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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cellular growth control requires the coordination and integration of multiple signaling pathways which are likely to be activated concomitantly. Mitogenic signaling initiated by thyrotropin (TSH) in thyroid cells seems to require two distinct signaling pathways, a cyclic AMP (cAMP)-dependent signaling pathway and a Ras-dependent pathway. This is a paradox, since activated cAMP-dependent protein kinase disrupts Ras-dependent signaling induced by growth factors such as epidermal growth factor and platelet-derived growth factor. This inhibition may occur by preventing Raf-1 protein kinase from binding to Ras, an event thought to be necessary for the activation of Raf-1 and the subsequent activation of the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinases (MEKs) and MAP kinase (MAPK)/ERKs. Here we report that serum-stimulated hyperphosphorylation of Raf-1 was inhibited by TSH treatment of Wistar rat thyroid cells, indicating that in this cell line, as in other cell types, increases in intracellular cAMP levels inhibit activation of downstream kinases targeted by Ras. Ras-stimulated expression of genes containing AP-1 promoter elements was similarly inhibited by TSH. On the other hand, stimulation of thyroid cells with TSH resulted in stimulation of DNA synthesis which was Ras dependent but both Raf-1 and MEK independent. We also show that Ras-stimulated DNA synthesis required the use of this kinase cascade in untreated quiescent cells but not in TSH-treated cells. These data suggest that in TSH-treated thyroid cells, Ras might be able to signal through effectors other than the well-studied cytoplasmic kinase cascade.
Mol Cell Biol 1995 Mar
PMID:Thyrotropin-induced mitogenesis is Ras dependent but appears to bypass the Raf-dependent cytoplasmic kinase cascade. 786 10

Insulin receptor substrate 1 (IRS-1) mediates the activation of a variety of signaling pathways by the insulin and insulin-like growth factor 1 receptors by serving as a docking protein for signaling molecules with SH2 domains. We and others have shown that in response to insulin stimulation IRS-1 binds GRB2/Sos and have proposed that this interaction is important in mediating Ras activation by the insulin receptor. Recently, it has been shown that the interleukin (IL)-4 receptor also phosphorylates IRS-1 and an IRS-1-related molecule, 4PS. Unlike insulin, however, IL-4 fails to activate Ras, extracellular signal-regulated kinases (ERKs), or mitogen-activated protein kinases. We have reconstituted the IL-4 receptor into an insulin-responsive L6 myoblast cell line and have shown that IRS-1 is tyrosine phosphorylated to similar degrees in response to insulin and IL-4 stimulation in this cell line. In agreement with previous findings, IL-4 failed to activate the ERKs in this cell line or to stimulate DNA synthesis, whereas the same responses were activated by insulin. Surprisingly, IL-4's failure to activate ERKs was not due to a failure to stimulate the association of tyrosine-phosphorylated IRS-1 with GRB2/Sos; the amounts of GRB2/Sos associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. Moreover, the amounts of phosphatidylinositol 3-kinase activity associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. In contrast to insulin, however, IL-4 failed to induce tyrosine phosphorylation of Shc or association of Shc with GRB2. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Previous studies have indicated that activation of ERks in this cell line is dependent upon Ras since a dominant-negative Ras (Asn-17) blocks ERK activation by insulin. Our findings, taken in the context of previous work, suggest that binding of GRB2/Sos to Shc may be the predominant mechanism whereby insulin as well as cytokine receptors activate Ras.
Mol Cell Biol 1995 Mar
PMID:Association between GRB2/Sos and insulin receptor substrate 1 is not sufficient for activation of extracellular signal-regulated kinases by interleukin-4: implications for Ras activation by insulin. 786 67

We have cloned a developmentally regulated mitogen-activated protein kinase (extracellular signal-regulated kinase) from Dictyostelium discoideum designated ERK1. Using anti-pTyr antibodies, we show that ERK1 is phosphorylated on tyrosine in vivo and that it will phosphorylate myelin basic protein. The gene expresses two transcripts, one that is preferentially expressed during vegetative growth and early development and one that is induced during the multicellular stages. Developmental Western blots (immunoblots) using anti-ERK1 antibodies indicate that ERK1 is present throughout development. ERK1/lacZ reporter constructs suggest that, in the multicellular stages, the gene is preferentially expressed in a subpopulation of cells scattered throughout the organism, similar to the pattern seen with anterior-like cell markers. Antisense mutagenesis from a derepressible promoter indicates that ERK1 is essential for vegetative growth. Overexpression of ERK1 from either the Actin 15 promoter or the ERK1 promoter results in abnormal morphogenesis starting at the slug stage. Overexpression of ERK1 in null mutants of the phosphotyrosine phosphatase PTP2 results in the production of large aggregation streams and subsequent abnormal morphogenesis that indicate a genetic interaction between ERK1 and PTP2. These cells produce very large aggregation streams that break up into very small mounds that undergo abnormal morphogenesis. The genetic interaction between ERK1 and PTP2 appears to be specific since overexpression of ERK1 in a ptp1- null mutant does not produce the same phenotype. Our results indicate that ERK1 plays an essential role during the growth and differentiation of D. discoideum.
Mol Cell Biol 1994 Oct
PMID:Identification and functional analysis of a developmentally regulated extracellular signal-regulated kinase gene in Dictyostelium discoideum. 793 16

MEK-1 is a dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK). MEK-1 is in turn activated by phosphorylation. Raf and MAPK/extracellular signal-regulated kinase kinase (MEKK) independently phosphorylate and activate MEK-1. Recombinant MEK-1 is also capable of autoactivation. Purified recombinant wild type MEK-1 and a mutant kinase inactive MEK-1 were used as substrates for MEKK, Raf, and autophosphorylation. MEK-1 phosphorylation catalyzed by Raf, MEKK, or autophosphorylation resulted in activation of MEK-1 kinase activity measured by phosphorylation of a mutant kinase inactive MAPK. Phosphoamino acid analysis and peptide mapping identified similar MEK-1 tryptic phosphopeptides after phosphorylation by MEK kinase, Raf, or MEK-1 autophosphorylation. MEK-1 is phosphorylated by MAPK at sites different from that for Raf and MEKK. Phosphorylation of MEK-1 by MAPK does not affect MEK-1 kinase activity. Several phosphorylation sites present in MEK-1 immunoprecipitated from 32P-labeled cells after stimulation with epidermal growth factor were common to the in vitro phosphorylated enzyme. The major site of MAPK phosphorylation in MEK-1 is threonine 292. Mutation of threonine 292 to alanine eliminates 90% of MAPK catalyzed phosphorylation of MEK-1 but does not influence MEK-1 activity. The results demonstrate that MEKK and Raf regulate MEK-1 activity by phosphorylation of common residues and thus, two independent protein kinases converge at MEK-1 to regulate the activity of MAPK.
Mol Biol Cell 1994 Feb
PMID:MEK-1 phosphorylation by MEK kinase, Raf, and mitogen-activated protein kinase: analysis of phosphopeptides and regulation of activity. 801 5

The extracellular signal-regulated kinase ERK2, a member of the protein kinase superfamily, phosphorylates a variety of cellular proteins in response to extracellular signals. ERK2 expressed in Escherichia coli as a fusion protein with the sequence Ala-His6 at the N terminus has low basal activity and very low levels of phosphate incorporation, but can be fully activated. The Ala-His6 ERK2 as expressed in the unphosphorylated form has been crystallized in space group P2(1). The cell constants are a = 49.32 A, b = 71.42 A, c = 61.25 A, and beta = 109.75 degrees, and the crystals diffract to better than 1.8 A resolution.
J Mol Biol 1993 Oct 05
PMID:Crystallization and preliminary X-ray studies of extracellular signal-regulated kinase-2/MAP kinase with an incorporated His-tag. 841 Nov 62

AML1 (also called PEBP2alphaB, CBFA2, or CBFalpha2) is one of the most frequently disrupted genes in chromosome abnormalities seen in human leukemias. It has been reported that AML1 plays several pivotal roles in myeloid hematopoietic differentiation and other biological phenomena, probably through the transcriptional regulation of various relevant genes. Here, we investigated the mechanism of regulation of AML1 functions through signal transduction pathways. The results showed that AML1 is phosphorylated in vivo on two serine residues within the proline-, serine-, and threonine-rich region, with dependence on the activation of extracellular signal-regulated kinase (ERK) and with interleukin-3 stimulation in a hematopoietic cell line. These in vivo phosphorylation sites of AML1 were phosphorylated directly in vitro by ERK. Although differences between wild-type AML1 and phosphorylation site mutants in DNA-binding affinity were not observed, we have shown that ERK-dependent phosphorylation potentiates the transactivation ability of AML1. Furthermore the phosphorylation site mutations reduced the transforming capacity of AML1 in fibroblast cells. These data indicate that AML1 functions are potentially regulated by ERK, which is activated by cytokine and growth factor stimuli. This study provides some important clues for clarifying unidentified facets of the regulatory mechanism of AML1 function.
Mol Cell Biol 1996 Jul
PMID:The extracellular signal-regulated kinase pathway phosphorylates AML1, an acute myeloid leukemia gene product, and potentially regulates its transactivation ability. 866 14

Expression of the ovine P-450 side-chain cleavage enzyme gene (CYP11A1) is stimulated by epidermal growth factor (EGF) through a pathway that involves c-Jun in JEG-3 placental cells. Growth factor signaling involves ras-dependent and ras-independent signaling pathways, which in turn regulate gene transcription through related but distinct mitogen-activated protein kinase pathways (MAPKs) including the extracellular signal-regulated kinases (ERKs) and the stress-activated protein kinases (SAPKs). We investigated the intracellular signaling pathways governing EGF induction of the CYP11A1 promoter. EGF stimulation of the CYP11A1 promoter (4-fold) was reduced 60% by a dominant negative mutant of ras (N17), and 30-40% by antisense ras. EGF induced both ERK and SAPK activity in JEG-3 cells. EGF-induced CYP11A1 promoter activity was reduced 60% by the MEK1 inhibitor PD098059 and 50% by a dominant negative mutant of the ERK-specific regulator MEK1. In contrast, dominant negative mutants of the SAPK-specific activator, SEK1, induced a further increase in EGF-induced CYP11A1 promoter activity. Constitutively active mutants of ras (V12 or L61) increased CYP11A1 promoter activity 6- to 8-fold. Deletion of the EGF response element (EGF-RE) between -92 and -77 bp reduced ras induction by 60%; however, a residual 3-fold induction remained through the proximal -77 bp. Mutation of the EGF-RE AP-1-like sequence in the context of the native promoter reduced CYP11A1 promoter activation by ras 60%. The EGF-RE sequence was sufficient for 6-fold activation by ras in the context of an heterologous thymidine kinase promoter. Candidate transcription factor targets (c-Jun, c-Ets-2) for the ras-signaling cascade were examined for their effects on CYP11A1 promoter activity. Overexpression of c-Jun induced the CYP11A1 promoter through the EGF-RE; however, c-Ets-2 activation of the CYP11A1 promoter (12-fold) required the proximal ras-responsive promoter sequences that are distinct from the EGF/MEK/c-Jun-responsive element. Induction of the CYP11A1 promoter by EGF involves a ras/MEK1/AP-1-dependent pathway that is distinct from induction by ras/c-Ets-2.
Mol Endocrinol 1996 Sep
PMID:Stimulation of the P-450 side chain cleavage enzyme (CYP11A1) promoter through ras- and Ets-2-signaling pathways. 888 43

Mutations in the gene for human fibroblast growth factor receptor 3 (hFGFR3) cause a variety of skeletal dysplasias, including the most common genetic form of dwarfism, achondroplasia (ACH). Evidence indicates that these phenotypes are not due to simple haploinsufficiency of FGFR3 but are more likely related to a role in negatively regulating skeletal growth. The effects of one of these mutations on FGFR3 signaling were examined by constructing chimeric receptors composed of the extracellular domain of human platelet-derived growth factor receptor beta (hPDGFR beta) and the transmembrane and intracellular domains of hFGFR3 or of an ACH (G375C) mutant. Following stable transfection in PC12 cells, which lack platelet-derived growth factor (PDGF) receptors, all clonal cell lines, with either type of chimera, showed strong neurite outgrowth in the presence of PDGF but not in its absence. Antiphosphotyrosine immunoblots showed ligand-dependent autophosphorylation, and both receptor types stimulated strong phosphorylation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase, an event associated with the differentiative response of these cells. In addition, ligand-dependent phosphorylation of phospholipase Cgamma and Shc was also observed. All of these responses were comparable to those observed from ligand activation, such as by nerve growth factor, of the native PC12 cells used to prepare the stable transfectants. The cells with the chimera bearing the ACH mutation were more rapidly responsive to ligand with less sustained MAPK activation, indicative of a preactivated or primed condition and consistent with the view that these mutations weaken ligand control of FGFR3 function. However, the full effect of the mutation likely depends in part on structural features of the extracellular domain. Although FGFR3 has been suggested to act as a negative regulator of long-bone growth in chrondrocytes, it produces differentiative signals similar to those of FGFR1, to which only positive effects have been ascribed, in PC12 cells. Therefore, its regulatory effects on bone growth likely result from cellular contexts and not the induction of a unique FGFR3 signaling pathway.
Mol Cell Biol 1997 Jul
PMID:Chimeras of the native form or achondroplasia mutant (G375C) of human fibroblast growth factor receptor 3 induce ligand-dependent differentiation of PC12 cells. 919 52

Byr2 protein kinase, a homolog of mammalian mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEKK) and Saccharomyces cerevisiae STE11, is required for pheromone-induced sexual differentiation in the fission yeast Schizosaccharomyces pombe. Byr2 functions downstream of Ste4, Ras1, and the membrane-associated receptor-coupled heterotrimeric G-protein alpha subunit, Gpa1. Byr2 has a distinctive N-terminal kinase regulatory domain and a characteristic C-terminal kinase catalytic domain. Ste4 and Ras1 interact with the regulatory domain of Byr2 directly. Here, we define the domains of Byr2 that bind Ste4 and Ras1 and show that the Byr2 regulatory domain binds to the catalytic domain in the two-hybrid system. Using Byr2 mutants, we demonstrate that these direct physical interactions are all required for proper signaling. In particular, the physical association between Byr2 regulatory and catalytic domains appears to result in autoinhibition, the loss of which results in kinase activation. Furthermore, we provide evidence that Shk1, the S. pombe homolog of the STE20 protein kinase, can directly antagonize the Byr2 intramolecular interaction, possibly by phosphorylating Byr2.
Mol Cell Biol 1997 Oct
PMID:Multiple regulatory domains on the Byr2 protein kinase. 931 45

In this report we show that extracellular signal-regulated kinase-1 and -2 (ERK-1 and -2) respond differently to signals that elicit proliferation and/or differentiation of myoblasts using the C2C12 cell line and nondifferentiating mutant NFB4 cells derived from them. Induction of differentiation by withdrawal of serum rendered ERKs in C2C12 myoblasts relatively insensitive to restimulation by serum. Instead, myogenic differentiation of C2C12 cells was associated with sustained activation of ERK-2 dependent on the insulin-like growth factor II (IGF-II) autocrine loop. By contrast, mutant NFB4 cells cultured under the same conditions remained proliferative and demonstrated robust activation of ERKs in response to serum. Similarly, a Gi-dependent signaling pathway induced activation of ERKs in NFB4 cells, but not in C2C12 cells, after stimulation by lysophosphatidic acid (LPA). In NFB4 cells partially rescued by prolonged IGF-I treatment, ERK activity remained responsive to Gi-dependent LPA stimulation, whereas rescue of NFB4 cells by constitutive expression of myogenin or MyoD, associated with activation of the IGF-II autocrine loop, rendered the Gi-signaling pathway refractory to LPA stimulation. Relatively high levels of G(alpha i2) were detected in NFB4 cells and IGF-I treated NFB4 cells, which correlated with responsive Gi signaling. Activation of the IGF-II autocrine loop in C2C12 and NFB4 myoblasts or treatment with IGF-II was associated with loss of G(alpha i2) and inhibition of Gi-dependent signaling. Thus, IGF-I and IGF-II activate distinct signaling cascades, with IGF-II eliciting a stronger differentiation effect correlated with down-regulation of G(alpha i2) protein. Short-term stimulation of NFB4 cells with IGF-I, a mitogenic signal for myoblasts, also induced ERK-1 and -2 activation. Transient stimulation of NFB4 cells with IGF-I while blocking activation of Gi-proteins is with pertussis toxin resulted in preferential activation of ERK-2 characteristic of differentiated C2C12 cells, suggesting that proliferation induced by IGF-I is Gi-dependent and separable from the IGF-I-signaling pathway that leads to differentiation.
Mol Endocrinol 1997 Dec
PMID:Extracellular signal-regulated kinase-1 and -2 respond differently to mitogenic and differentiative signaling pathways in myoblasts. 941 7


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