Mitogenic Signal Transduction Research Articles

Regulation of c-fos transcription by chemopreventive isoflavonoids and lignans in MDA-MB-468 breast cancer cells.

Isoflavonoids and lignans are diet constituents with chemopreventive properties. We compared the ability of the isoflavonoids genistein and equol, the lignans enterodiol, enterolactone and nordihydroguaiaretic acid (NDGA) and the lignan metabolite methyl p-hydroxyphenyllactate to interfere with mitogenic and tumour promotional signal transduction pathways. Their effects on c-fos mRNA levels after induction by either epidermal growth factor (EGF) or the tumour promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was measured in human breast cancer-derived MDA-MB-468 cells. Of the six agents, only genistein decreased EGF-induced, c-fos transcription (by 63% compared to control at 100 mumol/l). In contrast, both genistein and equol at 100 mumol/l decreased TPA-induced c-fos levels, by 75 and 67%, respectively. NDGA and methyl p-hydroxyphenyllactate did not inhibit TPA mediated c-fos transcription and enterolactone and enterodiol had only a weak inhibitory effect. NDGA at 0.1-10 mumol/l increased c-fos mRNA levels. None of the agents inhibited protein kinase C and only genistein inhibited EGF receptor-linked protein tyrosine kinase obtained from MDA-MB-468 cells, with an IC50 of 60 mumol/l. NDGA and genistein arrested cell colony formation potently, genistein was 15-fold more growth-inhibitory than equol. The results suggest that both genistein and equol interfere similarly with TPA-induced signal transduction pathways. Inhibition by genistein of EGF-induced c-fos mRNA transcription is probably related to its interruption of EGF receptor-linked protein tyrosine kinase, whereas genistein-induced growth arrest is not. If ability to antagonise phorbol ester effects is important for chemopreventive efficacy, equol and genistein might be equi-efficacious chemopreventors, whereas enterolactone, enterodiol and NDGA should be much less potent. If phorbol ester antagonism together with antimitogenic activity determine optimal chemopreventive activity of this type of agent, genistein would be more potent than equol.

Insulin-like Growth Factor I (IGF-I)-stimulated Pancreatic β-Cell Growth Is Glucose-dependent: SYNERGISTIC ACTIVATION OF INSULIN RECEPTOR SUBSTRATE-MEDIATED SIGNAL TRANSDUCTION PATHWAYS BY GLUCOSE AND IGF-I IN INS-1 CELLS

Nutrients and certain growth factors stimulate pancreatic beta-cell mitogenesis, however, the appropriate mitogenic signal transduction pathways have not been defined. In the glucose-sensitive pancreatic beta-cell line, INS-1, it was found that glucose (6-18 mM) independently increased INS-1 cell proliferation (>20-fold at 15 mM glucose). Insulin-like growth factor I (IGF-I)-induced INS-1 cell proliferation was glucose-dependent only in the physiologically relevant concentration range (6-18 mM glucose). The combination of IGF-I and glucose was synergistic, increasing INS-1 cell proliferation >50-fold at 15 mM glucose + 10 nM IGF-I. Glucose metabolism and phosphatidylinositol 3'-kinase (PI 3'-kinase) activation were necessary for both glucose and IGF-I-stimulated INS-1 cell proliferation. IGF-I and 15 mM glucose increased tyrosine phosphorylation mediated recruitment of Grb2/mSOS and PI 3'-kinase to IRS-2 and pp60. Glucose and IGF-I also induced Shc association with Grb2/mSOS. Glucose (3-18 mM) and IGF-I, independently of glucose, activated mitogen-activated protein kinase but this did not correlate with IGF-I-induced beta-cell proliferation. In contrast, p70(S6K) was activated with increasing glucose concentration (between 6 and 18 mM), and potentiated by IGF-I in the same glucose concentration range which correlated with INS-1 cell proliferation rate. Thus, glucose and IGF-I-induced beta-cell proliferation were mediated via a signaling mechanism that was facilitated by mitogen-activated protein kinase but dependent on IRS-mediated induction of PI 3'-kinase activity and downstream activation of p70(S6K). The glucose dependence of IGF-I mediated INS-1 cell proliferation emphasizes beta-cell signaling mechanisms are rather unique in being tightly linked to glycolytic metabolic flux.

A specific increased expression of insulin receptor substrate 2 in pancreatic beta-cell lines is involved in mediating serum-stimulated beta-cell growth.

Certain nutrients and growth factors can stimulate pancreatic beta-cell growth. However, the appropriate mitogenic signaling pathways in beta-cells have been relatively undefined. In this study, differential gene expression in NEDH rat insulinoma was compared with NEDH rat primary islet beta-cells. Differential mRNA display analysis revealed an elevated expression in insulinoma of VL30 transposons, S24 ribosomal protein, and cytochrome-C oxidaseVIIc that is typical for cells undergoing mitosis. A gene candidate approach revealed that mRNA levels of the oncogenes c-fos and c-jun were equivalently expressed in insulinoma and islet cells, as was the mRNA for the mitogenic signal transduction molecule insulin receptor substrate (IRS)-1. However, in contrast to that of IRS-1, IRS-2 gene expression was 60- to 70-fold higher in the insulinoma tissue compared with islets, which was reflected at the protein as well as the mRNA level. The specific elevated IRS-2 expression was a consistent observation across all rodent pancreatic beta-cell lines. To investigate whether IRS-2 was functional, serum-stimulated beta-cell proliferation was examined in isolated insulinoma cells. After a 48-h period of serum withdrawal, 24 h of serum refeeding rendered an 8- to 10-fold increase in [3H]thymidine incorporation into insulinoma cells. This serum-stimulated DNA synthesis was prevented by inhibitors of tyrosine protein kinase and phosphatidylinositol (PI) 3-kinase activities, as well as the activation of mitogen-activated protein (MAP) kinase and p70S6K. Examination of IRS-mediated signal transduction pathways indicated that after 10-15 min of serum refeeding, there was increased tyrosine phosphorylation of IRS-2 and pp60, and PI 3-kinase recruitment to IRS-2. Serum also increased the association of growth factor-bound protein 2/murine sons of sevenless 1 protein to a PI 3-kinase/IRS-2 protein complex. Moreover, serum also activated MAP-kinase (erk-1 and erk-2 isoforms) and 70 kD S6 kinase. Thus IRS-mediated signal transduction pathways are functional in pancreatic beta-cells. It is conceivable that IRS-2 expression in beta-cells contributes to maintaining the islet beta-cell population, complementary to observations in the IRS-2 knockout mouse in which beta-cell mass is markedly reduced.

Mapping of polyomavirus middle T domain that is responsible for AP-1 activation.

Cell transformation by Polyomavirus middle T (MT) oncoprotein involves binding and activation of several cytoplasmic proteins that participate in growth factors-induced mitogenic signal transduction to the nucleus. We have previously reported that the AP-1 transcriptional complex is a target for MT during cell transformation. To analyse the interactions between MT and cellular proteins that are required for constitutive AP-1 activation, we compared wild type and transformation-defective MT mutant cell lines. High AP-1 activity, assessed by gel mobility shift assays, displayed by MT-overexpressing cells, is dependent on MT binding to phosphatidylinositol-3 kinase (P13K). Treatment with wortmannin (a specific P13K inhibitor) leads to decreased AP-1 activity. Supershift and Western blot analysis with specific antisera, indicate that JunB and cJun, but not cFos or FosB are present in the AP-1 complex. The results confirm the AP-1 complex as a downstream MT target and indicate that AP-1 activation may not be sufficient for cell transformation, since two transformation-defective MT mutants (250phe and MT322) display high AP-1 activity.

Effects of LHRH-analogues on mitogenic signal transduction in cancer cells

The expression of luteinizing hormone-releasing hormone (LHRH) and its receptors has been demonstrated in a number of human malignant tumors, including cancers of the breast, ovary, endometrium and prostate. These findings suggest the presence of an autocrine regulatory system based on LHRH. Recent studies in our laboratory have demonstrated that the function of LHRH produced by ovarian cancer cells is the inhibition of their proliferation. Dose-dependent antiproliferative effects of LHRH-agonists have been observed by several laboratories in cell lines derived from the above cancers. Interestingly, also LHRH-antagonists have marked antiproliferative activity in most of the ovarian, breast and endometrial cancer cell lines tested so far, indicating that the dichotomy of LHRH-agonists/LHRH-antagonists is not valid for the LHRH-system in cancer cells. In addition, our data suggest that the classical LHRH receptor signal transduction mechanisms known from the pituitary (phospholipase-C, protein kinase C, adenylyl cyclase) are not involved in the mediation of LHRH effects in cancer cells. Data obtained by several groups, including ours, rather suggest that LHRH analogs interfere with the signal transduction of growth-factor receptors and related oncogene products associated with tyrosine-kinase activity. The mechanism of action is probably an LHRH-induced activation of a phosphotyrosine phosphatase, counteracting the effects of receptor associated tyrosine kinase. In our hands, LHRH analogs virtually blocked the EGF-induced MAP-kinase activity of ovarian and endometrial cancer cells. The pharmacological exploitation of this mechanism might provide promising new therapies for these cancers.

Hydrogen peroxide activates extracellular signal-regulated kinase via protein kinase C, Raf-1, and MEK1.

We have previously demonstrated that hydrogen peroxide (H2O2) treatment of bovine tracheal myocytes increases the activity of extracellular signal-regulated kinases (ERK), serine/threonine kinases of the mitogen-activated protein (MAP) kinase superfamily thought to play a key role in the transduction of mitogenic signals to the cell nucleus. Moreover, H2O2-induced ERK activation was partially reduced by pretreatment with phorbol 12,13-dibutyrate, which depletes protein kinase C (PKC). In this study, we further examined the signaling intermediates responsible for ERK activation by H2O2 in airway smooth muscle, focusing on MAP kinase/ERK kinase (MEK), a dual-function kinase which is required and sufficient for ERK activation in bovine tracheal myocytes; Raf-1, a serine/threonine kinase known to activate MEK; and PKC. Pretreatment of cells with inhibitors of MEK (PD98059), Raf-1 (forskolin), and PKC (chelerythrine) each reduced H2O2-induced ERK activity. In addition, H2O2 treatment significantly increased both MEK1 and Raf-1 activity. No activation of MEK2 was detected. Together these data suggest that H2O2 may stimulate ERK via successive activation of PKC, Raf-1, and MEK1.

Phosphatidylinositol 3-Kinase Is Involved in the Induction of Macrophage Growth by Oxidized Low Density Lipoprotein

Early atherosclerotic lesions are characterized by the presence of cholesterol-rich, macrophage-derived foam cells. It has recently been shown that macrophage proliferation occurs during the development of early lesions and that oxidized low density lipoprotein (LDL) stimulates macrophage growth. Possible mechanisms for this induction of macrophage growth include potentiation of mitogenic signal transduction by a component of oxidized LDL following internalization and degradation, interaction with integral plasma membrane proteins coupled to signaling pathways, or direct or indirect activation of growth factor receptors on the cell surface (e.g. GM-CSF receptor) through an autocrine/paracrine mechanism. The present study was undertaken to characterize some of the early intracellular signaling events by which oxidized LDL mediates macrophage cell growth. Extensively oxidized LDL increased protein-tyrosine phosphorylation and caused a 2-fold increase in phosphatidylinositol (PI) 3-kinase activity in phorbol ester-pretreated THP-1 cells (a human monocyte-like cell line). Similar concentrations of native LDL had no effect. Oxidized LDL also stimulated growth of resident mouse peritoneal macrophages, and this effect was reduced by 40-50% in cells treated with PI 3-kinase inhibitors (100 nM wortmannin or 20 microM LY294002). These results suggest that PI 3-kinase mediates part of the mitogenic effect of oxidized LDL, but parallel pathways involving other receptors and signal transduction pathways are likely also involved.

Interleukin-1 beta stimulates mitogen-activated protein kinase in U373 astrocytoma cells without the production of lipid second messengers.

IL-1 beta is one of the cytokines known to affect astroglial cells in normal brain development, brain injury and neurodegenerative diseases. IL-1 beta causes astrocytes to become more reactive, alter the expression and release of molecules and in some cases to proliferate. We have investigated the mitogenic effect and signal transduction pathway induced by IL-1 beta in U373 cells, a human astrocytoma cell-line. Recombinant human IL-1 beta induced mitogenesis of U373 cells in a dose-dependent fashion as assessed by tritiated thymidine incorporation. The following signal transduction mechanisms, reported to be induced in other systems by IL-1 beta, were investigated in U373 cells: (1) activation of phosphatidylcholine-specific phospholipase C as assayed by incorporation of tritiated choline into cellular phospholipids, (2) production of diacylglycerol, a lipid second messenger, (3) activation of sphingomyelinase, and (4) activation of mitogen-activated protein kinase (MAPK). Of these, IL-1 beta activated only MAPK. In cultured rat astrocytes, IL-1 beta caused activation of MAPK without inducing proliferation.

Cloning and characterization of cDNAs encoding chicken mitogen-activated protein kinase kinase type 2, MEK2: downregulation of MEK2 in response to inhibition of mitochondrial DNA expression.

The present work was initiated with the aim of identifying nuclear genes whose expression is sensitive to the mitochondrial DNA (mtDNA) status of transformed chicken DU24 cells. We cloned and sequenced cDNAs for the mitogen-activated protein kinase kinase type 2, MEK2, a protein involved in the mitogenic growth factor signal transduction pathway in vertebrates. Sequence comparisons between the chicken protein and its mammalian counterparts indicated that MEK2 proteins are highly conserved among vertebrates. Southern blot analysis of endonuclease-digested genomic DNA from primary chick embryo fibroblasts (CEF) suggested that MEK2 is a single-copy gene in this vertebrate species. The steady-state level of MEK2 transcripts is decreased in DUS3 mtDNA-less (rho0) cells developed by long-term exposure of DU24 rho+ cells to ethidium bromide (EtdBr). Run-on in vitro transcription assays and mRNA stability studies indicated that the decrease in MEK2 mRNA content is associated with post-transcriptional regulation. In parental DU24 cells, MEK2 mRNA content decreased after inhibition of mtDNA transcription by EtdBr and inhibition of translation on mitoribosomes by chloramphenicol (CAM). Cytoplasmic hybrids (cybrids) constructed by fusion of chicken rho0 cells with enucleated parental cells and CEF recovered a basal level of MEK2 expression. The MEK2 protein content is decreased in DUS3 rho0 cells and in parental DU24 rho+ cells treated with EtdBr and CAM for 6 days, while that of MEK1, a closely related kinase, remained unchanged. On the basis of these observations, we propose that mitochondria participate in the mitogenic signal transduction pathway in chicken cells through regulation of MEK2 expression.

Heparin suppresses sgk, an early response gene in proliferating vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) hyperplasia plays a central role in chronic and acute vascular pathology including arteriosclerosis and restenosis following vascular surgery. The glycosaminoglycans of the heparan sulfate class, including heparin, inhibit VSMC proliferation in animals and in culture. Heparin binds to high affinity sites on the cell surface, selectively modulates mitogenic signal transduction pathway(s), and rapidly alters transcription of several genes. To further explore the molecular mechanisms responsible for this growth inhibition, we have employed the differential display technique to identify heparin-regulated genes. Here we demonstrate that heparin inhibits the expression of the early response gene sgk (serum and glucocorticoid-regulated kinase). The expression of sgk is not inhibited by chondroitin sulfate, a nonantiproliferative glycosaminoglycan, suggesting that sgk suppression may play a functional role in the antiproliferative effect of heparin. This idea is strengthened by the finding that heparin does not inhibit sgk expression in VSMCs resistant to the antiproliferative effect of heparin or in vascular endothelial cells which are unresponsive to heparin. Expression of sgk mRNA diminishes with increasing concentrations of heparin. Finally, sgk expression is not suppressed by other growth inhibitors such as transforming growth factor-beta 1 (TGF-beta 1) and interferon-beta (IFN-beta), suggesting separate and distinct effects of these growth inhibitors on the mitogenic pathway.

Influence of ATM function on telomere metabolism.

The ATM gene product, which is defective in the cancer-prone disorder ataxia telangiectasia, has been implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination and cell cycle control. The ATM gene has homology with the TEL1 gene of yeast, mutations of which lead to shortened telomeres. To test the hypothesis that the ATM gene product is involved in telomere metabolism, we examined telomeric associations (TA), telomere length, and telomerase activity in human cells expressing either dominant-negative or complementing fragments of the ATM gene. The phenotype of RKO colorectal tumor cells expressing ATM fragments containing a leucine zipper (LZ) motif mimics that of ataxia telangiectasia (A-T) cells. These transfected RKO cells relative to transfected controls had a higher frequency of cells with TA and shortened telomeres, but no detectable change in telomerase activity. In addition, the percentage of cells with TA after gamma irradiation was higher in the transfected RKO cells with dominant negative activity of the ATM gene, compared to control cells. SV40 transformed fibroblasts derived from an A-T patient and transfected with a complementing carboxyl terminal kinase region of the ATM gene had a reduced frequency of cells with TA, with no effect on the telomere length or telomerase activity. The present studies using isogenic cells with manipulated ATM function demonstrate a role for the ATM gene product in telomere metabolism.

Growth-Inhibitory Actions of Analogues of Luteinizing Hormone Releasing Hormone on Tumor Cells

The expression of LHRH and its receptor has been demonstrated in a number of human malignant tumors, including cancers of the breast, ovary, endometrium, and prostate. These findings suggest the presence of an autocrine regulatory system based on LHRH. Dose-dependent antiproliferative effects of LHRH agonists in cell lines derived from these cancers have been observed by various investigators. LHRH antagonists also have marked antiproliferative activity in most of the ovarian, breast, and endometrial cancer cell lines tested, indicating that the dichotomy of LHRH agonists and antagonists might not apply to the LHRH system in cancer cells. Findings from our laboratories suggest that the classical LHRH receptor signal-transduction mechanisms, known to operate in the pituitary, are not involved in the mediation of antiproliferative effects of LHRH analogues in cancer cells. Results obtained by several groups, including ours, instead suggest that LHRH analogues interfere with the mitogenic signal transduction of growth-factor receptors and related oncogene products associated with tyrosine kinase activity. The pharmacological exploitation of these direct antiproliferative actions of LHRH analogues might provide new therapeutic approaches to these cancers. (Trends Endocrinol Metab 1997;8:355–362). © 1997, Elsevier Science Inc.

Molecular dynamics on complexes of ras-p21 and its inhibitor protein, rap-1A, bound to the ras-binding domain of the raf-p74 protein: identification of effector domains in the raf protein.

We have computed the average structures for the ras-p21 protein and its strongly homologous inhibitor protein, rap-1A, bound to the ras-binding domain (RBD) of the raf protein, using molecular dynamics. Our purpose is to determine the differences in structure between these complexes that would result in no mitogenic activity of rap-1A-RBD but full activity of p21-RBD. We find that despite the similarities of the starting structures for both complexes, the average structures differ considerably, indicating that these two proteins do not interact in the same way with this vital target protein. p21 does not undergo major changes in conformation when bound to the RBD, while rap-1A undergoes significant changes in structure on binding to the RBD, especially in the critical region around residue 61. The p21 and rap-1A make substantially different contacts with the RBD. For example, the loop region from residues 55-71 of rap-1a makes extensive hydrogen-bond contacts with the RBD, while the same residues of p21 do not. Comparison of the structures of the RBD in both complexes reveals that it undergoes considerable changes in structure when its structure bond to p21 is compared with that bound to rap-1A. These changes in structure are due to displacements of regular structure (e.g., alpha-helices and beta-sheets) rather than to changes in the specific conformations of the segments themselves. Three regions of the RBD have been found to differ significantly from one another in the two complexes: the binding interface between the two proteins at residues 60 and 70, the region around residues 105-106, and 118-120. These regions may constitute effector domains of the RBD whose conformations determine whether or not mitogenic signal transduction will occur.

Low-affinity thromboxane receptor mediates proliferation in cultured vascular smooth muscle cells of rats.

We examined the binding properties and mitogenic effects of U46619, using cultured vascular smooth muscle cells (VSMCs), by ligand-binding assay, measuring [3H]thymidine and [3H]leucine incorporation, checking with flow cytometry, and counting the cell number. The U46619-activated mitogenic signal-transduction pathway was assessed by measuring formation of inositol monophosphate (IP); [Ca2+]i; mitogen-activated protein kinase (MAPK), MAPK kinase (MAPKK), and p74raf-1 activities; and GTP-bound Ras. [3H]U46619 bound to cultured VSMCs from Wistar-Kyoto (WKY) rats at a single class of site (Kd: 15.5 +/- 2.6 nmol/L). However, it bound to VSMCs from spontaneously hypertensive rats (SHRs) at two classes of sites (Kd: 2.3 +/- 0.6 nmol/L and 1.4 +/- 0.5 mumol/L). U46619 increased DNA and protein synthesis, cell number, IP formation, [Ca2+]i, and MAPK and MAPKK activities, with EC50 values close to its Kd value for the low-affinity binding site in VSMCs from SHR. Prostaglandin (PG) E2 and PGF2 alpha showed little of such mitogenic effects. All these effects of U46619 were inhibited by SQ29548, staurosporine, or pretreatment of VSMCs with phorbol 12-myristate 13-acetate for 24 hours. However, U46619 stimulation did not lead to a significant increase in the Ras-GTP complex or p74raf-1 activity. In conclusion, the mitogenic effect of U46619 appears to be mediated via the activation of low-affinity thromboxane binding sites that trigger phosphoinositide hydrolysis and activate the MAPK pathway, leading to DNA synthesis and cell proliferation.

Diacylglycerol generated by exogenous phospholipase C activates the mitogen-activated protein kinase pathway independent of Ras- and phorbol ester-sensitive protein kinase C: dependence on protein kinase C-zeta.

The role of diacylglycerol (DG) formation from phosphatidylcholine in mitogenic signal transduction is poorly understood. We have generated this lipid at the plasma membrane by treating Rat-1 fibroblasts with bacterial phosphatidylcholine-specific phospholipase C (PC-PLC). This treatment leads to activation of mitogen-activated protein kinase (MAPK). However, unlike platelet-derived growth factor (PDGF) or epidermal growth factor (EGF), PC-PLC fails to activate Ras and to induce DNA synthesis, and activates MAPK only transiently (<45 min). Down-regulation of protein kinase C (PKC) -alpha, -delta and -epsilon isotypes has little or no effect on MAPK activation by either PC-PLC or growth factors. However, Ro 31-8220, a highly selective inhibitor of all PKC isotypes, including atypical PKC-zeta but not Raf-1, blocks MAPK activation by PDGF and PC-PLC, but not that by EGF, suggesting that atypical PKC mediates the PDGF and PC-PLC signal. In line with this, PKC-zeta is activated by PC-PLC and PDGF, but not by EGF, as shown by a kinase assay in vitro, using biotinylated epsilon-peptide as a substrate. Furthermore, dominant-negative PKC-zeta inhibits, while (wild-type) PKC-zeta overexpression enhances MAPK activation by PDGF and PC-PLC. The results suggest that DG generated by PC-PLC can activate the MAPK pathway independent of Ras and phorbol-ester-sensitive PKC but, instead, via PKC-zeta.

Mitotic percentage index: a new prognostic factor for childhood medulloblastoma.

We investigated the prognostic significance of a new method of mitotic figure quantitation, 'mitotic percentage index' (MPI), tumour S phase fraction (SPF) and DNA ploidy measured by flow cytometry, and various clinical prognostic factors including age, sex, tumour stage, degree of surgical resection, radiotherapy dose and adjuvant chemotherapy in 70 cases of childhood medulloblastoma diagnosed between 1968 and 1996. In univariate analysis, MPI (P < 0.0001), posterior fossa radiotherapy dose (P = 0.003), tumour stage (P = 0.014), craniospinal radiotherapy dose (P = 0.019), year of diagnosis (P = 0.024) and SPF (P = 0.048) were significantly related to survival. In multivariate analysis, including tumour c-erbB-2 oncogene product expression, only MPI (P < 0.0001), craniospinal radiotherapy dose (P = 0.003) and tumour stage (P = 0.035) retained independent prognostic significance, while age achieved significance (P = 0.039). A close relationship was observed between MPI and SPF (coeff = 0.8, P < 0.0001) and MPI and the percentage of tumour cells expressing the c-erbB-2 oncogene product (coeff = 0.416, P < 0.0001). This study has identified MPI as a new independent prognostic factor for childhood medulloblastoma. Its close relationship with tumour SPF confirms it as an accurate measure of tumour proliferation and its close relationship to expression of the c-erbB-2 oncogene supports a role for this growth factor receptor in the deregulation of normal mitogenic signal transduction in this malignancy.

Negative Modulation of Membrane Localization of the Raf-1 Protein Kinase by Hyperphosphorylation

The serine/threonine-specific protein kinase Raf-1 plays a key role in mitogenic signal transduction by coupling Ras to the mitogen-activated protein (MAP) kinase cascade. Ras-mediated translocation to the plasma membrane represents a crucial step in the process of serum-stimulated Raf-1 kinase activation. The exact role of the multisite phosphorylation in Raf regulation, however, is not clear. We have previously reported that the mobility shift-associated hyperphosphorylation of Raf correlates with a reduction of serum-stimulated Raf kinase activity (Wartmann, M., and Davis, R. J. (1994) J. Biol. Chem. 269, 6695-6701). Here we show that incubation of serum-starved CHO cells with D609, a purported inhibitor of phosphatidylcholine-specific phospholipase C, also results in a mobility shift of Raf-1 that is due to hyperphosphorylation on sites identical to those observed following mitogen stimulation. Subcellular fractionation analyses revealed that D609-induced mobility shift-associated hyperphosphorylation was paralleled by a decreased membrane association of Raf-1. Similar results were obtained in an in vitro reconstitution system. Furthermore, PD98059, a specific inhibitor of activation of the MAP kinase kinase MEK, prevented D609-induced Raf hyperphosphorylation and restored the amount of membrane-bound Raf to control levels. Taken together, these data suggest that mobility shift-associated hyperphosphorylation of Raf-1, by virtue of reducing the amount of plasma membrane-bound Raf-1, represents a negative feedback mechanism contributing to the desensitization of the MAP kinase signaling cascade.

A molecular model of the active site of dipeptidyl peptidase IV. Explanation of the substrate specificity and interaction with inhibitors.

The ectoenzyme dipeptidyl peptidase IV (DPP IV) is an integral plasma membrane glycoprotein abundantly expressed on a variety of cell surfaces.1 It is a serine peptidase of broad medical and biochemical significance. It plays a role in the degradations and post translational processing of bioactive peptides such as substance P, s-casomorphins, growth hormone-releasing hormone and promelittin.2–9 DPP IV has been identified as CD26.10 Recently, it could be demonstrated that it is not only a surface differentiation marker involved in the transduction of mitogenic signals in thymocytes and T lymphocytes in human but also a cofactor in AIDS expression.10–13

Serum- and Polypeptide Growth Factor-Inducible Gene Expression in Mouse Fibroblasts

Complex cellular processes such as proliferation, differentiation, and apoptosis are regulated in part by extracellular signaling molecules: for example, polypeptide growth factors, cytokines, and peptide hormones. Many polypeptide growth factors exert their mitogenic effects by binding to specific cell surface receptor protein tyrosine kinases. This interaction triggers numerous biochemical responses, including changes in phospholipid metabolism, the activation of a protein phosphorylation cascade, and the enhanced expression of specific immediate-early, delayed-early, or late response genes. In this review, I summarize the major findings obtained from studies investigating the effects of serum or individual polypeptide growth factors on gene expression in murine fibroblasts. Several experimental approaches, including differential hybridization screening of cDNA libraries and differential display, have been employed to identify mRNA species that are expressed at elevated levels in serum- or polypeptide growth factor-stimulated cells. These studies have demonstrated that serum- and growth factor-inducible genes encode a diverse family of proteins, including DNA-binding transcription factors, cytoskeletal and extracellular matrix proteins, metabolic enzymes, secreted chemokines, and serine-threonine kinases. Some of these gene products act as effectors of specific cell cycle functions (e.g., enzymes involved in nucleotide and DNA synthesis), others are required to successfully convert a metabolically inactive cell to a metabolically active cell that will eventually increase in size and then divide (e.g., glucose-metabolizing enzymes), and some actually function as positive or negative regulators of cell cycle progression. In conclusion, research conducted during the past 15 years on serum- and growth factor-regulated gene expression in murine fibroblasts has provided significant insight into mitogenic signal transduction and cell growth control.

Cloning and mutagenesis of the p110α subunit of human phosphoinositide 3′-hydroxykinase

Activation of phosphoinositide 3′-hydroxykinase (PI3K) is required for mitogenic signal transduction by several growth factors and oncogenes. PI3K is a heterodimer consisting of a p85 regulatory subunit and a p110 catalytic subunit. In the current study, we report the cloning and characterization of the p110α catalytic subunit of human PI3K. This clone is highly homologous (>99% amino acid identity) to bovine brain p110α, but contains 10 amino acid differences from the human p110α sequence previously reported. Comparison of this sequence with known Ser/Thr kinases and p110 homologs highlighted several conserved residues within the putative kinase domain. Mutational analysis of these residues (Asp915, (Asp933 + Phe934) yielded PI3K mutants with virtually complete loss of phosphoinositide phosphorylating activity. Expression of the wild-type p110α protein in CHO cells is sufficient to activate the serum response element derived from the promoter of c-fos, an immediate early gene product. In contrast, the catalytically impaired p110α mutants as well as the p85α subunit of PI3K were inactive in the fos assay. These studies suggest that the mitogenic signal transduction pathway mediated by PI3K is dependent upon the enzymatic activity of the p110α subunit of PI3K.