentiated and undifferentiated L6 myogenic cells

Differences in the cyclic AMP-dependent plasma membrane phosphorylation system of undifferentiated and differentiated L6 myogenic cells have been detected. Endogenous plasma membrane protein phosphorylation in undifferentiated L6 myoblasts was stimulated more than three fold by 5 x 10(-5) M cyclic AMP, whereas no statistically significant cyclic AMP-dependent phosphorylation of endogenous plasma membrane proteins was observed in differentiated L6 cells. In undifferentiated cells cyclic AMP promoted the phosphorylation of several proteins, the most prominent of which had a molecular weight of 110,000. In differentiated cells cyclic AMP did not selectively promote the phosphorylation of specific plasma membrane proteins. Both differentiated and undifferentiated L6 cells, however, contain a cyclic AMP-dependent protein kinase capable of catalyzing the phosphorylation of exogenous substrates, such as histone f2b. Therefore, the data show that differentiation in L6 cells is associated with a selective change in the activity of a plasma membrane cyclic AMP-dependent protein kinase which employs endogenous membrane proteins as substrate.     

SV40 immortalizes myogenic cells: DNA synthesis and mitosis in differentiating myotubes

Primary skeletal muscle myoblasts have a limited proliferative capacity in cell culture and cease to proliferate after several passages. We examined the effects of several oncogenes on the immortalization and differentiation of primary cultures of rat skeletal muscle myoblasts. Retroviruses containing a SV40 large T antigen (LT) gene very efficiently immortalize myogenic cells. The immortalized cell lines retain a very high differentiation capacity and form, in the appropriate culture conditions, a very dense network of muscle fibers. As in primary culture, cell fusion is associated with the synthesis of large amounts of muscle-specific proteins. However, unlike normal myoblasts (and previously established myogenic cell lines), nuclei in the multinucleated fibers of SV40-immortalized cells synthesize DNA and enter mitosis. Thus, withdrawal from DNA synthesis is not obligatory for cell fusion and biochemical differentiation. Using a retrovirus coding for a temperature-sensitive SV40 LT, myogenic cell lines were produced in which the SV40 LT could be inactivated by a shift from 33 degrees C to 39 degrees C. The inactivation of LT induced massive cell fusion and synthesis of muscle proteins. The nuclei in those fibers did not synthesize DNA, nor did they undergo mitosis. This approach enabled the reproducible establishment of myogenic cell lines from very small populations of myoblasts or single primary myogenic clones. Activated p53 also readily immortalized cells in primary muscle cultures, however the cells of eight out of the nine cell lines isolated had a fibroblastic morphology and could not be induced to form multinucleated fibers.     

Identification of a role for the sialomucin CD164 in myogenic differentiation by signal sequence trapping in yeast

Determination and differentiation of skeletal muscle precursors requires cell-cell contact, but the full range of cell surface proteins that mediate this requirement and the mechanisms by which they work are not known. To identify participants in cell contact-mediated regulation of myogenesis, genes that encode secreted proteins specifically upregulated during differentiation of C2C12 myoblasts were identified by the yeast signal sequence trap method (K. A. Jacobs, L. A. Collins-Racie, M. Colbert, M. Duckett, M. Golden-Fleet, K. Kelleher, R. Kriz, E. R. La Vallie, D. Merberg, V. Spaulding, J. Stover, M. J. Williamson, and J. M. McCoy, Gene 198:289-296, 1997), followed by RNA expression analysis. We report here the identification of CD164 as a gene expressed in proliferating C2C12 cells that is upregulated during differentiation. CD164 encodes a widely expressed cell surface sialomucin that has been implicated in regulation of cell proliferation and adhesion during hematopoiesis. Stable overexpression of CD164 in C2C12 and F3 myoblasts enhanced their differentiation, as assessed by both morphological and biochemical criteria. Furthermore, expression of antisense CD164 or soluble extracellular regions of CD164 inhibited myogenic differentiation. Treatment of C2C12 cells with sialidase or O-sialoglycoprotease, two enzymes previously reported to destroy functional epitopes on CD164, also inhibited differentiation. These data indicate that (i) CD164 may play a rate-limiting role in differentiation of cultured myoblasts, (ii) sialomucins represent a class of potential effectors of cell contact-mediated regulation of myogenesis, and (iii) carbohydrate-based cell recognition may play a role in mediating this phenomenon.     

Protein kinase B beta/Akt2 plays a specific role in muscle differentiation

Insulin-like growth factors positively regulate muscle differentiation through activation of the phosphatidylinositol 3-kinase/protein kinase B (PKB/Akt) signaling pathway. Here, we compare the role of the two closely related alpha (Akt1) and beta (Akt2) isoforms of PKB in muscle differentiation. During differentiation of C2.7 or L6D2 myoblasts, PKBbeta was up-regulated whereas expression of PKBalpha was unaltered. Although the two isoforms were found active in both myoblasts and myotubes, cell fractionation experiments indicated that they displayed distinct subcellular localizations in differentiated cells with only PKBbeta localized in the nuclei. In a transactivation assay, PKBbeta (either wild-type or constitutively active) was more efficient than PKBalpha in activating muscle-specific gene expression. Moreover, microinjection of specific antibodies to PKBbeta inhibited differentiation of muscle cells, whereas control or anti-PKBalpha antibodies did not. On the other hand, microinjection of the anti-PKBalpha antibodies caused a block in cell cycle progression in both non muscle and muscle cells, whereas anti-PKBbeta antibodies had no effect. Taken together, these results show that PKBbeta plays a crucial role in the commitment of myoblasts to differentiation that cannot be substituted by PKBalpha.     

Early changes in phosphoprotein patterns of Friend erythroleukaemia cells induced by dimethylsulphoxide or phorbol esters

Immediate changes in protein phosphorylation in Friend erythroleukaemia cells (FELC) were examined in response to stimulation with dimethylsulphoxide (DMSO), which triggers cell differentiation, 12-O-tetra-decanoyl-phorbol 13-acetate (TPA), a tumour promoter, and the Ca++ ionophore A23187. The effects of the cyclic nucleotides, cAMP and cGMP, on the patterns of phosphoproteins were also analysed. Autoradiographs of two-dimensional gels reveal that within 30 min of treatment by the various agents differences in the patterns of protein phosphorylation can be seen. Treatment with DMSO and TPA altered the phosphoprotein patterns in different ways. These patterns were distinct from those observed in untreated and cyclic nucleotide treated cells. Treatment with the calcium ionophore caused a unique phosphorylation pattern unlike any of the others. These data suggest that the very early changes seen in the patterns of protein phosphorylation in FELC may be related to the induction of differentiation.     

Molecular basis for impaired muscle differentiation in myotonic dystrophy

Differentiation of skeletal muscle is affected in myotonic dystrophy (DM) patients. Analysis of cultured myoblasts from DM patients shows that DM myoblasts lose the capability to withdraw from the cell cycle during differentiation. Our data demonstrate that the expression and activity of the proteins responsible for cell cycle withdrawal are altered in DM muscle cells. Skeletal muscle cells from DM patients fail to induce cytoplasmic levels of a CUG RNA binding protein, CUGBP1, while normal differentiated cells accumulate CUGBP1 in the cytoplasm. In cells from normal patients, CUGBP1 up-regulates p21 protein during differentiation. Several lines of evidence show that CUGBP1 induces the translation of p21 via binding to a GC-rich sequence located within the 5' region of p21 mRNA. Failure of DM cells to accumulate CUGBP1 in the cytoplasm leads to a significant reduction of p21 and to alterations of other proteins responsible for the cell cycle withdrawal. The activity of cdk4 declines during differentiation of cells from control patients, while in DM cells cdk4 is highly active during all stages of differentiation. In addition, DM cells do not form Rb/E2F repressor complexes that are abundant in differentiated cells from normal patients. Our data provide evidence for an impaired cell cycle withdrawal in DM muscle cells and suggest that alterations in the activity of CUGBP1 causes disruption of p21-dependent control of cell cycle arrest.