RhoE controls myoblast alignment prior fusion through RhoA and ROCK.

Differentiation of skeletal myoblasts into multinucleated myotubes is a multi-step process orchestrated by several signaling pathways. The Rho small G protein family plays critical roles both during myogenesis induction and myoblast fusion. We report here that in C2C12 myoblasts, expression of RhoE, an atypical member of this family, increases until the onset of myoblast fusion before resuming its basal level once fusion has occurred. We show that RhoE accumulates in elongated, aligned myoblasts prior to fusion and that its expression is also increased during injury-induced skeletal muscle regeneration. Moreover, although RhoE is not required for myogenesis induction, it is essential for myoblast elongation and alignment before fusion and for M-cadherin expression and accumulation at the cell-cell contact sites. Myoblasts lacking RhoE present with defective p190RhoGAP activation and RhoA inhibition at the onset of myoblast fusion. RhoE interacts also with the RhoA effector Rho-associated kinase (ROCK)I whose activity must be downregulated to allow myoblast fusion. Consistently, we show that pharmacological inactivation of RhoA or ROCK restores myoblast fusion in RhoE-deficient myoblasts. RhoE physiological upregulation before myoblast fusion is responsible for the decrease in RhoA and ROCKI activities, which are required for the fusion process. Therefore, we conclude that RhoE is an essential regulator of myoblast fusion.

Serum response factor p67SRF is expressed and required during myogenic differentiation of both mouse C2 and rat L6 muscle cell lines.

The 67-kD serum response factor (p67SRF) is a ubiquitous nuclear transcription factor that acts by direct binding to a consensus DNA sequence, the serum response element (SRE), present in the promoter region of numerous genes. Although p67SRF was initially implicated in the activation of mitogen-stimulated genes, the identification of a sequence similar to SRE, the CArG box motif, competent to interact with SRE binding factors in many muscle-specific genes, has led to speculation that, in addition to its function in cell proliferation, p67SRF may play a role in muscle differentiation. Indirect immunofluorescence using affinity-purified antibodies specifically directed against p67SRF reveals that this factor is constitutively expressed and localized in the nucleus of two skeletal muscle cell lines: rat L6 and mouse C2 myogenic cells during myogenic differentiation. This result was further confirmed through immunoblotting and Northern blot analysis. Furthermore, specific inhibition of p67SRF in vivo through microinjection of purified p67SRF antibodies prevented the myoblast-myotube transition and the expression of muscle-specific genes such as the protein troponin T. We further showed that anti-p67SRF injection also inhibited the expression of the myogenic factor myogenin, implying an early requirement for p67SRF in muscle differentiation. These results demonstrate that p67SRF is involved in the process of skeletal muscle differentiation. The potential action of p67SRF via CArG sequences is discussed.

Regulation of transcription factor localization: fine-tuning of gene expression.

Import of 'nuclear' proteins into the nucleus, in particular, transcription factors, is not a constitutive process; instead it appears to be modulated in response to external stimuli, cell-cycle progression and developmental cues. Examples of such regulation involve direct phosphorylation of the transported protein, masking of the nuclear localization signal(s), cytoplasmic retention by binding to an anchoring protein, modulation of the import machinery itself and possible interplay between these different mechanisms. As such, nucleo-cytoplasmic traffic constitutes an important regulatory checkpoint in the control of gene expression.

The small GTPases Cdc42Hs, Rac1 and RhoG delineate Raf-independent pathways that cooperate to transform NIH3T3 cells.

BACKGROUND: Ras-mediated transformation of mammalian cells has been shown to activate multiple signalling pathways, including those involving mitogen-activated protein kinases and the small GTPase Rho. Members of the Rho family affect cell morphology by controlling the formation of actin-dependent structures: specifically, filopodia are induced by Cdc42Hs, lamellipodia and ruffles by Rac, and stress fibers by RhoA. In addition, Rho GTPases are involved in progression through the G1 phase of the cell cycle, and Rac1 and RhoA have recently been directly implicated in the morphogenic and mitogenic responses to transformation by oncogenic Ras. In order to examine the cross-talk between Ras and Rho proteins, we investigated the effects on focus-forming activity and cell growth of the Rho-family members Cdc42Hs, Rac1 and RhoG by expressing constitutively active or dominant-negative forms in NIH3T3 cells. RESULTS: Expression of Rac1 or RhoG modulated the saturation density to which the cells grew, probably by affecting the level of contact inhibition. Although all three GTPases were required for cell transformation mediated by Ras but not by constitutively active Raf, the selective activation of each GTPase was not sufficient to induce the formation of foci. The coordinated activation of Cdc42Hs, RhoG and Rac1, however, elicited a high focus-forming activity, independent of the mitogen-activated ERK and JNK protein kinase pathways. CONCLUSIONS: Ras-mediated transformation induces extensive changes in cell morphology which require the activity of members of the Rho family of GTPases. Our data show that the pattern of coordinated Rho family activation that elicits a focus-forming activity in NIH3T3 cells is distinct from the regulatory cascade that has been proposed for the control of actin-dependent structures in Swiss 3T3 cells.

Kinectin is a key effector of RhoG microtubule-dependent cellular activity.

RhoG is a member of the Rho family of GTPases that activates Rac1 and Cdc42 through a microtubule-dependent pathway. To gain understanding of RhoG downstream signaling, we performed a yeast two-hybrid screen from which we identified kinectin, a 156-kDa protein that binds in vitro to conventional kinesin and enhances microtubule-dependent kinesin ATPase activity. We show that RhoG(GTP) specifically interacts with the central domain of kinectin, which also contains a RhoA binding domain in its C terminus. Interaction was confirmed by coprecipitation of kinectin with active RhoG(G12V) in COS-7 cells. RhoG, kinectin, and kinesin colocalize in REF-52 and COS-7 cells, mainly in the endoplasmic reticulum but also in lysosomes. Kinectin distribution in REF-52 cells is modulated according to endogenous RhoG activity. In addition, by using injection of anti-kinectin antibodies that challenge RhoG-kinectin interaction or by blocking anti-kinesin antibodies, we show that RhoG morphogenic activity relies on kinectin interaction and kinesin activity. Finally, kinectin overexpression elicits Rac1- and Cdc42-dependent cytoskeletal effects and switches cells to a RhoA phenotype when RhoG activity is inhibited or microtubules are disrupted. The functional links among RhoG, kinectin, and kinesin are further supported by time-lapse videomicroscopy of COS-7 cells, which showed that the microtubule-dependent lysosomal transport is facilitated by RhoG activation or kinectin overexpression and is severely stemmed upon RhoG inhibition. These data establish that kinectin is a key mediator of microtubule-dependent RhoG activity and suggest that kinectin also mediates RhoG- and RhoA-dependent antagonistic pathways.

The serum response factor nuclear localization signal: general implications for cyclic AMP-dependent protein kinase activity in control of nuclear translocation.

We have identified a basic sequence in the N-terminal region of the 67-kDa serum response factor (p67SRF or SRF) responsible for its nuclear localization. A peptide containing this nuclear localization signal (NLS) translocates rabbit immunoglobulin G (IgG) into the nucleus as efficiently as a peptide encoding the simian virus 40 NLS. This effect is abolished by substituting any two of the four basic residues in this NLS. Overexpression of a modified form of SRF in which these basic residues have been mutated confirms the absolute requirement for this sequence, and not the other basic amino acid sequences adjacent to it, in the nuclear localization of SRF. Since this NLS is in close proximity to potential phosphorylation sites for the cAMP-dependent protein kinase (A-kinase), we further investigated if A-kinase plays a role in the nuclear location of SRF. The nuclear transport of SRF proteins requires basal A-kinase activity, since inhibition of A-kinase by using either the specific inhibitory peptide PKIm or type II regulatory subunits (RII) completely prevents the nuclear localization of plasmid-expressed tagged SRF or an SRF-NLS-IgG conjugate. Direct phosphorylation of SRF by A-kinase can be discounted in this effect, since mutation of the putative phosphorylation sites in either the NLS peptide or the encoded full-length SRF protein had no effect on nuclear transport of the mutants. Finally, in support of an implication of A-kinase-dependent phosphorylation in a more general mechanism affecting nuclear import, we show that the nuclear transport of a simian virus 40-NLS-conjugated IgG or purified cyclin A protein is also blocked by inhibition of A-kinase, even though neither contains any potential sites for phosphorylation by A-kinase or can be phosphorylated by A-kinase in vitro.

Critical activities of Rac1 and Cdc42Hs in skeletal myogenesis: antagonistic effects of JNK and p38 pathways.

The Rho family of GTP-binding proteins plays a critical role in a variety of cellular processes, including cytoskeletal reorganization and activation of kinases such as p38 and C-jun N-terminal kinase (JNK) MAPKs. We report here that dominant negative forms of Rac1 and Cdc42Hs inhibit the expression of the muscle-specific genes myogenin, troponin T, and myosin heavy chain in L6 and C2 myoblasts. Such inhibition correlates with decreased p38 activity. Active RhoA, RhoG, Rac1, and Cdc42Hs also prevent myoblast-to-myotube transition but affect distinct stages: RhoG, Rac1, and Cdc42Hs inhibit the expression of all muscle-specific genes analyzed, whereas active RhoA potentiates their expression but prevents the myoblast fusion process. We further show by two different approaches that the inhibitory effects of active Rac1 and Cdc42Hs are independent of their morphogenic activities. Rather, myogenesis inhibition is mediated by the JNK pathway, which also leads to a cytoplasmic redistribution of Myf5. We propose that although Rho proteins are required for the commitment of myogenesis, they differentially influence this process, positively for RhoA and Rac1/Cdc42Hs through the activation of the SRF and p38 pathways, respectively, and negatively for Rac1/Cdc42Hs through the activation of the JNK pathway.

RhoG GTPase controls a pathway that independently activates Rac1 and Cdc42Hs.

RhoG is a member of the Rho family of GTPases that shares 72% and 62% sequence identity with Rac1 and Cdc42Hs, respectively. We have expressed mutant RhoG proteins fused to the green fluorescent protein and analyzed subsequent changes in cell surface morphology and modifications of cytoskeletal structures. In rat and mouse fibroblasts, green fluorescent protein chimera and endogenous RhoG proteins colocalize according to a tubular cytoplasmic pattern, with perinuclear accumulation and local concentration at the plasma membrane. Constitutively active RhoG proteins produce morphological and cytoskeletal changes similar to those elicited by a simultaneous activation of Rac1 and Cdc42Hs, i.e., the formation of ruffles, lamellipodia, filopodia, and partial loss of stress fibers. In addition, RhoG and Cdc42Hs promote the formation of microvilli at the cell apical membrane. RhoG-dependent events are not mediated through a direct interaction with Rac1 and Cdc42Hs targets such as PAK-1, POR1, or WASP proteins but require endogenous Rac1 and Cdc42Hs activities: coexpression of a dominant negative Rac1 impairs membrane ruffling and lamellipodia but not filopodia or microvilli formation. Conversely, coexpression of a dominant negative Cdc42Hs only blocks microvilli and filopodia, but not membrane ruffling and lamellipodia. Microtubule depolymerization upon nocodazole treatment leads to a loss of RhoG protein from the cell periphery associated with a reversal of the RhoG phenotype, whereas PDGF or bradykinin stimulation of nocodazole-treated cells could still promote Rac1- and Cdc42Hs-dependent cytoskeletal reorganization. Therefore, our data demonstrate that RhoG controls a pathway that requires the microtubule network and activates Rac1 and Cdc42Hs independently of their growth factor signaling pathways.

Expression and activity of serum response factor is required for expression of the muscle-determining factor MyoD in both dividing and differentiating mouse C2C12 myoblasts.

To understand the mechanism by which the serum response factor (SRF) is involved in the process of skeletal muscle differentiation, we have assessed the effect of inhibiting SRF activity or synthesis on the expression of the muscle-determining factor MyoD. Inhibition of SRF activity in mouse myogenic C2C12 cells through microinjection of either the SRE oligonucleotide (which acts by displacing SRF proteins from the endogenous SRE sequences), purified SRF-DB (a 30-kDa portion of SRF containing the DNA-binding domain of SRF, which acts as a dominant negative mutant in vivo), or purified anti-SRF antibodies rapidly prevents the expression of MyoD. Moreover, the rapid shutdown of MyoD expression after in vivo inhibition of SRF activity is observed not only in proliferating myoblasts but also in myoblasts cultured under differentiating conditions. Additionally, by using a cellular system expressing a glucocorticoid-inducible antisense-SRF (from aa 74 to 244) we have shown that blocking SRF expression by dexamethasone induction of antisense SRF results in the lack of MyoD expression as probed by both immunofluorescence and Northern blot analysis. Taken together these data demonstrate that SRF expression and activity are required for the expression of the muscle-determining factor MyoD.

Role of fos-AP-1 binding sequence (FAP) in the induction of c-fos expression by purified C-kinase and in c-fos down-regulation following serum induction.

Microinjection of purified calcium phospholipid-dependent protein kinase (C-kinase) resulted in the rapid and transient induction of c-fos in quiescent rat embryo fibroblats. This C-kinase-induced expression of c-fos was prevented by in vivo competition using co-injection of oligonucleotides corresponding to the sequence of either the serum response element (SRE) or the fos AP-1 binding sequence (FAP) adjacent to SRE. This indicates that both these sequences must be involved in the binding/activation of protein factors required for the induction of c-fos by C-kinase. In contrast, the induction of c-fos by serum or by casein kinase II microinjection, which is also inhibited by injection of SRE oligonucleotides, is only delayed and then markedly prolonged by injecting TRE/FAP sequence, demonstrating that the FAP site plays a prominent role in vivo in the down-regulation of the endogenous c-fos gene expression.

Comparative analysis of cellular and tissular expression of c-fos in human keratinocytes: evidence of its role in cell differentiation.

Recent studies on normal and pathological skin have suggested a role of the c-fos proto-oncogene in keratinocyte differentiation. To further elucidate this question we have used keratinocyte and skin culture models to study in vitro regulation of c-fos expression and attempted to correlate it with the keratinocyte maturation process. Our results show that c-fos expression is prolonged in keratinocyte monolayers both at the mRNA and protein level. Extracellular calcium which stimulate keratinocyte differentiation is able to induce c-fos expression in the presence of growth factors. However this c-fos expression cannot be maintained by these factors as seen in normal human skin in vivo. Conversely, spontaneous expression of c-fos can be seen in reconstituted skin when the neo-epidermis has completed its differentiation. All these data strongly support a role of c-fos as a switch between the early and late phases of keratinocyte differentiation allowing them to be definitively committed to their elimination process. Additionally, a differential regulation of c-fos seems to exist between keratinocyte culture and reconstituted epidermis, suggesting that tissular and serum factors are involved in the prolonged c-fos expression observed in human epidermis.

Microinjection strategies for the study of mitogenic signaling in mammalian cells.

First used in the analysis of dynamic changes in cell structure, microneedle microinjection allows in situ study of individual living cells as opposed to large scale metabolic analysis of heterogeneous cell culture. In addition, microinjection also offers the possibility to examine in vivo regulated processes by modulating the intracellular levels and activity of key regulatory proteins and genes in both a specific and controlled manner. A number of different strategies have been developed over the past 5 years to examine the pathways and effectors that are involved in mitogenic signaling as well as in the regulation of gene expression during the proliferative response to growth factors by normal fibroblasts. These strategies include: 1. Direct in vivo competition for various trans-activating DNA binding activities by microinjection of double-stranded oligonucleotides, microinjection of monospecific antibodies against transcription factors and microinjection of dominant negative mutants of transcription factors based upon their DNA binding domain. 2. Microinjection of purified enzymes (kinases and phosphatases) or peptides and antibodies that specifically inhibit these activities. 3. Microinjection of expression plasmids which encode various normal and epitope-tagged regulatory molecules. In many of the experiments described below, c-fos gene expression was monitored as an early marker of mitogenic response. The c-fos gene belongs to a family of genes whose transcription is activated very early after addition of growth factor (1-4). For in vivo studies, the c-fos promoter offers several unique advantages. Primarily, it is easy to manipulate. In practical terms, when mammalian fibroblasts are made quiescent (by replacing the normal growth media, with growth factors-depleted media) and subsequently activated by re-adding mitogen (growth factors, serum), c-fos RNA expression is restored within 15 minutes and the protein is specifically detected in the nuclei of cells after 90 minutes, but is no longer detectable after 3 hours. Secondly, results obtained with the c-fos promoter are directly applicable to cell growth since expression of c-fos is itself a prerequisite for proliferation as demonstrated by microinjection of anti-fos antibodies which prevented proliferation in mammalian cells (5). Thirdly, the c-fos promoter is exquisitely sensitive to agents which cause cell stress. In this respect, heat-shock, poor microinjection or microinjection in the presence of heavy metals or chelating agents in the culture media all rapidly stimulate c-fos expression. However, when compared to c-fos expression in the proliferative response, stress mediated c-fos expression is induced both more rapidly and strongly, reverses more slowly (the protein is still detectable after 5-6 hours) and does not result in cell proliferation (unpublished observation). As such, it provides an excellent internal control for identifying poor treatment and manipulation of cells . Finally, the c-fos promoter is subject to several levels of auto-regulation enabling the analysis of not only components involved in transcriptional activation , but also various aspects of transcriptional down regulation and shut-off.

Fos immunoreactivity assessment on human normal and pathological bronchial biopsies.

The transcription factor Fos is involved in cell proliferation and differentiation. Its expression in normal and pathological adult human tissues and cells has rarely been studied. We therefore studied bronchial biopsies obtained from 14 normal subjects (NS), 18 non-steroid-treated asthmatics, 10 corticosteroid-treated asthmatics and 10 patients with chronic bronchitis (CB), in addition to 34 patients with lung cancer (LC), by immunofluorescence for Fos immunoreactivity, using a highly specific polyclonal antibody. Bronchial tissue of 0/10 NS, 11/18 non-steroid-treated asthmatics, 1/10 steroid-treated asthmatics, 0/10 CB and 1/34 LC expressed Fos. In asthmatic patients, the expression was heterogeneous, localized to epithelial cells and correlated with the epithelium shedding (tau = 0.45, P = 0.0001). Corticosteroid-treated patients rarely expressed Fos, suggesting a role for this proto-oncogene in asthmatic bronchial inflammation. Fos was rarely expressed in the normal and pathological (CB, LC) proliferative compartment of the human bronchi, suggesting its low role in cell proliferation of the large airways.

P-cadherin is a direct PAX3-FOXO1A target involved in alveolar rhabdomyosarcoma aggressiveness.

Alveolar rhabdomyosarcoma (ARMS) is an aggressive childhood cancer of striated muscle characterized by the presence of the PAX3-FOXO1A or PAX7-FOXO1A chimeric oncogenic transcription factor. Identification of their targets is essential for understanding ARMS pathogenesis. To this aim, we analyzed transcriptomic data from rhabdomyosarcoma samples and found that P-cadherin expression is correlated with PAX3/7-FOXO1A presence. We then show that expression of a PAX3 dominant negative variant inhibits P-cadherin expression in ARMS cells. Using mouse models carrying modified Pax3 alleles, we demonstrate that P-cadherin is expressed in the dermomyotome and lies genetically downstream from the myogenic factor Pax3. Moreover, in vitro gel shift analysis and chromatin immunoprecipitation indicate that the P-cadherin gene is a direct transcriptional target for PAX3/7-FOXO1A. Finally, P-cadherin expression in normal myoblasts inhibits myogenesis and induces myoblast transformation, migration and invasion. Conversely, P-cadherin downregulation by small hairpin RNA decreases the transformation, migration and invasive potential of ARMS cells. P-cadherin also favors cadherin switching, which is a hallmark of metastatic progression, by controlling N- and M-cadherin expression and/or localization. Our findings demonstrate that P-cadherin is a direct PAX3-FOXO1A transcriptional target involved in ARMS aggressiveness. Therefore, P-cadherin emerges as a new and attractive target for therapeutic intervention in ARMS.

ras-induced c-fos expression and proliferation in living rat fibroblasts involves C-kinase activation and the serum response element pathway.

We have examined the early events involved in the proliferative activation of quiescent rat embryo fibroblasts by microinjection of oncogenic ras protein. Cells injected with ras show a transient expression of c-fos after 30-60 min visualized by immunofluorescence in the nucleus. This c-fos expression can be specifically suppressed by coinjection of a double-stranded oligonucleotide which corresponds to the serum response element (SRE) present in the c-fos promoter, implying that ras utilizes a pathway which activates the binding of serum response factor(s) (SRF) to SRE to induce c-fos transcription. Inhibition of this pathway also abolished ras-induced DNA synthesis indicating that the proliferative induction by ras requires expression of SRE-regulated genes. Both c-fos induction and DNA synthesis were prevented when ras oncoprotein was injected into quiescent cells together with either antibodies against calcium phospholipid-dependent protein kinase (C-kinase) or a synthetic peptide that specifically inhibits C-kinase. These data demonstrate the involvement of both functional C-kinase and the SRE pathway in the activation of quiescent cells by ras and suggest a potential relationship in their mechanism of action.

Cdc42Hs and Rac1 GTPases induce the collapse of the vimentin intermediate filament network.

In this study we show that expression of active Cdc42Hs and Rac1 GTPases, two Rho family members, leads to the reorganization of the vimentin intermediate filament (IF) network, showing a perinuclear collapse. Cdc42Hs displays a stronger effect than Rac1 as 90% versus 75% of GTPase-expressing cells show vimentin collapse. Similar vimentin IF modifications were observed when endogenous Cdc42Hs was activated by bradykinin treatment, endogenous Rac1 by platelet-derived growth factor/epidermal growth factor, or both endogenous proteins upon expression of active RhoG. This reorganization of the vimentin IF network is not associated with any significant increase in soluble vimentin. Using effector loop mutants of Cdc42Hs and Rac1, we show that the vimentin collapse is mostly independent of CRIB (Cdc42Hs or Rac-interacting binding)-mediated pathways such as JNK or PAK activation but is associated with actin reorganization. This does not result from F-actin depolymerization, because cytochalasin D treatment or Scar-WA expression have merely no effect on vimentin organization. Finally, we show that genistein treatment of Cdc42 and Rac1-expressing cells strongly reduces vimentin collapse, whereas staurosporin, wortmannin, LY-294002, R(p)-cAMP, or RII, the regulatory subunit of protein kinase A, remain ineffective. Moreover, we detected an increase in cellular tyrosine phosphorylation content after Cdc42Hs and Rac1 expression without modification of the vimentin phosphorylation status. These data indicate that Cdc42Hs and Rac1 GTPases control vimentin IF organization involving tyrosine phosphorylation events.

p67SRF is a constitutive nuclear protein implicated in the modulation of genes required throughout the G1 period.

Indirect immunofluorescence analysis, using antibodies directed against peptide sequences outside the DNA-binding domain of the 67-kDa serum response factor (p67SRF), revealed a punctuated nuclear staining, constant throughout the cell cycle and in all different cell lines tested. p67SRF was also tightly associated with chromatin through all stages of mitosis. Inhibition of p67SRF activity in vivo, through microinjection of anti-p67SRF antibodies, specifically suppressed DNA synthesis induced after serum addition or ras microinjection, suggesting that these antibodies were effective in preventing expression of serum response element (SRE)-regulated genes. A similar inhibition was also obtained in cells injected with oligonucleotides corresponding to the DNA binding sequence for p67SRF protein, SRE. Moreover, this inhibition of DNA synthesis by anti-p67SRF or SRE injection was still observed in cells injected during late G1, well after c-fos induction. These data imply that genes regulated by p67SRF are continuously involved in the proliferation pathway throughout G1 and that p67SRF forms an integral component of mammalian cell transcriptional control.

TrioGEF1 controls Rac- and Cdc42-dependent cell structures through the direct activation of rhoG.

Rho GTPases regulate the morphology of cells stimulated by extracellular ligands. Their activation is controlled by guanine exchange factors (GEF) that catalyze their binding to GTP. The multidomain Trio protein represents an emerging class of &Rgr; regulators that contain two GEF domains of distinct specificities. We report here the characterization of Rho signaling pathways activated by the N-terminal GEF domain of Trio (TrioD1). In fibroblasts, TrioD1 triggers the formation of particular cell structures, similar to those elicited by RhoG, a GTPase known to activate both Rac1 and Cdc42Hs. In addition, the activity of TrioD1 requires the microtubule network and relocalizes RhoG at the active sites of the plasma membrane. Using a classical in vitro exchange assay, TrioD1 displays a higher GEF activity on RhoG than on Rac1. In fibroblasts, expression of dominant negative RhoG mutants totally abolished TrioD1 signaling, whereas dominant negative Rac1 and Cdc42Hs only led to partial and complementary inhibitions. Finally, expression of a Rho Binding Domain that specifically binds RhoG(GTP) led to the complete abolition of TrioD1 signaling, which strongly supports Rac1 not being activated by TrioD1 in vivo. These data demonstrate that Trio controls a signaling cascade that activates RhoG, which in turn activates Rac1 and Cdc42Hs.

Promoter elements and transcriptional control of the chicken tropomyosin gene [corrected].

The chicken beta tropomyosin (beta TM) gene has two alternative transcription start sites (sk and nmCAP sites) which are used in muscle or non muscle tissues respectively. In order to understand the mechanisms involved in the tissue-specific and developmentally-regulated expression of the beta TM gene, we have analyzed the 5' regions associated with each CAP site. Truncated regions 5' to the nmCAP site were inserted upstream to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene and these constructs were transfected into avian myogenic and non myogenic cells. The maximum transcription is driven by the CAT construct (-168/ + 216 nt) in all cell types. Previous deletion analysis of the region 5' to the beta TMskCAP site has indicated that 805 nt confer myotube-specific transcription. In this work, we characterized an enhancer element (-201/-68 nt) which contains an E box (-177), a variant CArG box (-104) and a stretch of 7Cs (-147). Mutation of any of these motifs results in a decrease of the myotube-specific transcriptional activity. Electrophoretic mobility shift assays indicate that these cis-acting sequences specifically bind nuclear proteins. This enhancer functions in an orientation-dependent manner.

Nuclear import of the myogenic factor MyoD requires cAMP-dependent protein kinase activity but not the direct phosphorylation of MyoD.

MyoD is a nuclear phosphoprotein that belongs to the family of myogenic regulatory factors and acts in the transcriptional activation of muscle-specific genes. We have investigated the role of cAMP-dependent protein kinase (A-kinase) in modulating the nuclear locale of MyoD. Purified MyoD protein microinjected into the cytoplasm of rat embryo fibroblasts is rapidly translocated into the nucleus. Inhibition of A-kinase activity through injection of the specific inhibitory peptide PKI prevents this nuclear localisation. This inhibition of nuclear location is specifically reversed by injection of purified A-kinase catalytic subunit, showing the requirement for A-kinase in the nuclear import of MyoD. Site-directed mutagenesis of all the putative sites for A-kinase-dependent phosphorylation on MyoD, substituting serine or threonine residues for the non-phosphorylatable amino acid alanine, had no effect on nuclear import of mutated MyoD. These data exclude the possibility that the effect of A-kinase on the nuclear translocation of MyoD is mediated by direct phosphorylation of MyoD and imply that A-kinase operates through phosphorylation of components involved in the nuclear transport of MyoD.