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R J Davis

Publications and source records attributed to R J Davis.

At least 163 records · Page 9Linked to original sources
PubMed1995

Transcriptional regulation by MAP kinases.

Tyrosine kinase growth factor receptors activate MAP kinase by a complex mechanism involving the SH2/3 protein Grb2, the exchange protein Sos, and Ras. The GTP-bound Ras protein binds to the Raf kinase and initiates a protein kinase cascade that leads to MAP kinase activation. Three MAP kinase kinase kinases have been described--c-Raf, c-Mos, and Mekk--that phosphorylate and activate Mek, the MAP kinase kinase. Activated Mek phosphorylates and activates MAP kinase. Subsequently, the activated MAP kinase translocates into the nucleus where many of the physiological targets of the MAP kinase signal transduction pathway are located. These substrates include transcription factors that are regulated by MAP kinase phosphorylation (e.g., Elk-1, c-Myc, c-Jun, c-Fos, and C/EBP beta). Thus the MAP kinase pathway represents a significant mechanism of signal transduction by growth factor receptors from the cell surface to the nucleus that results in the regulation of gene expression. Three MAP kinase homologs have been identified in the rat: Erk1, Erk2, and Erk3. Human MAP kinases that are similar to the rat Erk kinases have also been identified by molecular cloning. The human Erk1 protein kinase has been shown to be widely expressed as a 44-kDa protein in many tissues. The human Erk2 protein kinase is a 41-kDa protein that is expressed ubiquitously. In contrast, a human Erk3-related protein kinase has been found to be expressed at a high level only in heart muscle and brain. The loci of these MAP kinase genes are widely distributed within the human genome: erk2 at 22q11.2; erk1 at 16p11.2; and ek3-related at 18q12-21. In the yeast Saccharomyces cerevisiae, five MAP kinase gene homologs have been described: smkl, mpk1, hog1, fus3, and kss1. Together, these kinases are a more diverse group than the human erks that have been identified. Thus the erks are likely to represent only one subgroup of a larger human MAP kinase gene family. A candidate for this extended family of MAP kinases is the c-Jun NH2-terminal kinase (Jnk), which binds to and phosphorylates the transcription factor c-Jun at the activating sites Ser-63 and Ser-73. Evidence is presented here to demonstrate that Jnk is a distant relative of the MAP kinase group that is activated by dual phosphorylation at Tyr and Thr.

Amino Acid Sequence↗
PubMed1995

Structural characterization of the FKHR gene and its rearrangement in alveolar rhabdomyosarcoma.

The FKHR gene, which contains a forkhead DNA-binding motif, is fused to either PAX3 or PAX7 by the t(2;13) or t(1;13) translocation in alveolar rhabdomyosarcoma,respectively. These tumors express chimeric transcripts encoding the N-terminal portion of either PAX protein fused to the C-terminal portion of FKHR. To understand the structural basis and functional consequences of these translocations, we characterized the wild-type FKHR gene and its rearrangement in alveolar rhabdomyosarcomas. By isolating and analyzing phage, cosmid and YAC clones, we determined that FKHR consists of three exons spanning 140 kb and that several highly similar loci are present in other genomic regions. Exon 1 encodes the N-terminus of the forkhead domain and is embedded within demethylated CpG island. RNA analyses reveal FKHR transcripts initiate from a TATA-less promoter within this island. Exon 2 encodes the C-terminus of the forkhead domain and a transcription activation domain, whereas exon 3 encodes a large 3' untranslated region. The intron 1-exon 2 boundary precisely matches the FHKR fusion point in the chimeric transcripts found in alveolar rhabdomyosarcomas. Using pulsed-field and fluorescence in situ hybridization analyses, we demonstrate that the 130kb FKHR intron 1 is rearranged in t(2;13)-containing alveolar rhabdomyosarcomas. Our findings indicate that FKHR intron 1 provides a large target for DNA rearrangemnt. Rearrangement of this intron with PAX3 produces two important functional consequences: in-frame fusion of N-terminal PAX3 sequences to the FKHR transcriptional activation domain and disruption of the FKHR DNA binding domain.

Amino Acid Sequence↗
PubMed1994

Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK.

Growth factors activate mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERKs) and Jun kinases (JNKs). Although the signaling cascade from growth factor receptors to ERKs is relatively well understood, the pathway leading to JNK activation is more obscure. Activation of JNK by epidermal growth factor (EGF) or nerve growth factor (NGF) was dependent on H-Ras activation, whereas JNK activation by tumor necrosis factor alpha (TNF-alpha) was Ras-independent. Ras activates two protein kinases, Raf-1 and MEK (MAPK, or ERK, kinase) kinase (MEKK). Raf-1 contributes directly to ERK activation but not to JNK activation, whereas MEKK participated in JNK activation but caused ERK activation only after overexpression. These results demonstrate the existence of two distinct Ras-dependent MAPK cascades--one initiated by Raf-1 leading to ERK activation, and the other initiated by MEKK leading to JNK activation.

3T3 Cells↗
PubMed1994

MAP kinase binds to the NH2-terminal activation domain of c-Myc.

The transcription factor c-Myc is a substrate for phosphorylation by MAP kinases. Here we demonstrate that MAP kinase binds to c-Myc. The NH2-terminal region (residues 1-100) is necessary and sufficient for this interaction. Binding to c-Myc is not dependent on the state of MAP kinase activation. However, the c-Myc/MAP kinase complex is disrupted by ATP. Together, these observations indicate that substrate binding interactions contribute to the specificity of phosphorylation by MAP kinases.

Adenosine Triphosphate↗
PubMed1994

An osmosensing signal transduction pathway in mammalian cells.

The osmotic balance between the cytoplasmic and extracellular compartments of cells is critical for the control of cell volume. A mammalian protein kinase, Jnk, which is a distant relative of the mitogen-activated protein kinase group, was activated by phosphorylation on threonine and tyrosine in osmotically shocked cells. The activation of Jnk may be relevant to the biological response to osmotic shock because the expression of human Jnk in the yeast Saccharomyces cerevisiae rescued a defect in growth on hyper-osmolar media. These data indicate that related protein kinases may mediate osmosensing signal transduction in yeast and mammalian cells.

Amino Acid Sequence↗
PubMed1994

Fusion of PAX7 to FKHR by the variant t(1;13)(p36;q14) translocation in alveolar rhabdomyosarcoma.

Although the t(2;13)(q35;q14) translocation has been found in most cases of the pediatric cancer alveolar rhabdomyosarcoma, several cases have been reported with a variant t(1;13)(p36;q14) translocation. Our findings indicate that this t(1;13) rearranges PAX7 on chromosome 1 and fuses it to FKHR on chromosome 13. This fusion results in a chimeric transcript consisting of 5' PAX7 and 3' FKHR regions, which is similar to the 5' PAX3-3' FKHR transcript formed by the t(2;13). The 5' PAX3 and PAX7 regions encode related DNA binding domains, and therefore we postulate that these translocations create similar chimeric transcription factors that alter expression of a common group of target genes.

Amino Acid Sequence↗
PubMed1994

JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain.

The ultraviolet (UV) response of mammalian cells is characterized by a rapid and selective increase in gene expression mediated by AP-1 and NF-kappa B. The effect on AP-1 transcriptional activity results, in part, from enhanced phosphorylation of the c-Jun NH2-terminal activation domain. Here, we describe the molecular cloning and characterization of JNK1, a distant relative of the MAP kinase group that is activated by dual phosphorylation at Thr and Tyr during the UV response. Significantly, Ha-Ras partially activates JNK1 and potentiates the activation caused by UV. JNK1 binds to the c-Jun transactivation domain and phosphorylates it on Ser-63 and Ser-73. Thus, JNK1 is a component of a novel signal transduction pathway that is activated by oncoproteins and UV irradiation. These properties indicate that JNK1 activation may play an important role in tumor promotion.

Amino Acid Sequence↗
PubMed1994

The native structure of the activated Raf protein kinase is a membrane-bound multi-subunit complex.

Raf is a mitogen-stimulated protein kinase that functions as a component of the signaling cascade that leads to the stimulation of mitogen-activated protein kinase. Here we show that the native structure of Raf is a large multi-subunit protein complex with an apparent mass of 300-500 kDa that interacts with Ras and the mitogen-activated protein kinase kinase Mek. Analysis of the structure of the Raf complex demonstrates that it contains a single Raf protein kinase together with the molecular chaperones hsp90 and p50. The Raf-hsp90-p50 complex was observed in starved cells and in cells activated with serum or phorbol ester. Thus, changes in complex formation with hsp90 and p50 are not required for activation of the Raf protein kinase. However, Raf activation caused by Ras was associated with the translocation of the cytoplasmic Raf-hsp90-p50 complex to the cell membrane. Significantly, it is only the membrane-bound complex that exhibits increased protein kinase activity. Thus, the Ras-activated Raf protein kinase functions as a membrane-bound multi-subunit complex.

Amino Acid Sequence↗
PubMed1994

Control of MAP kinase activation by the mitogen-induced threonine/tyrosine phosphatase PAC1.

Intracellular signalling following mitogenic stimulation of quiescent cells involves the initiation of a phosphorylation cascade that leads to the rapid and reversible activation of the mitogen-activated protein (MAP) kinases ERK1 and ERK2. MAP kinase activation is mediated by dual phosphorylation within the motif Thr-Glu-Tyr by MAP kinase kinase (MEK). Following activation, the MAP kinases translocate into the nucleus where they phosphorylate several transduction targets, including transcription factors. We have previously identified PAC1 as an immediate-early mitogen-inducible tyrosine phosphatase in nuclei of T cells. Here we present several lines of evidence indicating that PAC1 is a physiologically relevant MAP kinase phosphatase. Recombinant PAC1 in vitro is a dual-specific Thr/Tyr phosphatase with stringent substrate specificity for MAP kinase. Constitutive expression of PAC1 in vivo leads to inhibition of MAP kinase activity normally stimulated by epidermal growth factor, phorbol myristyl acetate, or T-cell receptor crosslinking. The inactivation of MAP kinase by PAC1 results in inhibition of MAP kinase-regulated reporter gene expression.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1994

MAPKs: new JNK expands the group.

Mitogen-activated protein kinases (MAPKs) are activated by dual phosphorylation on threonine and tyrosine in response to a wide array of extracellular stimuli. In the yeast Saccharomyces cerevisiae, a series of extracellular stimuli. In the yeast Saccharomyces cerevisiae, a series of MAPK signal transduction pathways have been demonstrated to control many cellular functions. By contrast, mammalian MAPKs are more poorly understood. However, recent studies have established important roles for three separate groups of mammalian MAPKs, which are characterized by distinct dual phosphorylation motifs. Together, these protein kinases mediate signal transduction in mammalian tissues and control many aspects of cellular physiology.

Amino Acid Sequence↗
PubMed1994

Mitogen-activated protein (MAP) kinase phosphorylation of MAP kinase kinase: determination of phosphorylation sites by mass spectrometry and site-directed mutagenesis.

Mitogen-activated protein kinase kinase (MKK) phosphorylates and activates mitogen-activated protein kinase (MAPK) in response to stimulation of various eukaryotic signaling pathways. Conversely, a recent report showed that MAPK phosphorylates MKK in vitro [Matsuda, S., Gotoh, Y., and Nishida, E. (1993) J. Biol. Chem. 268, 3277-3281]. To gain insight into the function of this feedback phosphorylation, we identified the major sites targeted for phosphorylation by MAPK and examined whether such a modification plays a role in regulating the basal and stimulated MKK activities. Two phosphopeptides generated by tryptic digestion of MAPK-phosphorylated MKK were identified by electrospray ionization mass spectrometry. Cyanogen bromide cleavage also yielded two phosphopeptides whose sequence overlapped with the tryptic phosphopeptides. Both sets of phosphopeptides contained candidate MAPK target sites at Thr292 and Thr386 that fit the consensus sequence ProXThr*Pro. Replacement of either Thr292 or Thr386 with alanine by site-directed mutagenesis reduced the phosphate incorporation respectively to 32 or 75% that of wild type MKK. Replacement of both threonine residues with alanine reduced phosphate incorporation to 2.5% that of wild type enzyme. Comparison of MAPK-phosphorylated vs. unphosphorylated MKK showed no significant differences in basal or Raf-1-stimulated MKK activity. We conclude that the phosphorylation of MKK at Thr292 and Thr386 does not interfere with catalysis in vitro.

Amino Acid Sequence↗
PubMed1994

Signal transduction by tumor necrosis factor mediated by JNK protein kinases.

JNK protein kinases are distantly related to mitogen-activated protein kinases (ERKs) and are activated by dual phosphorylation on Tyr and Thr. The JNK protein kinase group includes the 46-kDa isoform JNK1. Here we describe the molecular cloning of a second member of the JNK group, the 55-kDa protein kinase JNK2. The activities of both JNK isoforms are markedly increased by exposure of cells to UV radiation. Furthermore, JNK protein kinase activation is observed in cells treated with tumor necrosis factor. Although both JNK isoforms phosphorylate the NH2-terminal activation domain of the transcription factor c-Jun, the activity of JNK2 was approximately 10-fold greater than that of JNK1. This difference in c-Jun phosphorylation correlates with increased binding of c-Jun to JNK2 compared with JNK1. The distinct in vitro biochemical properties of these JNK isoforms suggest that they may have different functions in vivo. Evidence in favor of this hypothesis was obtained from the observation that JNK1, but not JNK2, complements a defect in the expression of the mitogen-activated protein kinase HOG1 in the yeast Saccharomyces cerevisiae. Together, these data indicate a role for the JNK group of protein kinases in the signal transduction pathway initiated by proinflammatory cytokines and UV radiation.

Amino Acid Sequence↗
PubMed1994

Genomic loci of human mitogen-activated protein kinases.

Mitogen-activated protein (MAP) kinases [also known as Erks] have been established to function as important mediators of signal transduction by growth factor receptors. Several components of the MAP kinase signal transduction pathway have been demonstrated to be oncogenically activated in malignant tumors. These include growth factor receptors, the GTP-binding protein Ras, and the protein kinase Raf. The genes that encode MAP kinases therefore represent potential targets of carcinogenic insults. Here, we report the genomic loci of three MAP kinase genes are widely distributed within the human genome: p41mapk (Erk2) at 22q11.2; p44mapk (Erk1) at 16p11.2; and p63mapk (Erk3-related) at 18q12-21.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1993

Transactivation of gene expression by Myc is inhibited by mutation at the phosphorylation sites Thr-58 and Ser-62.

The product of the human c-myc protooncogene (Myc) is a sequence-specific DNA binding protein. Here, we demonstrate that the placement of the specific Myc DNA binding site CACGTG upstream of a luciferase reporter gene conferred Myc-stimulated expression that was inhibited by the overexpression of the basic-helix-loop-helix/leucine zipper protein Max. It was observed that Myc was phosphorylated in vivo within the NH2-terminal domain at Thr-58 and Ser-62. Replacement of these phosphorylation sites with Ala residues caused a marked decrease in Myc-stimulated reporter gene expression. In contrast, the replacement of Thr-58 or Ser-62 with an acidic residue (Glu) caused only a small inhibition of transactivation. Together, these data demonstrate that the NH2-terminal phosphorylation sites Thr-58 and Ser-62 are required for high levels of transactivation of gene expression by Myc.

Amino Acid Sequence↗
PubMed1993

cPLA2 is phosphorylated and activated by MAP kinase.

Treatment of cells with agents that stimulate the release of arachidonic acid causes increased serine phosphorylation and activation of cytosolic phospholipase A2 (cPLA2). Here we report that cPLA2 is a substrate for mitogen-activated protein (MAP) kinase. Moreover, phosphorylation by MAP kinase increases the enzymatic activity of cPLA2. The site of cPLA2 phosphorylation by MAP kinase, Ser-505, is identical to the major site of cPLA2 phosphorylation observed in phorbol ester-treated cells. Replacement of Ser-505 with Ala resulted in a mutant cPLA2 that is not a substrate for MAP kinase and causes little or no enhanced agonist-stimulated arachidonate release from intact cells. Taken together, these data indicate that MAP kinase mediates, at least in part, the agonist-induced activation of cPLA2.

Amino Acid Sequence↗
PubMed1993

Mitogen-activated protein kinase stimulation by a tyrosine kinase-negative epidermal growth factor receptor.

Mutation of the epidermal growth factor receptor (EGF-R) within the ATP binding subdomain results in a receptor that lacks tyrosine kinase activity and is defective in signal transduction. However, this kinase-negative EGF-R is able to activate MAP kinase (Campos-Gonzalez, R., and Glenny, J. R. (1992) J. Biol. Chem. 267, 14535-14538). This observation suggests that signal initiation by the EGF-R can occur by a mechanism that is independent of the receptor tyrosine kinase activity. Here, we report that the kinase-negative EGF-R is phosphorylated on tyrosine in EGF-treated cells. The mechanism of tyrosine phosphorylation can be accounted for by the action of EGF to stimulate a protein kinase activity that is associated with the kinase-negative EGF-R. This protein kinase activity is not intrinsic to the receptor and can be separated from the EGF-R by incubation with 0.5 M NaCl. MAP kinase activation by the kinase-negative EGF-R may therefore occur by a mechanism that requires a receptor-associated tyrosine kinase. Thus, it is unnecessary to propose a novel kinase-independent mechanism of signal initiation to account for MAP kinase activation by the kinase-negative EGF-R.

Amino Acid Sequence↗