Monitoring of in-vitro ultrasonic stimulation of cells by numerical modeling.

Ultrasound stimulation of living tissues is a promising technique that can be safely applied for regenerative treatments. However, the ultrasound-induced mechanotransduction is still not well understood because of the large number of parameters involved at different scales and their difficult experimental accessibility. In this context, in-vitro studies may help to gain insight into the interaction between ultrasound and cells. Nevertheless, to conduct a reliable analysis of ultrasound effects on cell culture, the monitoring of the acoustic intensity delivered to the cells is of prime interest. Thanks to the development of an innovative custom experimental set-up inspired from ultrasound stimulation of bone regeneration conditions, major disturbing phenomena such as multiple reflections and standing wave formation inside the Petri dish are eliminated. Thus, the level of ultrasound stimulation, especially, in terms of spatial average temporal average intensity (ISATA), delivered to the cells can be monitored. Then, to properly estimate the level of ultrasound stimulation, a finite element model representing the experimental in-vitro configuration is developed. The numerical model manages on capturing the characteristics of the experimentally measured acoustic intensity distribution as illustrated by the experimental and numerical ISATA values of 42.3 and 45.8 mW/cm2 respectively, i.e. a relative difference of 8%. The numerical model would therefore allow exploring data inaccessible to experimental measurement and parametric studies to be carried out and facilitates the investigation of different virtual experimental configurations.

Gallium as a potential candidate for treatment of osteoporosis.

Gallium (Ga) is a semi-metallic element that displays antitumor, antiresorptive, anti-inflammatory and immunosuppressive properties. Among all these properties, antitumor properties were the most extensively applied and have shown efficacy in treatment of Paget's disease, myeloma and hypercalcemia in cases of malignancy. By contrast, no clinical trials have been conducted in prevention and/or treatment of osteoporosis. In this article I focus on Ga effects on bone tissue and cells, as well as on molecular mechanisms governing Ga internalization into cells. Eventually, the potential of Ga as an antiosteoporotic agent is discussed.

Molecular effects of gallium on osteoclastic differentiation of mouse and human monocytes.

We had previously reported that gallium (Ga) inhibited both the differentiation and resorbing activity of osteoclasts in a dose-dependent manner. To provide new insights into Ga impact on osteoclastogenesis, we investigated here the molecular mechanisms of Ga action on osteoclastic differentiation of monocytes upon Rankl treatment. We first observed that Ga treatment inhibited the expression of Rankl-induced early differentiation marker genes, while the same treatment performed subsequently did not modify the expression of late differentiation marker genes. Focusing on the early stages of osteoclast differentiation, we observed that Ga considerably disturbed both the initial induction as well as the autoamplification step of Nfatc1 gene. We next demonstrated that Ga strongly up-regulated the expression of Traf6, p62 and Cyld genes, and we observed concomitantly an inhibition of IκB degradation and a blockade of NFκB nuclear translocation, which regulates the initial induction of Nfatc1 gene expression. In addition, Ga inhibited c-Fos gene expression, and subsequently the auto-amplification stage of Nfatc1 gene expression. Lastly, considering calcium signaling, we observed upon Ga treatment an inhibition of calcium-induced Creb phosphorylation, as well as a blockade of gadolinium-induced calcium entry through TRPV-5 calcium channels. We identify for the first time Traf6, p62, Cyld, IκB, NFκB, c-Fos, and the calcium-induced Creb phosphorylation as molecular targets of Ga, this tremendously impacting the expression of the master transcription factor Nfatc1. In addition, our results strongly suggest that the TRPV-5 calcium channel, which is located within the plasma membrane, is a target of Ga action on human osteoclast progenitor cells.

Family of SRY/Sox proteins is involved in the regulation of the mouse Msh4 (MutS Homolog 4) gene expression.

The eukaryotic MutHLS-like system plays a crucial role in both mitosis and meiosis. Until now, a number of works have focused on the function of MutS and MutL homologs during mismatch DNA repair. Nevertheless, little is known about the role of these proteins during meiosis. MSH4 is a meiosis specific protein that is necessary for meiotic recombination in Saccharomyces cerevisiae. The human MSH4 protein is only found in testis and ovary. It is involved first in synapsis and second during recombination together with MLH1 (MutL Homolog 1). Here, we report the identification of the mouse Msh4 gene that is located on chromosome 3. We examined the expression of mMsh4 in testes of increasing developmental age and in elutriated germ cells. The pattern of expression during spermatogenesis is consistent with a role for MSH4 both during zygonema and pachynema. We demonstrated a promoter activity of the mMsh4 5'-flanking region by cell transfection experiments with a luciferase reporter gene. We found several SRY/Sox binding sites in this region and co-transfection experiments showed that SRY could down regulate mMsh4 promoter transcriptional activity. We propose that the regulation of mMsh4 expression could be one of the reasons for the persistence of SRY and/or SRY-related proteins in adult testis.

Gene trap analysis of germ cell signaling to Sertoli cells: NGF-TrkA mediated induction of Fra1 and Fos by post-meiotic germ cells.

Analysis of complex signalisation networks involving distinct cell types is required to understand most developmental processes. Differentiation of male germ cells in adult mammals involves such a cross-talk between Sertoli cells, the somatic component which supports and controls germinal differentiation, and germ cells at their successive maturation stages. We developed a gene trapping strategy to identify genes, which, in Sertoli cells, are either up- or down-regulated by signals emitted by the germinal component. A library of approximately 2,000 clones was constituted from colonies independently selected from the Sertoli line 15P-1 by growth in drug-containing medium after random integration of a promoter-less (beta)geo transgene (neo(r)-lacZ fusion), which will be expressed as a fusion transcript from a 'trapped' cellular promoter, different in each clone. A first screen conducted on 700 events identified six clones in which beta-galactosidase activity was increased and one in which it was repressed upon addition of germ cells. The targeted loci were identified by cloning and sequencing the genomic region 5' of the insert. One of them was identified as the gene encoding Fra1, a component of the AP1 transcription regulatory complex. Accumulation of Fra1 mRNA was induced, both in 15P-1 and in freshly explanted Sertoli cells, by addition of either round spermatids or nerve growth factor (NGF). The effect of NGF was mediated by the TrkA receptor and the ERK1-ERK2 kinase kinase pathway. Fos and Fra1 transcription were induced within the first hour after addition of the neurotrophin, but, unlike what is observed after serum induction in the same cells, a second wave of transcription of Fra1, but not of Fos, started 16 hours later and peaked at higher levels at about 20 hours. These results suggest that AP1 activation may be an important relay in the Sertoli-germ cell cross-talk, and validate the gene trapping approach as a tool for the identification of target genes in cell culture systems.

The gene encoding the mouse homologue of the human osteoclast-specific 116-kDa V-ATPase subunit bears a deletion in osteosclerotic (oc/oc) mutants.

Osteosclerosis (oc) is an autosomal recessive lethal mutation that impairs bone resorption by osteoclasts, and induces a general increase of bone density in affected mice. Genetic mapping of the oc mutation was used as a backbone in a positional cloning approach in the pericentromeric region of mouse chromosome 19. Perfect cosegregation of the osteopetrotic phenotype with polymorphic markers enabled the construction of a sequence-ready bacterial artificial chromosome (BAC) contig of this region. Genomic sequencing of a 200-kb area revealed the presence of the mouse homologue to the human gene encoding the osteoclast-specific 116-kDa subunit of the vacuolar proton pump. This gene was located recently on human 11q13, a genomic region conserved with proximal mouse chromosome 19. Sequencing of the 5' end of the gene in oc/oc mice showed a 1.6-kb deletion, including the translation start site, which impairs genuine transcription of this subunit. The inactivation of this osteoclast-specific vacuolar proton ATPase subunit could be responsible for the lack of this enzyme in the apical membranes of osteoclast cells in oc/oc mice, thereby preventing the resorption function of these cells, which leads to the osteopetrotic phenotype.

Cloning and recombinant expression of a novel mouse-secreted phospholipase A2.

Secreted phospholipases A2 (sPLA2s) form a class of structurally related enzymes that are involved in a variety of physiological and pathological effects including inflammation and associated diseases, cell proliferation, cell adhesion, and cancer, and are now known to bind to specific membrane receptors. Here, we report the cloning and expression of a novel sPLA2 isolated from mouse thymus. Based on its structural features, this sPLA2 is most similar to the previously cloned mouse group IIA sPLA2 (mGIIA sPLA2). As for mGIIA sPLA2, the novel sPLA2 is made up of 125 amino acids with 14 cysteines, is basic (pI = 8.71) and its gene has been mapped to mouse chromosome 4. However, the novel sPLA2 has only 48% identity with mGIIA and displays similar levels of identity with the other mouse group IIC and V sPLA2s, indicating that the novel sPLA2 is not an isoform of mGIIA sPLA2. This novel sPLA2 has thus been called mouse group IID (mGIID) sPLA2. In further contrast with mGIIA, which is found mainly in intestine, transcripts coding for mGIID sPLA2 are found in several tissues including pancreas, spleen, thymus, skin, lung, and ovary, suggesting distinct functions for the two enzymes. Recombinant expression of mGIID sPLA2 in Escherichia coli indicates that the cloned sPLA2 is an active enzyme that has much lower specific activity than mGIIA and displays a distinct specificity for binding to various phospholipid vesicles. Finally, recombinant mGIID sPLA2 did not bind to the mouse M-type sPLA2 receptor, while mGIIA was previously found to bind to this receptor with high affinity.

Structure, chromosome localization, and tissue distribution of the mouse twik K+ channel gene.

We have recently discovered a new class of potassium channels with two pore-forming domains and four membrane-spanning domains. When heterologously expressed, these channels produce time- and voltage-independent currents that classify them as background or leak channels. TWIK (for tandem of P domains in a weak inwardly rectifying K+ channel) was the first member of this family to be cloned. Here, we describe the genomic organization of TWIK in the mouse. The coding sequence as well as the untranslated sequences are contained in three exons. The twik gene (or KCNK1) has been mapped to chromosome 8, consistent with its localization to 1q42-43 in human. The twik gene is expressed in virtually all mouse tissues. It is most abundantly expressed in brain and moderately in other organs such as kidney. The level of expression is increased in brain and kidney from neonate to adult animals, but the TWIK message is also detected during embryogenesis, as early as day 7 post conception.

Essential role of calcium in the regulation of MAP kinase phosphatase-1 expression.

Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) is a dual-specificity protein phosphatase encoded by an immediate-early gene responsive to growth factors and stress. The MKP-1 protein selectively inactivates MAP kinases in vitro by dephosphorylation of the regulatory Thr and Tyr residues. Little is known on the mechanisms that regulate MKP-1 gene expression. Here, we demonstrate that Ca2+ is both necessary and sufficient for the induction of MKP-1 gene expression. Treatment of Rat1 fibroblasts with the Ca2+ chelating agent BAPTA completely suppressed serum-induced MKP-1 expression in a dose- and time-dependent manner. The inhibitory effect of BAPTA was observed at the level of the protein and the mRNA. Importantly, Ca2+ chelation blocked the induction of MKP-1 expression in response to all stimuli tested and in different cell types. Increasing the intracellular concentration of Ca2+ with the ionophore A23187 was sufficient to induce MKP-1 mRNA and protein expression in rat fibroblasts. We also provide evidence that activation of MAP kinases is not an absolute requirement for induction of the MKP-1 gene. Exposure of rat fibroblasts to A23187 induced MKP-1 expression without activating the JNK and p38 MAP kinase pathways. Also, inhibition of the ERK pathway with the selective MEK inhibitor PD98059 did not interfere with serum-stimulated MKP-1 mRNA expression. These results will help define the regulatory mechanisms that govern MKP-1 gene transcription in target cells.

Regulation of the MAP kinase cascade in PC12 cells: B-Raf activates MEK-1 (MAP kinase or ERK kinase) and is inhibited by cAMP.

In PC12 cells, cAMP stimulates the MAP kinase pathway by an unknown mechanism. Firstly, we examined the role of calcium ion mobilization and of protein kinase C in cAMP-stimulated MAP kinase activation. We show that cAMP stimulates p44mapk independently of these events. Secondly, we studied the role of B-Raf in this process. We observed that NGF, PMA and cAMP induce the phosphorylation of B-Raf as well as an upward shift in its electrophoretic mobility. We show that B-Raf is activated following NGF and PMA treatment of PC12 cells, and that it can phosphorylate and activate MEK-1. However, cAMP inhibits B-Raf autokinase activity as well as its ability to phosphorylate and activate MEK-1. This inhibition is likely to be due to a direct effect since we found that PKA phosphorylates B-Raf in vitro. Further, we show that B-Raf binds to p21ras, but more important, this binding to p21ras is virtually abolished with B-Raf from PC12 cells treated with CPT-cAMP. Hence, these data indicate that the PKA-mediated phosphorylation of B-Raf hampers its interaction with p21ras, which is responsible for the PKA-mediated decrease in B-Raf activity. Finally, our work suggests that in PC12 cells, cAMP stimulates MAP kinase through the activation of an unidentified MEK kinase and/or the inhibition of a MEK phosphatase.

Regulation of extracellular signal-regulated protein kinase-1 (ERK-1; pp44/mitogen-activated protein kinase) by epidermal growth factor and nerve growth factor in PC12 cells: implication of ERK1 inhibitory activities.

In PC12 cells, extracellular signal-regulated kinase-1 (ERK1 or pp44/mitogen-activated protein kinase) is stimulated in response to epidermal growth factor (EGF) and nerve growth factor (NGF). This stimulation is rapid and short-lived after EGF activation. In contrast, NGF promotes a swift, but persistent, ERK1 stimulation. We took advantage of this difference in activation pattern to study the negative regulation of ERK1. Using antibodies to the C-terminus of ERK1, we performed in vitro reconstitution experiments with immunoprecipitated ERK1 from stimulated cells and extracts from PC12 cells incubated with EGF or NGF for various periods of times. Using this approach, we showed that extracts from unstimulated cells reduce ERK1 activity. Upon exposure of cells to NGF or EGF, we found that the inhibitory activity had a pattern opposite that of ERK1 phosphorylation and activity. Indeed, the highest ERK1 activation was associated with the lowest ERK1-repressing activity and vice versa. This ERK1 inhibitory activity was found to be sensitive mainly to sodium orthovanadate and to a lesser extent to zinc acetate. Interestingly, okadaic acid decreased ERK1-repressing activity from unstimulated cells when tested with ERK1 from 5-min NGF-treated cells, but not with ERK1 from 5-min EGF-treated cells. Hence, ERK1 appears to be regulated differently after stimulation of cells with EGF compared to NGF. We show that cell extracts promote ERK1 dephosphorylation. Indeed, we were able to detect a phosphatase activity toward in vivo phosphorylated ERK1 that was regulated differently after NGF and EGF treatments of the cells, and that has a profile of regulation similar to that of the ERK1 inhibitory activity. This regulatable phosphatase activity was also observed using in vitro phosphorylated ERK1. Taken together, our data provide evidence that ERK1 is negatively controlled by a phosphatase(s) that can undergo differential modulation depending on the stimuli used.

Co-regulation of the mitogen-activated protein kinase, extracellular signal-regulated kinase 1, and the 90-kDa ribosomal S6 kinase in PC12 cells. Distinct effects of the neurotrophic factor, nerve growth factor, and the mitogenic factor, epidermal growth factor.

We recently characterized the association of the 44-kDa mitogen-activated protein kinase, also known as extracellular-regulated kinase 1 (ERK1), with the 90-kDa ribosomal S6 kinase (pp90rsk), one of its putative substrates in intact PC12 cells. Using antibodies to ERK1 that precipitate a functional ERK1.pp90rsk phosphoprotein complex, we demonstrate here the regulation of both kinases by various stimuli. In mouse fibroblasts expressing human insulin receptors, insulin and vanadate swiftly stimulated ERK1 activity within 5 min. While the hormonal effect was short-lived, vanadate led to a first peak followed by a progressively increasing second phase. In PC12 cells, epidermal growth factor, which is a growth promoting factor, provokes a rapid but evanescent activation of ERK1. In contrast, nerve growth factor (NGF), which acts as a neuronal differentiation factor for PC12 cells, induced a swift monophasic response followed by a sustained second phase. This strikingly different pattern of ERK1 stimulation by NGF and epidermal growth factor was associated to a contrasting effect on ERK1 cellular translocation. Thus, NGF induced a nuclear translocation of ERK1, while epidermal growth factor was without noticeable effect on ERK1 localization. In both cell systems all effectors tested stimulated ERK1 phosphorylation on both threonine and tyrosine residues in an 1:1 ratio. During ERK1 inactivation, phosphothreonine and phosphotyrosine were dephosphorylated in a similar fashion. Concurrent with ERK1 activation was the de novo appearance of phosphothreonine and an increase in phosphoserine on pp90rsk. The pp90rsk phosphothreonine content paralleled the ERK1 activity more closely than the phosphoserine level. These results provide compelling evidence that in fibroblasts and PC12 cells ERK1 plays a direct role in the phosphorylation of pp90rsk and that pp90rsk represents a physiologically relevant substrate of extracellular-regulated kinases. Finally, we would like to suggest that the differentiating action of NGF in PC12 cells might be due, at least in part, to the conjunction of its sustained and robust stimulation of ERK1 and pp90rsk, and of its induction of ERK1 nuclear translocation.

Differential activation of p44mapk (ERK1) by alpha-thrombin and thrombin-receptor peptide agonist.

alpha-Thrombin (thrombin), a potent mitogen for CCL39 hamster lung fibroblasts, stimulates phosphoinositide-specific phospholipase C (PI-PLC) and inhibits adenylate cyclase via cleavage of a specific G-protein-coupled receptor (TH-R), recently cloned from human and hamster cells. This action can be entirely mimicked by the synthetic peptide SFFLRNP, referred to here as TMP (thrombin-mimicking peptide). TMP corresponds to the first seven amino acids of the new N-terminus generated by thrombin cleavage of the hamster TH-R. Although thrombin and TMP apparently generate identical early transmembrane signals, only thrombin is mitogenic on its own. TMP needs to be associated with fibroblast growth factor (FGF), a tyrosine kinase-activating growth factor, to induce cell-cycle re-entry. Here, we have examined the early and late phase of p44 MAP kinase (p44mapk) activation in G0-arrested CCL39 cells after stimulation by thrombin, TMP, FGF or TMP+FGF. We found that: (i) both thrombin and TMP rapidly activate p44mapk in a dose-dependent manner with maximum activation at around 5 min, (ii) after the initial burst of activation, a second and long-lasting wave of activation is observed in response to thrombin (10-100 nM) but not to TMP (up to 300 microM), (iii) FGF alone (25 ng/ml), like thrombin, rapidly and persistently activates p44mapk (20-fold at 5 min and about 3-fold after 2 h), (iv) TMP added together with FGF strongly potentiates the second and sustained phase of p44mapk activation. From these results we propose that: (1) thrombin-induced mitogenesis is mediated only in part by the TH-R recently cloned and (2) activation of p44mapk, in particular the long-lasting phase that correlates with DNA synthesis, is an obligatory event for cell-cycle re-entry.

Nerve growth factor-induced phosphorylation cascade in PC12 pheochromocytoma cells. Association of S6 kinase II with the microtubule-associated protein kinase, ERK1.

Microtubule-associated protein (MAP) kinases form a group of serine/threonine kinases stimulated by various growth factors such as nerve growth factor (NGF) and hormones such as insulin. Interestingly, MAP kinases are thought to participate in a protein kinase cascade leading to cell growth as they have been shown to phosphorylate and activate ribosomal protein S6 kinase. To further evaluate the interactions between the different components of this cascade, we looked at the possible coprecipitation of MAP kinase activator(s) or MAP kinase substrate(s) with MAP kinase. Using antipeptides to the C terminus of the M(r) 44,000 MAP kinase, ERK1, and cell extracts from unstimulated or NGF-treated PC12 cells, we obtained in addition to MAP kinase itself coprecipitation of a protein with a M(r) in the 90,000 range. We further show that this protein is a protein kinase since it becomes phosphorylated on serine residues, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to a polyvinylidene difluoride membrane. In vitro phosphorylation performed before sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates NGF-sensitive phosphorylation of this 90-kDa protein on both serine and threonine; the serine phosphorylation is likely to be due to autophosphorylation, and the threonine phosphorylation due to phosphorylation by the copurifying MAP kinase. Furthermore, immunoprecipitation of this 90-kDa protein was obtained with antibodies to S6 kinase II. Finally, using in situ chemical cross-linking, we were able to demonstrate in intact cells the occurrence of an anti-ERK1 immunoreactive species with a molecular mass of approximately 125,000 compatible with a complex between ERK1 and a 90-kDa S6 kinase. Taken together, our observations demonstrate that the 44-kDa MAP kinase is associated, in intact PC12 cells, with a protein kinase which is very likely to be S6 kinase II. In conclusion, our data represent strong evidence for a physiological role of the MAP kinase-S6 kinase cascade in PC12 cells. Finally, our antipeptides provide us with a powerful tool to search for additional physiologically relevant substrates for MAP kinase, a key integrator enzyme for growth factors and hormones.

Insulin and orthovanadate stimulate multiple phosphotyrosine-containing serine kinases.

Using the synthetic peptide substrate Kemptide and cytosolic extracts of mouse fibroblasts transfected with a human insulin receptor cDNA construct, we have studied an insulin-sensitive serine kinase activity. This activity is rapidly stimulated by insulin (maximum within 5 min) and also by orthovanadate. During cell extract preparation, para-nitrophenylphosphate and phosphotyrosine are able to preserve the enzyme activity, while phosphothreonine and phosphoserine fail to do so. Using antiphosphotyrosine antibodies, specific immunoprecipitation of this insulin- and orthovanadate-sensitive serine kinase was obtained. We then analysed by gel filtration chromatography eluates containing tyrosine-phosphorylated proteins obtained from unstimulated, insulin- and vanadate-treated cells. We found that several activities, with molecular weights estimated to be 30 kDa and smaller, are stimulated by both, insulin and orthovanadate. As a whole, our data indicate that insulin and orthovanadate enhance the cytosolic content in at least 2 or 3 phosphotyrosine-containing serine kinase activities.

Activation and regulation of the insulin receptor kinase.

For the insulin receptor and the EGF receptor it is believed that ligand occupancy results in interactions within the heterotetrameric alpha 2 beta 2 insulin receptor or between monomeric EGF receptors. These interactions then activate the intracellular receptor tyrosine kinase which induces receptor autophosphorylation and phosphorylation of cellular substrates. In the present study we have approached the nature of this receptor activation and autophosphorylation. We have investigated whether these phenomena occur via an intra--or an intermolecular process. To this end the following receptor model system consisting of two receptors was co-expressed in NIH 3T3 cells: a kinase inactive human insulin receptor (HIR K1018A) and a chimeric (EIR) receptor corresponding to the extracellular and transmembrane domains of the human EGF receptor and the cytosolic domain of the human insulin receptor beta subunit. Using this system we found that stimulation of the cells with EGF induced tyrosine autophosphorylation of the EGF-insulin receptor chimera (150 kd) and tyrosine phosphorylation of the beta-subunit of the kinase-deficient insulin receptor (95 kd). The phosphopeptides of the autophosphorylated cytoplasmic domain of the EGF-insulin receptor chimera were comparable to those of the transphosphorylated beta subunit of the kinase-deficient insulin receptor and the wild type human insulin receptor. When immunoaffinity purified EGF-insulin receptor hybrids and kinase-deficient insulin receptors were used in a cell lysate phosphorylation assay, it was found that addition of EGF produced [32P]-labeling of both receptor species. In conclusion, we have shown that tyrosine transphosphorylation can occur between homologous receptor domains. This transphosphorylation and transactivation could be a possible mechanism for signal amplification.2+ domain could influence interactions between the receptor and cellular structures and, as such, play a key role in signal transduction.

Phenylarsine oxide stimulates a cytosolic tyrosine kinase activity and glucose transport in mouse fibroblasts.

In the present report we further approach the mechanism by which insulin and phenylarsine oxide (PAO), a trivalent arsenical compound, regulate glucose transport in mouse fibroblasts (NIH3T3). First, we show that PAO is a powerful stimulatory agent on glucose transport. Second, at least three series of observations indicate that this action of PAO is not mediated through the insulin receptor: (i) the same effect of PAO is observed in NIH3T3 and in transfected cells expressing 6 x 10(6) insulin receptors, while the effect of insulin is markedly increased in the transfected cells; (ii) PAO does not affect the tyrosine phosphorylation of the insulin receptor; (iii) the tyrosine kinase activity of the insulin receptor toward exogenous substrates is not increased by PAO. Since PAO appears to act on glucose transport by a different mechanism than insulin, we have compared the effect of PAO and insulin on tyrosine phosphorylation of cellular proteins. Using Western blot analysis we did not detect common substrates in PAO- and insulin-treated cells. However, we found in cell extracts from both PAO- and insulin-treated cells a 50-kDa protein that is immunoprecipitated by antiphosphotyrosine antibody. In addition, PAO activates a cytosolic tyrosine kinase capable of poly(Glu/Tyr) phosphorylation. As a whole, our data suggest that the 50-kDa protein found in cells incubated with PAO and insulin could be the convergence point of the insulin and PAO signaling pathways.

Tyrosine and threonine phosphorylation of an immunoaffinity-purified 44-kDa MAP kinase.

We have approached the functioning of a MAP kinase, which is thought to be a "switch kinase" in the phosphorylation cascade initiated from various receptor tyrosine kinases including the insulin receptor. To do so, antipeptide antibodies were raised against the C-terminal portion of ERK1 (extracellular signal-regulated kinase 1), a protein kinase belonging to the family of MAP kinases. With these antipeptide antibodies, we observed the following: (i) a 44-kDa protein can be specifically recognized both under native and denaturing conditions; (ii) a 44-kDa phosphoprotein can be revealed in 32P-labeled cells; its phosphorylation is stimulated by insulin, sodium orthovanadate, and okadaic acid; (iii) a MBP kinase activity can be precipitated, which phosphorylates MBP on threonine residues, and which is stimulated by insulin, sodium orthovanadate, okadaic acid, and fetal calf serum; (iv) this MBP kinase activity appears to be correlated with the in vivo induced phosphorylation of the 44-kDa protein. We next studied the in vitro phosphorylation of this 44-kDa/ERK1-immunoreactive protein. A time- and manganese-dependent phosphorylation was stimulated by the in vitro addition of sodium orthovanadate. Phosphoamino acid analysis of the in vitro phosphorylated 44-kDa protein revealed both threonine and tyrosine phosphorylation. Importantly, this in vitro phosphorylation of MAP kinase results in activation of phosphorylation of added MBP substrate. As a whole, our data indicate that the 44-kDa phosphoprotein identified by our antipeptide antibodies very likely corresponds to a MAP kinase.(ABSTRACT TRUNCATED AT 250 WORDS)