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At least 991 records · Page 55Linked to original sources
PubMed2002

The proteoglycan NG2 is complexed with alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors by the PDZ glutamate receptor interaction protein (GRIP) in glial progenitor cells. Implications for glial-neuronal signaling.

The proteoglycan NG2 is expressed by immature glial cells in the developing and adult central nervous system. Using the COOH-terminal region of NG2 as bait in a yeast two-hybrid screen, we identified the glutamate receptor interaction protein GRIP1, a multi-PDZ domain protein, as an interacting partner. NG2 exhibits a PDZ binding motif at the extreme COOH terminus which binds to the seventh PDZ domain of GRIP1. In addition to the published expression in neurons, GRIP1 is expressed by immature glial cells. GRIP1 is known to bind to the GluRB subunit of the AMPA glutamate receptor expressed by subpopulations of neurons and immature glial cells. In cultures of primary oligodendrocytes, cells coexpress GluRB and NG2. A complex of NG2, GRIP1, and GluRB can be precipitated from transfected mammalian cells and from cultures of primary oligodendrocytes. Furthermore, NG2 and GRIP can be coprecipitated from developing brain tissue. These data suggest that GRIP1 acts as a scaffolding molecule clustering NG2 and AMPA receptors in immature glia. In view of the presence of synaptic contacts between neurons and NG2-positive glial cells in the hippocampus and the close association of NG2-expressing glial cells with axons, we suggest a role for the NG2.AMPA receptor complex in glial-neuronal recognition and signaling.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Is an ATPase involved in uncoating of plasma membrane adaptor complex AP-2?

The coat of clathrin-coated vesicles mostly consists of clathrin and adaptor complexes AP-1 or AP-2. Clathrin is released from the vesicles in an ATP-dependent fashion prior to their fusion with endosomes. In the present study we found that ATP strongly inhibits in vitro binding of cytosolic AP-2 to membranes of stripped vesicles, and promotes the release of endogenous AP-2 from clathrin-deprived coated vesicles. Both effects required hydrolysis of ATP. In contrast, binding of AP-1 to stripped vesicles was not affected by ATP, but was enhanced by GTP-gamma-S. These results point to an ATPase that promotes the release of AP-2 from clathrin-coated vesicles.

Acyl Coenzyme A↗
PubMed2001

Mapping the Zap-70 phosphorylation sites on LAT (linker for activation of T cells) required for recruitment and activation of signalling proteins in T cells.

T-cell-receptor (TCR)-mediated LAT (linker for activation of T cells) phosphorylation is critical for the membrane recruitment of signalling complexes required for T-cell activation. Although tyrosine phosphorylation of LAT is required for recruitment and activation of signalling proteins, the molecular mechanism associated with this event is unclear. In the present study we reconstituted the LAT signalling pathway by demonstrating that a direct tyrosine phosphorylation of LAT with activated protein-tyrosine kinase Zap70 is necessary and sufficient for the association and activation of signalling proteins. Zap-70 efficiently phosphorylates LAT on tyrosine residues at positions 226, 191, 171, 132 and 127. By substituting these tyrosine residues in LAT with phenylalanine and by utilizing phosphorylated peptides derived from these sites, we mapped the tyrosine residues in LAT required for the direct interaction and activation of Vav, p85/p110alpha and phospholipase Cgamma1 (PLCgamma1). Our results indicate that Tyr(226) and Tyr(191) are required for Vav binding, whereas Tyr(171) and Tyr(132) are necessary for association and activation of phosphoinositide 3-kinase activity and PLCgamma1 respectively. Furthermore, by expression of LAT mutants in LAT-deficient T cells, we demonstrate that Tyr(191) and Tyr(171) are required for T-cell activation and Tyr(132) is required for the activation of PLCgamma1 and Ras signalling pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Short-chain fatty acids induce gamma-globin gene expression by displacement of a HDAC3-NCoR repressor complex.

High-level induction of fetal (gamma) globin gene expression for therapy of beta-hemoglobinopathies likely requires local chromatin modification and dissociation of repressor complexes for gamma-globin promoter activation. A novel gamma-globin-inducing short-chain fatty acid derivative (SCFAD), RB7, which was identified through computational modeling, produced a 6-fold induction in a reporter assay that detects only strong inducers of the gamma-globin gene promoter and in cultured human erythroid progenitors. To elucidate the molecular mechanisms used by high-potency SCFADs, chromatin immunoprecipitation (ChIP) assays performed at the human gamma- and beta-globin gene promoters in GM979 cells and in erythroid progenitors demonstrate that RB7 and butyrate induce dissociation of HDAC3 (but not HDAC1 or HDAC2) and its adaptor protein NCoR, specifically from the gamma-globin gene promoter. A coincident and proportional recruitment of RNA polymerase II to the gamma-globin gene promoter was observed with exposure to these gamma-globin inducers. Knockdown of HDAC3 by siRNA induced transcription of the gamma-globin gene promoter, demonstrating that displacement of HDAC3 from the gamma-globin gene promoter by the SCFAD is sufficient to induce gamma-globin gene expression. These studies demonstrate new dynamic alterations in transcriptional regulatory complexes associated with SCFAD-induced activation of the gamma-globin gene and provide a specific molecular target for potential therapeutic intervention.

Adult↗
PubMed1997

Mapping of the molecular determinants involved in the interaction between eps15 and AP-2.

eps15, a substrate for the epidermal growth factor receptor and other receptor tyrosine kinases, possesses a discrete domain structure with protein-binding properties. It interacts with a number of cellular proteins through an evolutionarily conserved protein-binding domain, the eps15 homology domain, located in its NH2-terminal region. In addition, a proline-rich region, located in the COOH-terminal portion of eps15, can bind to the Src homology 3 domain of the crk proto-oncogene product in vitro. Recently, coimmunoprecipitation between eps15 and AP-2, a major component of coated pits, was reported. Here, we characterize the molecular determinants of the eps15/AP-2 interaction. The AP-2 binding region of eps15 is localized in its COOH-terminal region and spans approximately 80 amino acids. At least three molecular determinants, located at residues 650-660, 680-690, and 720-730, are involved in the binding. AP-2 binds to eps15 through its alpha subunit (alpha-adaptin); in particular, the COOH-terminal region of alpha-adaptin, the so-called alpha-ear, contains the eps15 binding region.

3T3 Cells↗
PubMed1999

Activation of nuclear factor of activated T cells-(NFAT) and activating protein 1 (AP-1) by oncogenic 70Z Cbl requires an intact phosphotyrosine binding domain but not Crk(L) or p85 phosphatidylinositol 3-kinase association.

The Cbl proto-oncogene product is a complex adapter protein that functions as a negative regulator of protein tyrosine kinases. It is rapidly tyrosine-phosphorylated and associates with Crk(L) and p85 phosphatidylinositol 3-kinase (PI3K) upon engagement of numerous receptors linked to tyrosine kinases. Elucidation of the mechanism(s) underlying Cbl deregulation is therefore of considerable interest. The 70Z Cbl oncoprotein shows increased baseline tyrosine phosphorylation in fibroblasts and enhances nuclear factor of activated T cells (NFAT) activity in Jurkat T cells. Its transforming ability has been proposed to relate to its increased phosphotyrosine content. We demonstrate that 70Z Cbl shows increased basal and activation-induced tyrosine phosphorylation and association with Crk(L) and p85 PI3K in Jurkat T cells. 70Z Cbl, however, retains the ability to enhance NFAT and activating protein 1 (AP1) activity in the absence of Crk(L)/p85 PI3K association. In contrast, the G306E mutation, which inactivates the phosphotyrosine binding domain of Cbl, blocks NFAT/AP1 activation by 70Z Cbl. We conclude that 70Z Cbl-induced NFAT/AP1 activation requires the phosphotyrosine binding domain but not Crk(L)/p85 PI3K association. We hypothesize that 70Z Cbl acts as a dominant negative by blocking the negative regulatory function of the Cbl phosphotyrosine binding domain on protein-tyrosine kinases.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Glucocorticoids modulate amino acid-induced translation initiation in human skeletal muscle.

Amino acids are unique anabolic agents in that they nutritively signal to mRNA translation initiation and serve as substrates for protein synthesis in skeletal muscle. Glucocorticoid excess antagonizes the anabolic action of amino acids on protein synthesis in laboratory animals. To examine whether excessive glucocorticoids modulate mixed amino acid-signaled translation initiation in human skeletal muscle, we infused an amino acid mixture (10% Travasol) systemically to 16 young healthy male volunteers for 6 h in the absence (n = 8) or presence (n = 8) of glucocorticoid excess (dexamethasone 2 mg orally every 6 h for 3 days). Vastus lateralis muscles were biopsied before and after amino acid infusion, and the phosphorylation of eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1), ribosomal protein S6 kinase (p70(S6K)), and eIF2alpha and the guanine nucleotide exchange activity of eIF2B were measured. Systemic infusion of mixed amino acids significantly stimulated the phosphorylation of 4E-BP1 (P < 0.04) and p70(S6K) (P < 0.001) and the dephosphorylation of eIF2alpha (P < 0.003) in the control group. Dexamethasone treatment did not alter the basal phosphorylation state of 4E-BP1, p70(S6K), or eIF2alpha; however, it abrogated the stimulatory effect of amino acid infusion on the phosphorylation of 4E-BP1 (P = 0.31) without affecting amino acid-induced phosphorylation of p70(S6K) (P = 0.002) or dephosphorylation of eIF2alpha (P = 0.003). Neither amino acid nor dexamethasone treatment altered the guanine nucleotide exchange activity of eIF2B. We conclude that changes of amino acid concentrations within the physiological range stimulate mRNA translation by enhancing the binding of mRNA to the 43S preinitiation complex, and the activity of p70(S6K) and glucocorticoid excess blocks the former action in vivo in human skeletal muscle.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Organization of the mouse Ruk locus and expression of isoforms in mouse tissues.

Ruk is a recently identified gene with a complex pattern of expression in mammalian cells and tissues. Multiple Ruk transcripts and several protein isoforms have been detected in various types of cells. Ruk proteins have multidomain organization characteristic of adapter proteins involved in regulation of signal transduction. Interaction of some Ruk isoforms with several signalling proteins, including the p85 regulatory subunit of the Class IA PI 3-kinase, c-Cbl and Grb2, has been demonstrated. Ruk(l), an isoform with three SH3 domains, inhibits lipid kinase activity of the PI 3-kinase in vitro; overexpression of this protein induces apoptotic cell death of primary neurons in culture and changes in membrane trafficking in other cultured cells. However, shorter isoforms of Ruk block pro-apoptotic effect of Ruk(l), suggesting that expression of different combinations of Ruk proteins in cells could be involved in the regulation of their survival and other intracellular processes. To understand the mechanism of differential expression of Ruk proteins we studied organization of the mouse Ruk gene and its transcripts. Twenty-four exons of the Ruk gene span over 320 kb of the mouse chromosome X. Analysis of cDNA clones, ESTs and products of RT-PCR amplifications with different combinations of primers revealed how alternative splicing and promoter usage generate a variety of Ruk transcripts and encoded protein isoforms in different mouse tissues.

Adaptor Proteins, Signal Transducing↗
PubMed1996

The role of DNA mismatch repair in platinum drug resistance.

Loss of DNA mismatch repair occurs in many types of tumors. The effect of the loss of DNA mismatch repair activity on sensitivity to cisplatin and a panel of analogues was tested using two pairs of cell lines proficient or deficient in this function. HCT116+ch2, a human colon cancer cell line deficient in hMLH1, was 2.1-fold resistant to cisplatin and 1.3-fold resistant to carboplatin when compared to a subline complemented with chromosome 3 expressing a wild-type copy of hMLH1. Likewise, the human endometrial cancer cell line HEC59, which is deficient in hMSH2, was 1.8-fold resistant to cisplatin and 1.5-fold resistant to carboplatin when compared to a subline complemented with chromosome 2 with a wild-type hMSH2. In contrast to cisplatin and carboplatin, which form the same types of adducts in DNA, there was no difference in sensitivity between the DNA mismatch repair-proficient and -deficient cell lines for oxaliplatin, tetraplatin, transplatin, JM335, or JM216. The formation of protein-DNA complexes that contained hMSH2 and hMLH1 was documented by mobility shift assay when nuclear extracts were incubated with DNA platinated with cisplatin but not with oxaliplatin. These results demonstrate a correlation between failure of the DNA mismatch repair proteins to recognize the platinum adduct and low-level resistance, suggesting a role for the DNA mismatch repair system in generating signals that contribute to the generation of apoptotic activity. They also identify the use of drugs whose adducts are not recognized as a strategy for circumventing resistance due to loss of DNA mismatch repair.

Adaptor Proteins, Signal Transducing↗
PubMed2002

IRAK-M is a negative regulator of Toll-like receptor signaling.

Toll-like receptors (TLRs) detect microorganisms and protect multicellular organisms from infection. TLRs transduce their signals through MyD88 and the serine/threonine kinase IRAK. The IRAK family consists of two active kinases, IRAK and IRAK-4, and two inactive kinases, IRAK-2 and IRAK-M. IRAK-M expression is restricted to monocytes/macrophages, whereas other IRAKs are ubiquitous. We show here that IRAK-M is induced upon TLR stimulation and negatively regulates TLR signaling. IRAK-M prevented dissociation of IRAK and IRAK-4 from MyD88 and formation of IRAK-TRAF6 complexes. IRAK-M(-/-) cells exhibited increased cytokine production upon TLR/IL-1 stimulation and bacterial challenge, and IRAK-M(-/-) mice showed increased inflammatory responses to bacterial infection. Endotoxin tolerance, a protection mechanism against endotoxin shock, was significantly reduced in IRAK-M(-/-) cells. Thus, IRAK-M regulates TLR signaling and innate immune homeostasis.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Insulin regulation of protein translation repressor 4E-BP1, an eIF4E-binding protein, in renal epithelial cells.

BACKGROUND: Augmented protein translation by insulin involves activation of eukaryotic initiation factor 4E (eIF4E) that follows release of eIF4E from a heterodimeric complex by phosphorylation of its inhibitory binding protein, 4E-BP1. We examined insulin regulation of 4E-BP1 phosphorylation in murine proximal tubular epithelial cells. METHODS AND RESULTS: Insulin (1 nmol/L) increased de novo protein synthesis by 58 +/- 11% (P < 0.001). Insulin also augmented 4E-BP1 phosphorylation and phosphatidylinositol 3-kinase (PI 3-kinase) activity in antiphosphotyrosine immunoprecipitates. This could be prevented by PI 3-kinase inhibitors, Wortmannin, and LY294002. Insulin also activated Akt that lies downstream of PI 3-kinase. Rapamycin abrogated 4E-BP1 phosphorylation in response to insulin, suggesting involvement of mammalian target of rapamycin (mTOR), a kinase downstream of Akt. Insulin-stimulated phosphorylation of 4E-BP1 was also inhibited by PD098059, implying involvement of Erk-1/-2 mitogen-activated protein (MAP) kinase. An increase in Erk-1/-2 type MAP kinase activity by insulin was directly confirmed in an immunokinase assay and was found to be PI 3-kinase dependent. CONCLUSIONS: In proximal tubular epithelial cells, insulin augments 4E-BP1 phosphorylation, which is PI 3-kinase and mTOR dependent. The requirement for Erk-1/-2 MAP kinase activation for 4E-BP1 phosphorylation by insulin suggests a cross-talk between PI 3-kinase and Erk-1/-2-type MAP kinase pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Dexamethasone protects primary cultured hepatocytes from death receptor-mediated apoptosis by upregulation of cFLIP.

Dexamethasone (DEX) pretreatment protected hepatocytes from TNF-alpha plus actinomycin D (ActD)-induced apoptosis by suppressing caspase-8 activation and the mitochondria-dependent apoptosis pathway. DEX treatment upregulated cellular FLICE inhibitory protein (cFLIP) expression, but did not alter the protein levels of Bcl-2, Bcl-xL, Mcl-1, and cIAP as well as Akt activation. The increased cFLIP mRNA level by DEX was inhibited by ActD, indicating that DEX upregulates cFLIP expression at the transcriptional step. DEX also inhibited Jo2-mediated hepatocyte apoptosis by blocking the formation of the death-inducing signaling complex and caspase-8 activation. Specific downregulation of cFLIP expression using siRNA reversed the antiapoptotic effect of DEX by increasing caspase-8 activation. Moreover, DEX administration into mice increased cFLIP expression in the liver and prevented Jo2-induced hepatic injury by inhibiting caspase-8 and -3 activities. Our results indicate that DEX exerts a protective role in death receptor-induced in vitro and in vivo hepatocyte apoptosis by upregulating cFLIP expression.

Animals↗
PubMed2002

Fibroblast growth factor homologous factors and the islet brain-2 scaffold protein regulate activation of a stress-activated protein kinase.

Fibroblast growth factor homologous factors (FHFs) form native intracellular complexes with the mitogen-activated protein kinase (MAPK) scaffold protein islet-brain 2 (IB2) in adult brain. FHF binding to IB2 facilitates recruitment of the MAPK p38delta (SAPK4), while failing to stimulate binding of JNK, the preferred kinase of the related scaffold IB1 (JIP-1). We now report further biochemical evidence supporting FHFs as regulators of IB2 scaffold activity. Mixed lineage kinase 3 (MLK3) and IB2 synergistically activate p38delta but not the MAPKs JNK-1 and p38alpha. Binding of p38delta to IB2 is mediated by the carboxyl-terminal half of the scaffold (IB2(Delta1-436)). FHF2 also binds weakly to IB2(Delta1-436) and can thereby increase p38delta interaction with IB2(Delta1-436). FHF-induced recruitment of p38delta to IB2 is accompanied by increased levels of activated p38delta, and synergistic activation of p38delta by MLK3 and IB2 is further enhanced by FHF2. Consistent with a role for FHFs as signaling molecules, FHF2 isolated from rat brain is serine/threonine-phosphorylated, and FHF can serve as a substrate for p38delta in vitro. These results support the existence of a signaling module in which IB2 scaffolds a MLK3/MKK/p38delta kinase cascade. FHFs aid in recruitment of p38 to IB2 and may serve as kinase substrates.

Adaptor Proteins, Signal Transducing↗
PubMed2003

TIR-containing adapter molecule (TICAM)-2, a bridging adapter recruiting to toll-like receptor 4 TICAM-1 that induces interferon-beta.

Lipopolysaccharide (LPS) is an agonist for Toll-like receptor (TLR) 4 and expresses many genes including NF-kappaB- and interferon regulatory factor (IRF)-3/IFN-inducible genes in macrophages and dendritic cells (DCs). TICAM-1/TRIF was identified as an adapter that facilitates activation of IRF-3 followed by expression of interferon (IFN)-beta genes in TLR3 signaling, but TICAM-1 does not directly bind TLR4. Although MyD88 and Mal/TIRAP adapters functions downstream of TLR4, DC maturation and IFN-beta induction are independent of MyD88 and Mal/TIRAP. In this investigation, we report the identification of a novel adapter, TICAM-2, that physically bridges TLR4 and TICAM-1 and functionally transmits LPS-TLR4 signaling to TICAM-1, which in turn activates IRF-3. In its structural features, TICAM-2 resembled Mal/TIRAP, an adapter that links TLR2/4 and MyD88. However, TICAM-2 per se exhibited minimal ability to activate NF-kappaB and the IFN-beta promoter. Hence, in LPS signaling TLR4 recruits two types of adapters, TIRAP and TICAM-2, to its cytoplasmic domain that are indirectly connected to two effective adapters, MyD88 and TICAM-1, respectively. We conclude that for LPS-TLR4-mediated activation of IFN-beta, the adapter complex of TICAM-2 and TICAM-1 plays a crucial role. This results in the construction of MyD88-dependent and -independent pathways separately downstream of the two distinct adapters.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Localization of dynein light chains 1 and 2 and their pro-apoptotic ligands.

The dynein and myosin V motor complexes are multi-protein structures that function to transport molecules and organelles within the cell. DLC (dynein light-chain) proteins, found as components of both dynein and myosin V motor complexes, connect the complexes to their cargoes. One of the roles of these motor complexes is to selectively sequester the pro-apoptotic 'BH3-only' (Bcl-2 homology 3-only) proteins, Bim (Bcl-2-interacting mediator of cell death) and Bmf (Bcl-2-modifying factor), and so regulate their cell death-inducing function. In vivo DLC2 is found exclusively as a component of the myosin V motor complex and Bmf binds DLC2 selectively. On the other hand, Bim interacts with DLC1 (LC8), an integral component of the dynein motor complex. The two DLCs share 93% sequence identity yet show unambiguous in vivo specificity for their respective BH3-only ligands. To investigate this specificity the three-dimensional solution structure of DLC2 was elucidated using NMR spectroscopy. In vitro structural and mutagenesis studies show that Bmf and Bim have identical binding characteristics to recombinant DLC2 or DLC1. Thus the selectivity shown by Bmf and Bim for binding DLC1 or DLC2, respectively, does not reside in their DLC-binding domains. Remarkably, mutational analysis of DLC1 and DLC2 indicates that a single surface residue (residue 41) determines the specific localization of DLCs with their respective motor complexes. These results suggest a molecular mechanism for the specific compartmentalization of DLCs and their pro-apoptotic cargoes and implicate other protein(s) in defining the specificity between the cargoes and the DLC proteins.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Regulation of Ly49D/DAP12 signal transduction by Src-family kinases and CD45.

Activating, DAP12-coupled members of the Ly-49 family of NK cell receptors help control viral infections in mice. However, the kinases and/or phosphatases mediating tyrosine phosphorylation of Ly-49D-associated DAP12 have not been elucidated. In this study, we show for the first time that Src family tyrosine kinases are physically and functionally associated with Ly-49D/DAP12 signaling in murine NK cells. Specifically, we demonstrate the following: 1) inhibition of Src family kinases suppresses DAP12 phosphorylation and downstream DAP12 signals; 2) both Fyn and Lck are capable of phosphorylating DAP12; and 3) both kinases coimmunoprecipitate with the Ly-49D/DAP12 complex in NK cells. Although we detect enhanced phosphorylation of Fyn upon Ly-49D cross-linking in NK cells, Ly-49D-mediated events in both Fyn-/- and Fyn/Lck-/- mice appear normal, reinforcing the theme of redundancy in the ability of Src family kinases to initiate activation events. In contrast to disruption of specific Src family enzymes, Ly-49D/DAP12-mediated calcium mobilization and cytokine production by CD45 null NK cells are defective. Although others have ascribed the effects of CD45 mutation solely on the suppression of Src family activity, we demonstrate in this study that DAP12 is hyperphosphorylated in CD45 null NK cells, resulting in uncoordinated tyrosine-mediated signaling upon Ly-49D ligation. Therefore, although our data are consistent with a Src kinase activity proximally within DAP12 signaling, DAP12 also appears to be a substrate of CD45, suggesting a more complex role for this phosphatase than has been reported previously.

Adaptor Proteins, Signal Transducing↗
PubMed2003

The inhibitory gamma subunit of the type 6 retinal cGMP phosphodiesterase functions to link c-Src and G-protein-coupled receptor kinase 2 in a signaling unit that regulates p42/p44 mitogen-activated protein kinase by epidermal growth factor.

The inhibitory gamma subunit of the retinal photoreceptor type 6 cGMP phosphodiesterase (PDEgamma) is phosphorylated by G-protein-coupled receptor kinase 2 on threonine 62 and regulates the epidermal growth factor- dependent stimulation of p42/p44 mitogen-activated protein kinase in human embryonic kidney 293 cells. We report here that PDEgamma is in a pre-formed complex with c-Src and that stimulation of cells with epidermal growth factor promotes the association of GRK2 with this complex. c-Src has a critical role in the stimulation of the p42/p44 mitogen-activated protein kinase cascade by epidermal growth factor, because c-Src inhibitors block the activation of this kinase by the growth factor. Mutation of Thr-62 (to Ala) in PDEgamma produced a GRK2 phosphorylation-resistant mutant that was less effective in associating with GRK2 in response to epidermal growth factor and did not potentiate the stimulation of p42/p44 mitogen-activated protein kinase by this growth factor. The transcript for a short splice variant version of PDEgamma lacking the Thr-62 phosphorylation site is also expressed in certain mammalian cells and, in common with the Thr-62 mutant, failed to potentiate the stimulatory effect of epidermal growth factor on p42/p44 mitogen-activated protein kinase. The mutation of Thr-22 (to Ala) in PDEgamma, which is a site for phosphorylation by p42/p44 mitogen-activated protein kinase, resulted in a prolonged activation of p42/p44 mitogen-activated protein kinase by epidermal growth factor, suggesting a role for this phosphorylation event in the negative feedback control of PDEgamma.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1998

Two CD95 (APO-1/Fas) signaling pathways.

We have identified two cell types, each using almost exclusively one of two different CD95 (APO-1/Fas) signaling pathways. In type I cells, caspase-8 was activated within seconds and caspase-3 within 30 min of receptor engagement, whereas in type II cells cleavage of both caspases was delayed for approximately 60 min. However, both type I and type II cells showed similar kinetics of CD95-mediated apoptosis and loss of mitochondrial transmembrane potential (DeltaPsim). Upon CD95 triggering, all mitochondrial apoptogenic activities were blocked by Bcl-2 or Bcl-xL overexpression in both cell types. However, in type II but not type I cells, overexpression of Bcl-2 or Bcl-xL blocked caspase-8 and caspase-3 activation as well as apoptosis. In type I cells, induction of apoptosis was accompanied by activation of large amounts of caspase-8 by the death-inducing signaling complex (DISC), whereas in type II cells DISC formation was strongly reduced and activation of caspase-8 and caspase-3 occurred following the loss of DeltaPsim. Overexpression of caspase-3 in the caspase-3-negative cell line MCF7-Fas, normally resistant to CD95-mediated apoptosis by overexpression of Bcl-xL, converted these cells into true type I cells in which apoptosis was no longer inhibited by Bcl-xL. In summary, in the presence of caspase-3 the amount of active caspase-8 generated at the DISC determines whether a mitochondria-independent apoptosis pathway is used (type I cells) or not (type II cells).

Adaptor Proteins, Signal Transducing↗