Biomedical subjects

G R Guy

Publications and source records attributed to G R Guy.

At least 19 recordsLinked to original sources

PubMed2006

BNIP-Salpha induces cell rounding and apoptosis by displacing p50RhoGAP and facilitating RhoA activation via its unique motifs in the BNIP-2 and Cdc42GAP homology domain.

Changes in cell morphology are linked to many cellular events including cytokinesis, differentiation, migration and apoptosis. We recently showed that BNIP-Salpha induced cell rounding that leads to apoptosis via its BNIP-2 and Cdc42GAP Homology (BCH) domain, but the underlying mechanism has not been determined. Here, we have identified a unique region (amino acid 133-177) of the BNIP-Salpha BCH domain that targets RhoA, but not Cdc42 or Rac1 and only the dominant-negative form of RhoA could prevent the resultant cell rounding and apoptotic effect. The RhoA-binding region consists of two parts; one region (residues 133-147) that shows some homology to part of the RhoA switch I region and an adjacent sequence (residues 148-177) that resembles the REM class I RhoA-binding motif. The sequence 133-147 is also necessary for its heterophilic interaction with the BCH domain of the Rho GTPase-activating protein, p50RhoGAP/Cdc42GAP. These overlapping motifs allow tripartite competition such that overexpression of BNIP-Salpha could reduce p50RhoGAP binding to RhoA and restore RhoA activation. Furthermore, BNIP-Salpha mutants lacking the RhoA-binding motif completely failed to induce cell rounding and apoptosis. Therefore, via unique binding motifs within its BCH domain, BNIP-Salpha could interact and activate RhoA while preventing its inhibition by p50RhoGAP. This concerted mechanism could allow effective propagation of the RhoA pathway for cell rounding and apoptosis.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Identification and characterization of mErk5-T, a novel Erk5/Bmk1 splice variant.

Extracellular regulated kinase 5 (ERK5) is an unusually large member of the MAP kinase family of signaling molecules that plays an important role in cellular proliferation, differentiation and survival. Recently, three transcriptional variants of murine Erk5 were described (mErk5-a, -b and -c) that result from alternate splicing across introns 1 and/or 2, the net effect of which is translation of a peptide that lacks the kinase domain. It has been demonstrated that expression of mErk5-b and -c impinge on the function of the full length mErk5 protein product via a dominant negative effect. Here, we report the identification of another murine Erk5 splice variant and the orthologous human transcript that arise due to alternate splicing of intron 4. Failure to splice out intron 4 introduces a premature in-frame stop codon that directs translation of a peptide lacking the nuclear localization signal (NLS) and proline-rich region (PR). Experimental characterization demonstrated that like mERK5, mERK5-T becomes phosphorylated by co-expression with a constitutively active mMEK5 (mMEK5DD), and is able to coimmunoprecipitate with both itself and mERK5. Unlike mERK5, however, activated ERK5-T is unable to translocate from the cytoplasm to the nucleus in HeLaS3 cells, causing the retention of active mERK5 in the cytoplasm. Taken together with previous reports of domain content modification of ERK5 via alternate splicing, these observations add to the suggestion that regulation of ERK5 signaling may be mediated, at least in part, at the level of RNA processing.

Alternative Splicing↗

PubMed2001

Stimulation of phosphatidylinositol 3-kinase by fibroblast growth factor receptors is mediated by coordinated recruitment of multiple docking proteins.

The docking protein FRS2 is a major downstream effector that links fibroblast growth factor (FGF) and nerve growth factor receptors with the Ras/mitogen-activated protein kinase signaling cascade. In this report, we demonstrate that FRS2 also plays a pivotal role in FGF-induced recruitment and activation of phosphatidylinositol 3-kinase (PI3-kinase). We demonstrate that tyrosine phosphorylation of FRS2alpha leads to Grb2-mediated complex formation with the docking protein Gab1 and its tyrosine phosphorylation, resulting in the recruitment and activation of PI3-kinase. Furthermore, Grb2 bound to tyrosine-phosphorylated FRS2 through its SH2 domain interacts primarily via its carboxyl-terminal SH3 domain with a proline-rich region in Gab1 and via its amino-terminal SH3 domain with the nucleotide exchange factor Sos1. Assembly of FRS2alpha:Grb2:Gab1 complex induced by FGF stimulation results in activation of PI3-kinase and downstream effector proteins such as the S/T kinase Akt, whose cellular localization and activity are regulated by products of PI3-kinase. These experiments reveal a unique mechanism for generation of signal diversity by growth factor-induced coordinated assembly of a multidocking protein complex that can activate the Ras/mitogen-activated protein kinase cascade to induce cell proliferation and differentiation, and PI3-kinase to activate a mediator of a cell survival pathway.

3T3 Cells↗

PubMed2000

The BNIP-2 and Cdc42GAP homology domain of BNIP-2 mediates its homophilic association and heterophilic interaction with Cdc42GAP.

We recently showed that BNIP-2 is a putative substrate of the fibroblast growth factor receptor tyrosine kinase and it possesses GTPase-activating activity toward the small GTPase, Cdc42. The carboxyl terminus of BNIP-2 shares high homology to the non-catalytic domain of Cdc42GAP, termed BCH (for BNIP-2 and Cdc42GAP homology) domain. Despite the lack of obvious homology to any known catalytic domains of GTPase-activating proteins (GAPs), the BCH domain of BNIP-2 bound Cdc42 and stimulated the GTPase activity via a novel arginine-patch motif similar to that employed by one contributing partner in a Cdc42 homodimer. In contrast, the BCH domain of Cdc42GAP, although it can bind Cdc42, is catalytically inactive. This raises the possibility that these domains might have other roles in the cell. Using glutathione S-transferase recombinant proteins, immunoprecipitation studies, and yeast two-hybrid assays, it was found that BNIP-2 and Cdc42GAP could form homo and hetero complexes via their conserved BCH domains. Molecular modeling of the BNIP-2 BCH homodimer complex and subsequent deletion mutagenesis helped to identify the region (217)RRKMP(221) as the major BCH interaction site within BNIP-2. In comparison, deletion of either the arginine-patch (235)RRLRK(239) (necessary for GAP activity) or region (288)EYV(290) (a Cdc42 binding sequence) had no effect on BCH-BCH interaction. Extensive data base searches showed that the BCH domain is highly conserved across species. The results suggest that BCH domains of BNIP-2 and Cdc42GAP represent a novel protein-protein interaction domain that could potentially determine and/or modify the physiological roles of these molecules.

Amino Acid Sequence↗

PubMed2000

Evidence for direct interaction between Sprouty and Cbl.

Sprouty (SPRY) was first identified in a genetic screen in Drosophila as an antagonist of fibroblast and epidermal growth factor receptors and Sevenless signaling, seemingly by inhibiting the receptor tyrosine kinase (RTK)/Ras/MAPK pathway. To date, four mammalian Sprouty genes have been identified; the primary sequences of the gene products share a well conserved cysteine-rich C-terminal domain with their Drosophila counterpart. The N-terminal regions do not, however, exhibit a large degree of homology. This study was aimed at identifying proteins with which human SPRY2 (hSPRY2) interacts in an attempt to understand the mechanism by which Sprouty proteins exert their down-regulatory effects. Here, we demonstrate that hSPRY2 associates directly with c-Cbl, a known down-regulator of RTK signaling. A short sequence in the N terminus of hSPRY2 was found to bind directly to the Ring finger domain of c-Cbl. Parallel binding was apparent between the Drosophila homologs of Sprouty and Cbl, with cross-species associations occurring at least in vitro. Coexpression of hSPRY2 abrogated an increase in the rate of epidermal growth factor receptor internalization induced by c-Cbl, whereas a mutant hSPRY2 protein unable to bind c-Cbl showed no such effect. Our results suggest that one function of hSPRY2 in signaling processes downstream of RTKs may be to modulate c-Cbl physiological function such as that seen with receptor-mediated endocytosis.

Amino Acid Sequence↗

PubMed2000

Sprouty proteins are targeted to membrane ruffles upon growth factor receptor tyrosine kinase activation. Identification of a novel translocation domain.

Sprouty (Spry) was first identified in a genetic screen in Drosophila to be an antagonist of fibroblast growth factor and epidermal growth factor (EGF) signaling, seemingly by inhibiting the Ras/MAP kinase pathway. Data base searches lead to the identification and cloning of, to date, four mammalian sprouty genes. The primary sequences of the mammalian sprouty gene products share a well conserved cysteine-rich C-terminal domain with the Drosophila protein. The N-terminal regions, however, do not exhibit significant homology. This study aimed at determining the disposition of Spry proteins in intact cells before and after stimulation of the EGF receptor tyrosine kinase. Full-length or deletion mutants of Spry, tagged at the N termini with the FLAG-epitope, were expressed in COS-1 cells by transient transfection and analyzed by immunofluorescence microscopy before and after EGF stimulation of the cells. In unstimulated cells, the Spry proteins were distributed throughout the cytosol except for human Sprouty2 (hSpry2), which, although generally located in the cytosol, co-localized with microtubules. In all cases, the Spry proteins underwent rapid translocation to membrane ruffles following EGF stimulation. The optimal translocation domain was identified by deletion and immunofluorescence analysis to be a highly conserved 105-amino acid domain in the C-terminal half of the hSpry2 protein. The translocation of this conserved domain, based on hSpry2 data, was independent of the activation of phosphatidylinositol-3 kinase.

Amino Acid Sequence↗

PubMed2000

Evidence for a novel Cdc42GAP domain at the carboxyl terminus of BNIP-2.

We recently identified BNIP-2, a previously cloned Bcl-2- and E1B-associated protein, as a putative substrate of the FGF receptor tyrosine kinase and showed that it possesses GTPase-activating activity toward Cdc42 despite the lack of homology to previously described catalytic domains of GTPase-activating proteins (GAPs). BNIP-2 contains many arginine residues at the carboxyl terminus, which includes the region of homology to the noncatalytic domain of Cdc42GAP, termed BNIP-2 and Cdc42GAP homology (BCH) domain. Using BNIP-2 glutathione S-transferase recombinants, it was found that its BCH bound Cdc42, and contributed the GAP activity. This domain was predicted to fold into alpha-helical bundles similar to the topology of the catalytic GAP domain of Cdc42GAP. Alignment of exposed arginine residues in this domain helped to identify Arg-235 and Arg-238 as good candidates for catalysis. Arg-238 matched well to the arginine "finger" required for enhanced GTP hydrolysis in homodimerized Cdc42. Site-directed mutagenesis confirmed that an R235K or R238K mutation severely impaired the BNIP-2 GAP activity without affecting its binding to Cdc42. From deletion studies, a region adjacent to the arginine patch ((288)EYV(290) on BNIP-2) and the Switch I and Rho family-specific "Insert" region on Cdc42 are involved in the binding. The results indicate that the BCH domain of BNIP-2 represents a novel GAP domain that employs an arginine patch motif similar to that of the Cdc42-homodimer.

Amino Acid Sequence↗

PubMed2000

FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors.

The docking protein FRS2 was implicated in the transmission of extracellular signals from the fibroblast growth factor (FGF) or nerve growth factor (NGF) receptors to the Ras/mitogen-activated protein kinase signaling cascade. The two members of the FRS2 family, FRS2alpha and FRS2beta, are structurally very similar. Each is composed of an N-terminal myristylation signal, a phosphotyrosine-binding (PTB) domain, and a C-terminal tail containing multiple binding sites for the SH2 domains of the adapter protein Grb2 and the protein tyrosine phosphatase Shp2. Here we show that the PTB domains of both the alpha and beta isoforms of FRS2 bind directly to the FGF or NGF receptors. The PTB domains of the FRS2 proteins bind to a highly conserved sequence in the juxtamembrane region of FGFR1. While FGFR1 interacts with FRS2 constitutively, independent of ligand stimulation and tyrosine phosphorylation, NGF receptor (TrkA) binding to FRS2 is strongly dependent on receptor activation. Complex formation with TrkA is dependent on phosphorylation of Y490, a canonical PTB domain binding site that also functions as a binding site for Shc (NPXpY). Using deletion and alanine scanning mutagenesis as well as peptide competition assays, we demonstrate that the PTB domains of the FRS2 proteins specifically recognize two different primary structures in two different receptors in a phosphorylation-dependent or -independent manner. In addition, NGF-induced tyrosine phosphorylation of FRS2alpha is diminished in cells that overexpress a kinase-inactive mutant of FGFR1. This experiment suggests that FGFR1 may regulate signaling via NGF receptors by sequestering a common key element which both receptors utilize for transmitting their signals. The multiple interactions mediated by FRS2 appear to play an important role in target selection and in defining the specificity of several families of receptor tyrosine kinases.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Tyrosine phosphorylation of the Bcl-2-associated protein BNIP-2 by fibroblast growth factor receptor-1 prevents its binding to Cdc42GAP and Cdc42.

Fibroblast growth factor (FGF) receptor tyrosine kinases are involved in the regulation of cell growth, development, and differentiation in a variety of tissues. To isolate potential signaling molecules in the FGF signaling pathway, we have initiated a yeast two-hybrid screening using the cytosolic domain of FGF receptor-1 (Flg). Here we report the identification of BNIP-2, a previously cloned Bcl-2- and adenovirus E1B-associated protein, as a putative substrate of the receptor. When cotransfected in 293T cells, BNIP-2 was tyrosine-phosphorylated via Flg, but their interaction was transient and could only be seen by "capture" experiments with catalytically inert kinase mutants. When responsive cells were challenged with basic FGF, endogenous tyrosine-phosphorylated BNIP-2 could be precipitated with a BNIP-2 antibody. In addition, the recombinant BNIP-2 expressed in bacteria could be phosphorylated by active Flg in vitro. BNIP-2 shares a region of homology with the noncatalytic domain of Cdc42GAP, a GTPase-activating protein for the small GTP-binding molecule, Cdc42. We show here that BNIP-2 and Cdc42GAP could directly bind to each other and they also compete for the binding to the same target, Cdc42. Unexpectedly, BNIP-2, either produced as a bacterial recombinant protein or expressed in 293T cells, could stimulate the intrinsic GTPase activity of Cdc42. In all cases, tyrosine phosphorylation of BNIP-2 severely impaired its association with Cdc42GAP and its induced GTPase-activating protein-like activity toward Cdc42. These findings should allow us to further characterize the integration of signaling between receptor tyrosine kinases, GTP-binding molecules, and apoptotic pathways.

Amino Acid Sequence↗

PubMed1999

Association of atypical protein kinase C isotypes with the docker protein FRS2 in fibroblast growth factor signaling.

FRS2 is a docker protein that recruits signaling proteins to the plasma membrane in fibroblast growth factor signal transduction. We report here that FRS2 was associated with PKC lambda when Swiss 3T3 cells were stimulated with basic fibroblast growth factor. PKC zeta, the other member of the atypical PKC subfamily, could also bind FRS2. The association between FRS2 and PKC lambda is likely to be direct as shown by yeast two-hybrid analysis. The C-terminal fragments of FRS2 (amino acid residues 300-508) and SNT2 (amino acids 281-492), an isoform bearing 50% identity to FRS2, interacted with PKC lambda at a region (amino acids 240-562) that encompasses the catalytic domain. In vitro kinase assays revealed neither FRS2 nor SNT2 was a substrate of PKC lambda or zeta. Mutation of the alanine residue (Ala-120) to glutamate in the pseudo-substrate region of PKC lambda results in a constitutively active kinase that exhibited more than 2-fold greater binding to FRS2 in vitro than its "closed" wild-type counterpart. Tyrosine phosphorylation of FRS2 did not affect its binding to the constitutively active PKC lambda mutant, suggesting that the activation of PKC lambda is necessary and sufficient for its association with FRS2. It is likely that FRS2 serves as an anchoring protein for targeting activated atypical PKCs to the cell plasma membrane in signaling pathways.

3T3 Cells↗

PubMed1997

Growth factors stimulate tyrosine dephosphorylation of p75 and its dissociation from the SH2 domain of Grb2.

The growth factor receptor-binding protein (Grb2) has a key role in initiating the mitogen-activated protein kinase signaling cascade in major cell regulatory pathways. The binding of proteins to the SH2 domain of Grb2 has been reported to occur mainly after they are tyrosine-phosphorylated following receptor activation. Using an in vitro binding assay, immunoprecipitation, and Far Western techniques, we report that in quiescent cells a 75-kDa protein binds directly to the SH2 domain of Grb2. All of the tyrosine-phosphorylated p75 protein co-localizes with Grb2.Sos complex in the cytosolic fraction of the cell in vivo and undergoes tyrosine dephosphorylation when cells are treated with mitogenic ligands such as epidermal, platelet-derived, and fibroblast growth factors, endothelin-1, and bombesin but not tumor necrosis factor-alpha, interferon-alpha and -gamma, interleukein-6, and leukemic inhibitory factor, which are either cell growth inhibitory or not significantly mitogenic. The dephosphorylation of p75 and the ensuing dissociation from Grb2 is rapid, occurring within 30 s following mitogenic stimulation by ligands such as epidermal growth factor, suggesting p75 to be an early component in the signal transduction pathways involving Grb2.

3T3 Cells↗

PubMed1997

SHP2 associates directly with tyrosine phosphorylated p90 (SNT) protein in FGF-stimulated cells.

In a number of cell lines responsive to basic fibroblast growth factor (bFGF), two major tyrosine phosphorylated proteins, of molecular weights around 120kDa and 90kDa, are precipitated along with the tyrosine phosphatase SHP2 from the lysates of stimulated cells. The docker protein Gab-1 represents at least part of the 120kDa protein(s). The p90 protein was identified as the SNT protein. The two SH2 domains of SHP2 bind directly and synergistically to tyrosine phosphorylated SNT. Tyrosine phosphorylated SNT does not bind SHP1 and does not appear to be an in vivo substrate of SHP2 but is likely to function as an adapter protein in FGF-signalling.

3T3 Cells↗

PubMed1996

Suc1-associated neurotrophic factor target (SNT) protein is a major FGF-stimulated tyrosine phosphorylated 90-kDa protein which binds to the SH2 domain of GRB2.

In Swiss 3T3 fibroblasts, basic fibroblast growth factor (bFGF) stimulates the unique tyrosine phosphorylation of a protein complex around 90 kDa, as ascertained by high resolution 2-D PAGE and anti-phosphotyrosine blotting. The majority of this complex consists of the protein(s) designated previously as SNT (suc1-associated neurotrophic factor target). Tyrosine phosphorylated SNT binds to both p13suc1 protein and the SH2 domain of Grb2. Binding of SNT to Grb2 is likely to be mediated through the consensus binding motif, pYXN, on SNT. The binding of SNT to p13suc1 is independent of the pYXN motif. Tyrosine phosphorylated SNT is localised in the plasma membrane where it could form a complex with Grb2 and Sos, enabling the initiation of a novel FGF-specific signalling pathway.

3T3 Cells↗

PubMed1996

Casein kinase II associates with Egr-1 and acts as a negative modulator of its DNA binding and transcription activities in NIH 3T3 cells.

Although the activation domains within early growth response gene protein 1 (Egr-1) have been mapped, little is known of the kinases which phosphorylate Egr-1 and how phosphorylation correlates with the transcriptional activity of Egr-1. In this study we report that casein kinase II (CKII) co-immunoprecipitates with Egr-1 from NIH 3T3 cell lysates. The association of Egr-1 and CKII requires the C terminus of Egr-1 and CKII phosphorylates Egr-1 in vitro. The in vitro phosphorylation of Egr-1 by CKII and that induced by serum in vivo was compared by examining the CNBr-digested fragments of the phosphorylated Egr-1. CKII strongly phosphorylates fragments 7 and 10 which cover part of the activation/nuclear localization and DNA binding domains of Egr-1. CKII also phosphorylates, albeit weakly, fragments 5 and 8 which cover part of activation domain and the entire repression domain of Egr-1, respectively. Strong phosphorylation on fragment 10 as well as fragment 5 was also observed in Egr-1 immunoprecipitated from serum-induced, 32P-labeled cells. CKII phosphorylation of Egr-1 resulted in a decrease of its DNA binding as well as its transcriptional activities.

3T3 Cells↗

PubMed1996

Identification of p90, a prominent tyrosine-phosphorylated protein in fibroblast growth factor-stimulated cells, as 80K-H.

Tyrosine phosphorylation of cellular proteins occurs rapidly upon treatment of fibroblasts with acidic or basic fibroblast growth factors (aFGF, bFGF), suggesting a role for protein phosphorylation in the FGF signaling pathway. Stimulation of Swiss 3T3 cells and MRC-5 fibroblasts with bFGF results in the tyrosine phosphorylation of several proteins, of which the most prominent has been designated as p90. The phosphorylation of p90 is observed within 30 s of treating the cells with FGF but not with other growth factors. Microsequencing of p90 resolved on two-dimensional polyacrylamide gel electrophoresis indicated an N-terminal amino acid sequence which corresponded to a protein previously named as 80K-H. Polyclonal antibodies raised against the predicted C terminus of 80K-H recognized p90 on all Western blots. p90 was found to bind specifically to GRB-2-glutathione S-transferase fusion protein and to be immunoreactive with 80K-H antibody. In addition, anti-phosphotyrosine antibodies immunoprecipitated 80K-H from cell lysates of FGF-stimulated but not from control fibroblasts. The biological function of 80K-H is yet unknown. However, from this study and a previous observation of the obligatory dependence of p90 phosphorylation on FGF receptor occupation, it appears that 80K-H is involved in FGF signaling.

3T3 Cells↗

PubMed1996

Activation and association of Stat3 with Src in v-Src-transformed cell lines.

STAT proteins are a group of latent cytoplasmic transcription factors which function as signal transducers and activators of transcription. Stat1 and -2 were originally identified to function in interferon signaling, and Stat1 was also found to be activated by epidermal growth factor (EGF) and other cytokines. New members of the STAT gene family are identified. Among them, Stat3 has 52.5% amino acid sequence homology with Stat1 and is activated by platelet-derived growth factor (PDGF), colony-stimulating factor 1 (CSF-1), EGF, interleukin-6, and other cytokines. Treatment of cells with EGF activates Stat1 and Stat3, which become phosphorylated on tyrosine residues to form homo - or heterodimers and translocate into the nucleus, binding to the sis-inducible element (SIE) in the c-fos promoter. Somatic cell genetic analyses demonstrated that Jaks, a family of nontransmembrane protein tyrosine kinases, are required for the activation of Stat1 and Stat2 in interferon-treated cells. However, little is known about the activation of Stat3 by growth factors. Here we report that in all v-Src-transformed cell lines examined, Stat3 is constitutively activated to bind to DNA and the phosphorylation of tyrosine on Stat3 is enhanced by the induction of v-Src expression. We also report that Src is shown to be associated with Stat3 in vivo, as well as in vitro, and phosphorylates Stat3 in vitro. Stat3 is also activated by CSF-1, possibly through CSF-1 receptor-c Src association in NIH 3T3 cells overexpressing CSF-1 receptors. Together, the data suggest that Src is involved in activation of Stat3 in growth factor signal transduction.

3T3 Cells↗

PubMed1995

Involvement of a putative protein-tyrosine phosphatase and I kappa B-alpha serine phosphorylation in nuclear factor kappa B activation by tumor necrosis factor.

Inhibitors of phosphotyrosyl protein phosphatases, pervanadate and phenylarsine oxide, abrogate tumor necrosis factor (TNF)-induced nuclear factor kappa B (NF-kappa B) nuclear translocation in transformed cell lines (U-937 and Jurkat) and primary fibroblasts (MRC-5 and REF). The inhibitors also abrogate NF-kappa B activation by the phosphoseryl/threonyl protein phosphatase inhibitor okadaic acid in U-937 cells. Inhibition of NF-kappa B activation is not due to a general inhibitory effect since neither pervanadate nor phenylarsine oxide treatment affected the constitutive DNA-binding activity of the transcription factors octamer-1 and cAMP response element-binding protein in U-937 cells, nor did these compounds inhibit the TNF-induced phosphorylation of proteins, viz. hsp-27, eukaryotic initiation factor 4e, and pp19, in MRC-5 fibroblasts. Overexpression of the protein-tyrosine phosphatase HPTP alpha resulted in a constitutive nuclear NF-kappa B-like DNA-binding activity in REF cells. Conversely, treatment of human protein-tyrosine phosphatase alpha-overexpressing cells with phenylarsine oxide led to a loss of the constitutive NF-kappa B activity. The presence of a tyrosine phosphorylation site on the inhibitor of NF-kappa B (I kappa B-alpha) suggested that it could be a target for TNF/okadaic acid-induced tyrosine dephosphorylation. However, no tyrosine phosphorylation was detected on I kappa B-alpha fron unstimulated cells, while TNF/okadaic acid-treated cells showed increased phosphorylation of I kappa B-alpha exclusively at serine residue(s). Treatment of cells with pervanadate inhibited TNF-induced I kappa B-alpha phosphorylation and degradation, whereas the serine protease inhibitors tosylphenylalanyl chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone prevented TNF-induced I kappa B-alpha degradation and NF-kappa B nuclear translocation, but not the TNF-induced phosphorylation of I kappa B-alpha. The data suggest that TNF and okadaic acid induce the activation of a putative protein-tyrosine phosphatase(s), leading to I kappa B-alpha serine phosphorylation and degradation and NF-kappa B nuclear translocation.

Amino Acid Sequence↗

PubMed1995

Activation of protein kinases and the inactivation of protein phosphatase 2A in tumour necrosis factor and interleukin-1 signal-transduction pathways.

We report the identification of 16 of the 30 cellular proteins which are rapidly phosphorylated in tumour-necrosis-factor-(TNF)-treated or interleukin-1-(IL-1)-treated primary human fibroblasts. Phosphorylation assays of proteins found in the cytosolic extract of human fibroblasts by in vitro assays indicate that at least 12 of these proteins are likely to be substrates for mitogen-activated protein kinase(s) (MAP kinase), mitogen-activated protein-kinase-activated protein kinase 2 (MAPKAP kinase 2), a pp60c-src-like tyrosine kinase as well as for a putative dual nucleotide protein kinase (DNK) in TNF-treated or IL-1-treated cells. Comparison of the phosphorylation of cytosolic proteins in vitro by exogenously added protein kinases with that observed in cells treated with TNF or IL-1 enabled the identification of cellular substrates of TNF-activated and IL-1-activated cellular protein kinases. Comparison of protein kinase activities of cytosolic extracts derived from TNF-treated or IL-1-treated and control fibroblasts also show the activation of MAP kinase, MAPKAP kinase 2, a putative DNK and a pp60src-like tyrosine kinase 3-19 fold. The data suggest TNF or IL-1 signal transduction may involve the phosphorylation of protein phosphatase type 2A by a pp60src-like tyrosine kinase, followed by the activation of MAP kinase, MAPKAP kinase 2 and the putative DNK. However, the activation of MAP kinase and MAPKAP kinase 2 may be independent of the earlier activation of pp60src-like tyrosine kinase and the inactivation of protein phosphatase type 2A.

Calcium-Calmodulin-Dependent Protein Kinases↗