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L C Mahadevan

Publications and source records attributed to L C Mahadevan.

16 recordsLinked to original sources
PubMed1995

Parallel signal processing among mammalian MAPKs.

The intracellular signalling field is dominated by the mitogen-activated protein kinase (MAPK) cascade and its control, which involves the small GTPase Ras and sequential kinase activation. Until recently, ERK1 and ERK2 were the only cloned and well-characterized mammalian MAPKs; diverse ligand-stimulated, proline-directed protein phosphorylation events were attributed to these kinases. The recent discovery of two other MAPK subtypes, the JNK/SAPK subfamily and p38/RK (mammalian equivalents of HOG1 in yeast), reveals extreme complexity within the family and, most intriguingly, the existence in mammalian cells of parallel MAPK cascades that can be activated simultaneously.

Amino Acid Sequence↗
PubMed1994

A mitogen- and anisomycin-stimulated kinase phosphorylates HMG-14 in its basic amino-terminal domain in vivo and on isolated mononucleosomes.

The rapid, transient induction of 80-100 immediate-early (IE) genes upon mitogenic stimulation occurs irrespective of protein synthesis and is mediated by modification of existing proteins. Two mechanisms, not mutually exclusive, involving modification either of sequence-specific transcription factors or of structural chromatin proteins primed by pre-association with responsive effectors are conceivable. Here, we show that upon IE gene induction, the non-histone high-mobility-group protein HMG-14, but not the related protein HMG-17, becomes serine phosphorylated in its basic, amino-terminal region close to where it binds nucleosomal DNA. Phosphorylation, normally transient, occurs independent of transcription and is quantitative and prolonged during superinduction. Brief micrococcal nuclease digestion substantially releases HMG-14 from nuclei in the mononucleosome-bound state. Finally, mononucleosomes prepared from mitogen-stimulated, but not control, cells contain a mitogen-activated kinase that phosphorylates HMG-14 in vitro on the same site(s) as in intact cells. The association of HMG-14 and its mitogen-activated kinase with nuclease-sensitive mononucleosomes has implications for models of mitogen-stimulated IE gene induction.

Amino Acid Sequence↗
PubMed1994

Anisomycin-activated protein kinases p45 and p55 but not mitogen-activated protein kinases ERK-1 and -2 are implicated in the induction of c-fos and c-jun.

Independent of its ability to block translation, anisomycin intrinsically initiates intracellular signals and immediate-early gene induction [L. C. Mahadevan and D. R. Edwards, Nature (London) 349:747-749, 1991]. Here, we characterize further its action as a potent, selective signalling agonist. In-gel kinase assays show that epidermal growth factor (EGF) transiently activates five kinases: the mitogen-activated protein (MAP) kinases ERK-1 and -2, and three others, p45, p55, and p80. Anisomycin, at inhibitory and subinhibitory concentrations, does not activate ERK-1 and -2 but elicits strong sustained activation of p45 and p55, which are unique in being serine kinases whose detection is enhanced with poly-Glu/Tyr or poly-Glu/Phe copolymerized in these gels. Translational arrest using emetine or puromycin does not activate p45 and p55 but does prolong EGF-stimulated ERK-1 and -2 activation. Rapamycin, which blocks anisomycin-stimulated p70/85S6k activation without affecting nuclear responses, has no effect on p45 or p55 kinase. p45 and p55 are activable by okadaic acid or UV irradiation, and both kinases phosphorylate the c-Jun NH2-terminal peptide 1-79, putatively placing them within c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) subfamily of MAP kinases. Thus, the EGF- and anisomycin-activated kinases p45 and p55 are strongly implicated in signalling to c-fos and c-jun, whereas the MAP kinases ERK-1 and -2 are not essential for this process.

Animals↗
PubMed1992

Protein synthesis inhibitors differentially superinduce c-fos and c-jun by three distinct mechanisms: lack of evidence for labile repressors.

Protein synthesis inhibitors strongly augment and prolong the usually transient induction of c-fos and c-jun by growth factors, phorbol esters etc., a phenomenon termed superinduction which is conventionally regarded as a secondary consequence of translational arrest. Our recent demonstration that some inhibitors can act positively as nuclear signalling agonists compromises this view and necessitates a re-evaluation of superinduction. First, we show that labile repressors, widely postulated to act negatively on diverse superinducible genes, are not involved in regulating c-fos and c-jun. Secondly, two components of c-fos and c-jun superinduction, namely the delay in shutting off transcription and stabilization of their mRNAs, arise from translational arrest and are common to all protein synthesis inhibitors. Thirdly, the recently described capacity to act positively as nuclear signalling agonists to stimulate pp33/pp15 phosphorylation is restricted to compounds such as anisomycin and cycloheximide; these, but not emetine or puromycin, will induce c-fos/c-jun on their own. Fourthly, the translational arrest-related components of superinduction are dissociable from the signalling agonist effects at sub-inhibitory concentrations of anisomycin, under which conditions a new type of c-fos/c-jun superinduction with 'spike' kinetics is observed. Finally, we show that in response to EGF plus anisomycin, the nuclear signalling responses are themselves augmented and prolonged in a manner that corresponds to c-fos/c-jun superinduction under these conditions.

Animals↗
PubMed1992

Intracellular signalling pathways involved in mesoderm induction by FGF.

We have examined the possible role of two signal transducing mechanisms, tyrosine phosphorylation and activation of protein kinase C (PKC), during fibroblast growth factor (FGF)-induced mesoderm induction in Xenopus. Tyrosine phosphorylation was examined through the use of a monoclonal anti-phosphotyrosine antibody. This antibody was shown to recognize the FGF receptor crosslinked to radioiodinated FGF. We also studied the response of Xenopus ectodermal explants to sodium orthovanadate, a compound that has been shown to elevate intracellular phosphotyrosine levels. Thirty percent of explants cultured in 100 microM vanadate were induced. In addition, vanadate synergized with FGF to give inductions that were more dorsal in nature than either vanadate or FGF alone. The role of PKC was evaluated by measuring PKC activity during mesoderm induction by FGF and by examining the effect of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on explants. TPA did not induce mesoderm, however, activation of PKC was detected in FGF-treated explants. Therefore, activation of the PKC pathway alone is not sufficient for mesoderm induction. Simultaneous treatment with TPA and FGF resulted in a significant inhibition of mesoderm induction by FGF, suggesting that activation of PKC could be part of a negative feedback mechanism. In contrast, TPA had no effect on induction by activin A.

Activins↗
PubMed1991

Rapid histone H3 phosphorylation in response to growth factors, phorbol esters, okadaic acid, and protein synthesis inhibitors.

When quiescent cells are stimulated with growth factors, phorbol esters, okadaic acid, or protein synthesis inhibitors, the early-response genes, which include c-fos and c-jun, are rapidly induced. The earliest growth factor- and phorbol ester-stimulated nuclear signaling events concomitant with proto-oncogene induction are the rapid phosphorylation of two chromatin-associated proteins, pp33 and pp15. We show here that the tumor promoter okadaic acid, which inhibits protein phosphatases 1 and 2A, and the protein synthesis inhibitors anisomycin and cycloheximide also stimulate pp33 and pp15 phosphorylation. Using transcriptional inhibitors, we show that this response is not a consequence of early gene induction. By peptide mapping and microsequencing, chromatin-associated pp15 is identified as histone H3. Upon stimulation, histone H3 is rapidly phosphorylated on serine residues within its highly charged, basic N-terminal domain. Thus, these diverse agents elicit a common early nuclear signal modulating nucleosomal structure or function, potentially contributing to conformational regulation of proto-oncogene induction.

Amanitins↗
PubMed1990

2-Aminopurine abolishes EGF- and TPA-stimulated pp33 phosphorylation and c-fos induction without affecting the activation of protein kinase C.

Epidermal Growth Factor (EGF) and Tetradecanoyl Phorbol Acetate (TPA) initiate signalling cascades in C3H 10T1/2 fibroblasts by primarily activating distinct protein kinases, the EGF receptor tyrosine kinase and protein kinase C respectively; there is no signal crossover at the initiation of signalling. Nevertheless, we show here that both agents rapidly elicit common intracellular responses, including the phosphorylation of complexed and chromatin-associated forms of a 33 kDa phosphoprotein (pp33), that of a 15 kDa chromatin-associated phosphoprotein (pp15), as well as the transcriptional activation of a common subset of genes including the c-fos proto-oncogene. 2-aminopurine specifically abolishes complexed and chromatin-associated pp33 phosphorylation in response to EGF and TPA, as well as the induction of c-fos by both agents. The activation of protein kinase C and the levels of transcription factors that bind to the serum response element (SRE), TPA response element (TRE) or NFkB sites in stimulated cells are relatively unaffected by 2-aminopurine. This, to our knowledge, is the first demonstration that it is possible, by using 2-aminopurine which selectively blocks TPA-stimulated pp33 phosphorylation, to block c-fos induction in TPA-treated cells although protein kinase C remains fully active. Further, we show here that although EGF- and TPA-stimulated induction of c-fos is abolished by 2-aminopurine, the appearance of TRE-binding activity in nuclear extracts of stimulated cells is unaffected, suggesting that EGF- and TPA-stimulated induction of TRE-binding activity utilises existing proteins and is not dependent on fresh c-FOS synthesis. These results imply that 2-aminopurine-sensitive complexed and chromatin-associated pp33 phosphorylation may be crucial to c-fos induction in response to EGF and TPA.

2-Aminopurine↗
PubMed1989

2-Aminopurine abolishes epidermal growth factor-stimulated phosphorylation of complexed and chromatin-associated forms of a 33 kDa phosphoprotein.

We have recently reported that several purified polypeptide mitogens such as epidermal growth factor, embryonal carcinoma-derived growth factor, basic fibroblast growth factor and Bombesin induce the rapid appearance of a 33 kDa chromatin-associated phosphoprotein in the nuclei of murine fibroblasts. We show here that in both mouse and human cell lines, a second form of the 33 kDa phosphoprotein exists in a detergent-extractable complexed form which may be pelleted by ultracentrifugation. When quiescent [32P]-labelled cells are treated with EGF, both complexed and chromatin-associated forms of the labelled phosphoprotein are detectable within 10 min, the response peaking at about 1 h and being substantially over 3 h after EGF stimulation. By chymotryptic and cyanogen bromide phosphopeptide mapping studies, the two forms of the 33 kDa phosphoprotein are indistinguishable, as are the mouse and human forms of the protein. The protein kinase inhibitor 2-aminopurine, which has recently been shown to block growth factor-stimulated c-fos and c-myc induction, specifically abolishes the mitogen-stimulated appearance of both forms of the 33 kDa phosphoprotein without affecting the phosphorylation of other cellular proteins. The 33 kDa protein has been purified from Hela cells by a combination of sucrose density gradient centrifugation, preparative electrophoresis and reverse-phase HPLC during which the protein is resolved into two closely-eluting peaks which differ markedly in their specific activity. These results are discussed in relation to the possible role of these events in coupling growth factor-receptor interaction at the cell surface to the early responses of transcriptional activation in the nucleus.

2-Aminopurine↗
PubMed1988

Rapid appearance of novel phosphoproteins in the nuclei of mitogen-stimulated fibroblasts.

A sequential extraction procedure has been used to prepare three protein fractions from control and mitogen-stimulated [32P]-PO4-labelled fibroblasts, the first containing soluble and membrane-bound proteins, the second mainly the cytoskeletal proteins vimentin and actin, and the third, a chromatin-derived fraction enriched in histones. We describe here an analysis of the mitogen-stimulated changes in the [32P] labelled phosphoprotein composition of these fractions. The most obvious changes in response to epidermal growth factor, embryonal carcinoma-derived growth factor, basic fibroblast growth factor and bombesin were the rapid appearance of 33 and 15 kDa phosphoproteins in the chromatin-derived fraction. The epidermal growth factor- and basic fibroblast growth factor-stimulated 33 kDa phosphoprotein produced similar chymotryptic peptides and was phosphorylated on serine residues. DNAse/RNAse treatment of the lysates was essential for the extraction of the 33 kDa phosphoprotein. Further, its presence could be demonstrated in preparations of conventionally purified nuclei. An in situ extraction procedure has been used to provide morphological verification of the sequential extraction data. The final structure containing these phosphoproteins is clearly derived from nuclei, enriched in histones, stains for DNA and appears by electron microscopy, to be homogenously composed of chromatin-like material. Thus, we describe here the rapid mitogen-induced appearance of novel phosphoproteins in the nucleus, raising the possibility that they may be involved in orchestrating early nuclear responses to polypeptide growth factors.

Animals↗
PubMed1987

Calcium-dependant binding proteins associated with human placental syncytiotrophoblast microvillous cytoskeleton.

Isolated human placental syncytiotrophoblast microvillous plasma membrane vesicles were extracted with Triton X-100 to yield a detergent-insoluble residue. The residue contained approx. 50% of the total membrane protein and was qualitatively different from untreated trophoblast on SDS-polyacrylamide gel electrophoresis, Western blots and dot-immunobinding assay. Three major proteins, with molecular weights of 68, 36 and 34 kDa, dissociated from this non-ionic detergent-insoluble submembranous cytoskeletal fraction in the presence of calcium chelators. They were immunologically related to human lymphocyte cytoskeletal calcium-binding proteins, and the 36 kDa component reacted with antisera to the phospholipase A2 inhibitor, lipocortin II. Anti-lipocortin I sera did not recognise the 34 kDa protein, but did react with a series of trophoblast cytoskeletal proteins in the 34-37 kDa region. Incubation of epidermal growth factor with isolated trophoblast membrane vesicles stimulated the phosphorylation of a 36 kDa protein on tyrosine residues. Immunoprecipitation studies further showed there was no phosphorylation of the 34 kDa protein, but the 68 kDa protein was a major phosphorylated component of isolated syncytiotrophoblast membranes. p68 was principally phosphorylated on serine with slight tyrosine phosphorylation which showed an apparent increase after epidermal growth factor treatment. These results indicate a family of calcium-dependant binding proteins, some of which are phosphorylated, associated with the submembranous cytoskeleton of syncytiotrophoblast microvilli.

Calcium-Binding Proteins↗
PubMed1987

Embryonal carcinoma-derived growth factor activates protein kinase C in vivo and in vitro.

We have recently reported that a polypeptide mitogen, the embryonal carcinoma-derived growth factor (ECDGF), induces phosphorylation of the epidermal growth factor (EGF) receptor in intact C3H 10T 1/2 mouse fibroblasts with concomittant loss of high affinity EGF binding sites. This phenomenon appears to be mediated through an activation of protein kinase C. Several groups have described an acidic 80,000 dalton protein substrate of protein kinase C. In this paper, we demonstrate that the addition of ECDGF or the phorbol ester TPA to intact C3H 10T 1/2 cells results in the enhanced phosphorylation of this 80 kd protein in vivo. Furthermore, this response is demonstrable in vitro. Thus the addition of ECDGF, the phorbol ester TPA, protein kinase C or phosphoinositidase C to crude membranes prepared from C3H 10T 1/2 cells resulted in the enhanced phosphorylation of this protein. Data obtained by phosphopeptide mapping of the 80 kd protein show that the ECDGF-induced activation of protein kinase C in our membrane preparations is comparable with that obtained in vivo. The availability of an in vitro system in which this response is preserved should now allow a detailed biochemical analysis of the steps between binding of a mitogen to its receptor and the activation of protein kinase C.

Animals↗
PubMed1984

The brain isoform of a key ATP-regulating enzyme, creatine kinase, is a phosphoprotein.

Two-dimensional electrophoretic analysis of crude microtubule preparations from the rat brain revealed the presence of three polypeptides in positions corresponding to those of the isovariants of purified rat brain creatine kinase (CK-BB). By the use of [gamma-32P]ATP, the two more acidic forms of these polypeptides were shown to be phosphorylated. Their identity as phosphorylated forms of CK-BB was established by using various peptide mapping techniques. Thus CK-BB is a phosphoprotein and its isoelectric variation may be attributed to phosphorylation.

Adenosine Triphosphate↗
PubMed1982

The polyadenylated RNA directing the synthesis of the rat myelin basic proteins is present in both free and membrane-bound forebrain polyribosomes.

Free and membrane-bound polyribosomes were isolated from the forebrain of actively myelinating 24-day-old rats. The poly(A)+ RNA (polyadenylated RNA) extracted from both fractions was translated in vitro in reticulocyte lysates [Hall & Lim (1981) Biochem. J. 196. 327-336] in the presence or absence of a heterologous microsomal membrane fraction from dog pancreas. The rat myelin basic proteins synthesized in vitro were isolated by CM-cellulose chromatography and by immunoprecipitation with purified anti-(myelin basic protein) antibody. The large (mol.wt. 18 500) and small (mol.wt. 16 000) myelin basic proteins were translational products of poly(A)+ RNA from both free and membrane-bound polyribosomes. The identity of the myelin basic proteins was verified by analysis of peptides generated by the cathepsin D digestion of the immunoprecipitated proteins synthesized in vitro, in comparison with authentic rat myelin basic proteins. Although several other translational products of membrane-bound polyribosomal poly(A)+ RNA were modified when microsomal membranes were present during translation, molecular weights of the myelin basic proteins themselves were unchanged. The myelin basic proteins synthesized in vitro also did not differ significantly in size from the authentic myelin basic proteins, indicating that these membrane proteins are unlikely to be synthesized as substantially larger precursor molecules. The presence of the specific mRNA species on both free and membrane-bound polyribosomes is compatible with the extrinsic location of the myelin basic proteins on the cytoplasmic surface of the myelin membrane.

Animals↗
PubMedDate not supplied

Abelson-transformed fibroblasts contain nuclear phosphotyrosyl-proteins which preferentially bind to murine DNA.

Protein-tyrosine kinases, either in the form of growth-factor receptors or as the polypeptide products of oncogenes, appear to be important in the regulation of cell growth and transformation. A major question, however, is how their substrates mediate changes in gene expression (reviewed in refs 6-8). Because binding of proteins to specific DNA sequences represents the most direct mechanism for regulating transcription, we have investigated the possibility that some DNA-binding proteins may be substrates of protein-tyrosine kinases. Here, we present evidence for nuclear phosphotyrosyl-proteins in murine fibroblasts transformed by the v-abl protein-tyrosine kinase. Furthermore, we have found that these proteins are not significantly phosphorylated in normal NIH 3T3 cells. Finally, using affinity competition chromatography with bacterial and mouse DNA, we have demonstrated that some of these proteins preferentially bind to mouse DNA. The identification of phosphotyrosyl-proteins with selective DNA-binding properties suggests a possible mechanism through which protein-tyrosine kinases may effect changes in gene transcription.

Abelson murine leukemia virus↗