Biomedical subjects

J Blenis

Publications and source records attributed to J Blenis.

At least 37 records · Page 2Linked to original sources

PubMed1999

Caspase-8 is required for cell death induced by expanded polyglutamine repeats.

We show here that caspase-8 is required for the death of primary rat neurons induced by an expanded polyglutamine repeat (Q79). Expression of Q79 recruited and activated caspase-8. Inhibition of caspase-8 blocked polyglutamine-induced cell death. Coexpression of Q79 with the caspase inhibitor CrmA, a dominant-negative mutant of FADD (FADD DN), Bcl-2, or Bcl-xL, but not an N-terminally tagged Bcl-xL, prevented the recruitment of caspase-8 and inhibited polyglutamine-induced cell death. Furthermore, Western blot analysis revealed the presence of activated caspase-8 in the insoluble fraction of affected brain regions from Huntington's disease (HD) patients but not in those from neurologically unremarkable controls, suggesting the relocation and activation of caspase-8 during the pathogenesis of HD. These results suggest an essential role of caspase-8 in HD-related neural degenerative diseases.

Adaptor Proteins, Signal Transducing↗

PubMed1999

p70 S6 kinase is regulated by protein kinase Czeta and participates in a phosphoinositide 3-kinase-regulated signalling complex.

p70 S6 kinase (p70S6K) is an important regulator of cell proliferation. Its activation by growth factor requires phosphorylation by various inputs on multiple sites. Data accumulated thus far support a model whereby p70S6K activation requires sequential phosphorylations at proline-directed residues in the putative autoinhibitory pseudosubstrate domain, as well as threonine 389. Threonine 229, a site in the catalytic loop is phosphorylated by phosphoinositide-dependent kinase 1 (PDK-1). Experimental evidence suggests that p70S6K activation requires a phosphoinositide 3-kinase (PI3-K)-dependent signal(s). However, the intermediates between PI3-K and p70S6K remain unclear. Here, we have identified PI3-K-regulated atypical protein kinase C (PKC) isoform PKCzeta as an upstream regulator of p70S6K. In coexpression experiments, we found that a kinase-inactive PKCzeta mutant antagonized activation of p70S6K by epidermal growth factor, PDK-1, and activated Cdc42 and PI3-K. While overexpression of a constitutively active PKCzeta mutant (myristoylated PKCzeta [myr-PKCzeta]) only modestly activated p70S6K, this mutant cooperated with PDK-1 activation of p70S6K. PDK-1-induced activation of a C-terminal truncation mutant of p70S6K was also enhanced by myr-PKCzeta. Moreover, we have found that p70S6K can associate with both PDK-1 and PKCzeta in vivo in a growth factor-independent manner, while PDK-1 and PKCzeta can also associate with each other, suggesting the existence of a multimeric PI3-K signalling complex. This work provides evidence for a link between a phorbol ester-insensitive PKC isoform and p70S6K. The existence of a PI3-K-dependent signalling complex may enable efficient activation of p70S6K in cells.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed1999

FADD is required for multiple signaling events downstream of the receptor Fas.

To identify essential components of the Fas-induced apoptotic signaling pathway, Jurkat T lymphocytes were chemically mutagenized and selected for clones that were resistant to Fas-induced apoptosis. We obtained five cell lines that contain mutations in the adaptor FADD. All five cell lines did not express FADD by immunoblot analysis and were completely resistant to Fas-induced death. Complementation of the FADD mutant cell lines with wild-type FADD restored Fas-mediated apoptosis. Fas activation of caspase-2, caspase-3, caspase-7, and caspase-8 and the proteolytic cleavage of substrates such as BID, protein kinase Cdelta, and poly(ADP-ribose) polymerase were completely defective in the FADD mutant cell lines. In addition, Fas activation of the stress kinases p38 and c-Jun NH2 kinase and the generation of ceramide in response to Fas ligation were blocked in the FADD mutant cell lines. These data indicate that FADD is essential for multiple signaling events downstream of Fas.

Adaptor Proteins, Signal Transducing↗

PubMed1998

The germinal center kinase (GCK)-related protein kinases HPK1 and KHS are candidates for highly selective signal transducers of Crk family adapter proteins.

Adapter proteins function by mediating the rapid and specific assembly of multi-protein complexes during the signal transduction which guards proliferation, differentiation and many functions of higher eukaryotic cells. To understand their functional roles in different cells it is important to identify the selectively interacting proteins in these cells. Two novel candidates for signalling partners of Crk family adapter proteins, the hematopoietic progenitor kinase 1 (HPK1) and the kinase homologous to SPS1/STE20 (KHS), were found to bind with great selectivity to the first SH3 domains of c-Crk and CRKL. While KHS bound exclusively to Crk family proteins, HPK1 also interacted with both SH3 domains of Grb2 and weakly with Nck, but not with more than 25 other SH3 domains tested. The interaction of HPK1 with c-Crk and CRKL was studied in more detail. HPK1-binding to the first SH3 domain of CRKL is direct and occurs via proline-rich motifs in the C-terminal, non-catalytic portion of HPK1. In vitro complexes were highly stable and in vivo complexes of c-Crk and CRKL with HPK1 were detectable by co-immunoprecipitation with transiently transfected cells but also with endogenous proteins. Furthermore, c-Crk II and, to a lesser extent, CRKL were substrates for HPK1. These results make it likely that HPK1 and KHS participate in the signal transduction of Crk family adapter proteins in certain cell types.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Essential requirement for caspase-8/FLICE in the initiation of the Fas-induced apoptotic cascade.

BACKGROUND: Fas (APO-1/CD95) is a member of the tumor necrosis factor receptor (TNF-R) family and induces apoptosis when crosslinked with either Fas ligand or agonistic antibody (Fas antibody). The Fas-Fas ligand system has an important role in the immune system where it is involved in the downregulation of immune responses and the deletion of peripheral autoreactive T lymphocytes. The intracellular domain of Fas interacts with several proteins including FADD (MORT-1), DAXX, RIP, FAF-1, FAP-1 and Sentrin. The adaptor protein FADD can, in turn, interact with the cysteine protease caspase-8 (FLICE/MACH/Mch5). RESULTS: In a genetic screen for essential components of the Fas-mediated apoptotic cascade, we isolated a Jurkat T lymphocyte cell line deficient in caspase-8 that was completely resistant to Fas-induced apoptosis. Complementation of this cell line with wild-type caspase-8 restored Fas-mediated apoptosis. Fas activation of multiple caspases and of the stress kinase p38 and c-Jun NH2-terminal kinase (JNK) was completely blocked in the caspase-8-deficient cell line. Furthermore, the cell line was severely deficient in cell death induced by TNF-alpha and was partially deficient in cell death induced by ultraviolet irradiation, adriamycin and etoposide. CONCLUSIONS: This study provides the first genetic evidence that caspase-8 occupies an essential and apical position in the Fas signaling pathway and suggests that caspase-8 may participate broadly in multiple apoptotic pathways.

Apoptosis↗

PubMed1998

pp90rsk1 regulates estrogen receptor-mediated transcription through phosphorylation of Ser-167.

The estrogen receptor alpha (ER), a member of the steroid receptor superfamily, contains an N-terminal hormone-independent transcriptional activation function (AF-1) and a C-terminal hormone-dependent transcriptional activation function (AF-2). Here, we used in-gel kinase assays to determine that pp90rsk1 activated by either epidermal growth factor (EGF) or phorbol myristate acetate specifically phosphorylates Ser-167 within AF-1. In vitro kinase assays demonstrated that pp90rsk1 phosphorylates the N terminus of the wild-type ER but not of a mutant ER in which Ser-167 was replaced by Ala. In vivo, EGF stimulated phosphorylation of Ser-167 as well as Ser-118. Ectopic expression of active pp90rsk1 increased the level of phosphorylation of Ser-167 compared to that of either a mutant pp90rsk1, which is catalytically inactive in the N-terminal kinase domain, or to that of vector control. The ER formed a stable complex with the mutant pp90rsk1 in vivo. Transfection of the mutant pp90rsk1 depressed ER-dependent transcription of both a wild-type ER and a mutant ER that had a defective AF-2 domain (ER TAF-1). Furthermore, replacing either Ser-118 or Ser-167 with Ala in ER TAF-1 showed similar decreases in transcription levels. A double mutant in which both Ser-118 and Ser-167 were replaced with Ala demonstrated a further decrease in transcription compared to either of the single mutations. Taken together, our results strongly suggest that pp90rsk1 phosphorylates Ser-167 of the human ER in vivo and that Ser-167 aids in regulating the transcriptional activity of AF-1 in the ER.

Animals↗

PubMed1997

Cloning and characterization of a human STE20-like protein kinase with unusual cofactor requirements.

We cloned and characterized a novel human member of the STE20 serine/threonine protein kinase family named mst-3. Based on its domain structure, mst-3 belongs to the SPS1 subgroup of STE20-like proteins, which includes germinal center (GC) kinase, hematopoietic progenitor kinase (HPK), kinase homologous to STE20/SPS-1 (KHS), kinases responsive to stress (KRS1/2), the mammalian STE20-like kinases (mst1/2), and the recently published STE20/oxidant stress response kinase SOK-1. mst-3 is most closely related to SOK-1, with 88% amino acid similarity in the kinase domain. The similarity of the mst-3 kinase domain to STE20 is 42%. The mst-3 transcript is ubiquitously expressed, and the protein was found in all human, mouse, and monkey cell lines tested. An in vitro kinase assay showed that mst-3 can phosphorylate basic exogenous substrates as well as itself. Interestingly, mst-3 prefers Mn2+ to Mg2+ as a divalent cation and can use both GTP and ATP as phosphate donors. Like SOK-1, mst-3 is activated by autophosphorylation. However, a physiological stimulus of mst-3 activity was not identified. mst-3 activity does not change upon exposure to several mitogenic and stress stimuli. Overexpression of mst-3 wild-type or kinase dead protein affects neither the extracellular signal-regulated kinases (ERK1/2 or ERK6), c-Jun N-terminal kinase (JNK), p38, nor pp70S6 kinase, suggesting that mst-3 is part of a novel signaling pathway.

3T3 Cells↗

PubMed1997

Evidence for MEK-independent pathways regulating the prolonged activation of the ERK-MAP kinases.

The mitogen-activated protein kinases (MAPKs) ERK-1 and ERK-2 are activated by a wide variety of oncogenes and extracellular stimuli. The MAPKs participate in a signalling cascade downstream of growth factor/cytokine receptors, Ras, Raf, and MEK. However, MAPK activation is more complicated than a simple linear pathway, and the evidence presented here supports a model of multiple, temporally distinct pathways converging on MAPK which are differentially utilized by various stimuli and cell types. In addition to MEK-dependent MAPK activation, we provide evidence for MEK-independent regulation of the MAPKs. Our results suggest that phosphatidylinositol-3-kinases (PI(3)K) or conventional protein kinase C isoforms (cPKCs) partially contribute to MEK-dependent activation. Importantly, we also find that PI3K and cPKCs play a major role in the MEK-independent, prolonged MAPK activation by platelet-derived growth factor signalling. This finding is of interest as the maintained activation of MAPK has been correlated by others to the regulation of cell proliferation and differentiation.

3T3 Cells↗

PubMed1997

Neurotransmitter- and growth factor-induced cAMP response element binding protein phosphorylation in glial cell progenitors: role of calcium ions, protein kinase C, and mitogen-activated protein kinase/ribosomal S6 kinase pathway.

To understand how extracellular signals may produce long-term effects in neural cells, we have analyzed the mechanism by which neurotransmitters and growth factors induce phosphorylation of the transcription factor cAMP response element binding protein (CREB) in cortical oligodendrocyte progenitor (OP) cells. Activation of glutamate receptor channels by kainate, as well as stimulation of G-protein-coupled cholinergic receptors by carbachol and tyrosine kinase receptors by basic fibroblast growth factor (bFGF), rapidly leads to mitogen-activated protein kinase (MAPK) phosphorylation and ribosomal S6 kinase (RSK) activation. Kainate and carbachol activation of the MAPK pathway requires extracellular calcium influx and is accompanied by protein kinase C (PKC) induction, with no significant increase in GTP binding to Ras. Conversely, growth factor-stimulated MAPK phosphorylation is independent of extracellular calcium and is accompanied by Ras activation. Both basal and stimulated MAPK activity in OP cells are influenced by cytoplasmic calcium levels, as shown by their sensitivity to the calcium chelator bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid. The kinetics of CREB phosphorylation in response to the various agonists corresponds to that of MAPK activation. Moreover, CREB phosphorylation and MAPK activation are similarly affected by calcium ions. The MEK inhibitor PD 098059, which selectively prevents activation of the MAPK pathway, strongly reduces induction of CREB phosphorylation by kainate, carbachol, bFGF, and the phorbol ester TPA. We propose that in OPs the MAPK/RSK pathway mediates CREB phosphorylation in response to calcium influx, PKC activation, and growth factor stimulation.

Animals↗

PubMed1997

A novel human SPS1/STE20 homologue, KHS, activates Jun N-terminal kinase.

STE20-homologous proteins have been implicated in mammalian MAP kinase pathways as important transducers of signals from p21 family GTPases. We have cloned a novel STE20 family member, which we call KHS for kinase homologous to SPS1/STE20, that encodes a kinase of 95 kD which is expressed in a variety of tissues. Transiently expressed fusion protein GST-KHS exhibits phosphotransferase activity toward a panel of test substrates, including myelin basic protein (MBP), which is phosphorylated by all known STE20 homologues. KHS is most closely related to another human STE20, GC kinase (74% similar in the catalytic domain), which has recently been placed upstream of the stress-activated MAP kinases (SAPKs/JNKs). KHS also activates JNK in transient coexpression experiments, suggesting a role for KHS in the stress response of fibroblasts. Characterization and comparison of the regulation of these two kinases will be important in elucidating MAP kinase signalling cascades.

3T3 Cells↗

PubMed1997

Fas activation of the p38 mitogen-activated protein kinase signalling pathway requires ICE/CED-3 family proteases.

The Fas receptor mediates a signalling cascade resulting in programmed cell death (apoptosis) within hours of receptor cross-linking. In this study Fas activated the stress-responsive mitogen-activated protein kinases, p38 and JNK, within 2 h in Jurkat T lymphocytes but not the mitogen-responsive kinase ERK1 or pp70S6k. Fas activation of p38 correlated temporally with the onset of apoptosis, and transfection of constitutively active MKK3 (glu), an upstream regulator of p38, potentiated Fas-induced cell death, suggesting a potential involvement of the MKK3/p38 activation pathway in Fas-mediated apoptosis. Fas has been shown to require ICE (interleukin-1 beta-converting enzyme) family proteases to induce apoptosis from studies utilizing the cowpox ICE inhibitor protein CrmA, the synthetic tetrapeptide ICE inhibitor YVAD-CMK, and the tripeptide pan-ICE inhibitor Z-VAD-FMK. In this study, crmA antagonized, and YVAD-CMK and Z-VAD-FMK completely inhibited, Fas activation of p38 kinase activity, demonstrating that Fas-dependent activation of p38 requires ICE/CED-3 family members and conversely that the MKK3/p38 activation cascade represents a downstream target for the ICE/CED-3 family proteases. Intriguingly, p38 activation by sorbitol and etoposide was resistant to YVAD-CMK and Z-VAD-FMK, suggesting the existence of an additional mechanism(s) of p38 regulation. The ICE/CED-3 family-p38 regulatory relationship described in the current work indicates that in addition to the previously described destructive cleavage of substrates such as poly(ADP ribose) polymerase, lamins, and topoisomerase, the apoptotic cysteine proteases also function to regulate stress kinase signalling cascades.

Amino Acid Chloromethyl Ketones↗

PubMed1997

STAT3 serine phosphorylation by ERK-dependent and -independent pathways negatively modulates its tyrosine phosphorylation.

Recent studies have indicated that serine phosphorylation regulates the activities of STAT1 and STAT3. However, the kinase(s) responsible and the role of serine phosphorylation in STAT function remain unresolved. In the present studies, we examined the growth factor-dependent serine phosphorylation of STAT1 and STAT3. We provide in vitro and in vivo evidence that the ERK family of mitogen-activated protein (MAP) kinases, but not JNK or p38, specifically phosphorylate STAT3 at serine 727 in response to growth factors. Evidence for additional mitogen-regulated serine phosphorylation is also provided. STAT1 is a relatively poor substrate for all MAP kinases tested both in vitro and in vivo. STAT3 serine phosphorylation, not its tyrosine phosphorylation, results in retarded mobility of the STAT3 protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Importantly, serine 727 phosphorylation negatively modulates STAT3 tyrosine phosphorylation, which is required for dimer formation, nuclear translocation, and the DNA binding activity of this transcriptional regulator. Interestingly, the cytokine interleukin-6 also stimulates STAT3 serine phosphorylation, but in contrast to growth factors, this occurs by an ERK-independent process.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1996

A Drosophila gene structurally and functionally homologous to the mammalian 70-kDa s6 kinase gene.

cDNAs encoding the Drosophila 70-kDa S6 kinase (S6K) were isolated by low-stringency hybridization with mammalian p70S6k probes. Conceptual translation of S6k cDNA sequences yields a product containing all of the canonical features typical of serine/threonine kinases and has 78% amino acid identity in the catalytic domain with the human p70S6k homologue. The S6k gene, located at polytene chromosome site 65D, gives rise to two predominant transcripts of 3.0 and 5.0 kb and at least two smaller transcripts (< 3.0 kb) that are found in whole-animal RNAs at all stages of development. Blood cells derived from the hematopoietic organs of ribosomal protein S6 (RpS6air8) mutant animals express higher levels of the smaller S6k transcripts, suggesting tissue- or genotype-specific differences in the regulation of the S6k gene. Drosophila S6K expressed in COS or NIH 3T3 cells phosphorylates mammalian RPS6 in a mitogen-dependent wortmannin- and rapamycin-sensitive manner, suggesting that its regulation is similiar to mammalian p70S6k.

3T3 Cells↗

PubMed1996

The serine protease inhibitors, tosylphenylalanine chloromethyl ketone and tosyllysine chloromethyl ketone, potently inhibit pp70s6k activation.

pp70(s6k) is a mitogen-regulated serine/threonine kinase involved in the G1 to S phase transition of the cell cycle. We have analyzed its regulation in several cell lines by a variety of agonists and have found that pp70(s6k) activation by all stimuli tested is completely blocked by the serine protease inhibitors tosylphenylalanine chloromethyl ketone (TPCK) and tosyllysine chloromethyl ketone (TLCK). TPCK inhibition of the pp70(s6k) signaling pathway resembles that of the immunosuppressant rapamycin; however, we demonstrate that their methods of inhibition differ. We find that TPCK and TLCK are not general signaling inhibitors since the activation of the mitogen-activated protein kinase pathway is not abrogated. The demonstration that these protease inhibitors prevent signaling via the pp70(s6k) pathway will help in understanding the variety of physiological processes that TPCK and TLCK have been shown to effect.

3T3 Cells↗

PubMed1996

The signal-dependent coactivator CBP is a nuclear target for pp90RSK.

We have examined the mechanism by which growth factor-mediated induction of the Ras pathway interferes with signaling via the second messenger cAMP. Activation of cellular Ras with insulin or NGF stimulated recruitment of the S6 kinase pp90RSK to the signal-dependent coactivator CBP. Formation of the pp90RSK-CBP complex occurred with high stoichiometry and persisted for 6-8 hr following growth factor addition. pp90RSK specifically recognized the E1A-binding domain of the coactivator CBP. In addition, like E1A, binding of pp90RSK to CBP was sufficient to repress transcription of cAMP-responsive genes via the cAMP-inducible factor CREB. By contrast with its effects on the cAMP pathway, formation of the pp90RSK-CBP complex was required for induction of Ras-responsive genes. These results provide a demonstration of cross-coupling between two signaling pathways that occurs at the level of a signal-dependent coactivator.

Adenovirus E1A Proteins↗

PubMed1996

The 70 kDa S6 kinase complexes with and is activated by the Rho family G proteins Cdc42 and Rac1.

The 70 kDa ribosomol S6 kinase (pp70S6k) plays an important role in the progression of cells through G1 phase of the cell cycle. However, little is known of the signaling molecules that mediate its activation. We demonstrate that Rho family G proteins regulate pp70S6k activity in vivo. Activated alleles of Cdc42 and Rac1, but not RhoA, stimulate pp70S6k activity in multiple cell types. Activation requires an intact effector domain and isoprenylation of Cdc42 and Rac1. Coexpression of Dbl, an exchange factor for Cdc42, also activates pp70S6k. Growth factor-induced activation of pp70S6k is abrogated by dominant negative alleles of Cdc42 and Rac1. In addition, Cdc42 and Rac1 form GTP-dependent complex with the catalytically inactive form of pp70S6k in vitro and in vivo, suggesting a mechanism by which these G proteins activate pp70S6k.

3T3 Cells↗

PubMed1996

Phosphorylation of c-Fos at the C-terminus enhances its transforming activity.

c-Fos is phosphorylated by MAP kinase and the 90 kDa-ribosomal S6 kinase (RSK) in vitro at serines 362 and 374 (rat) which we demonstrate are major in vivo phosphorylation sites in early G1. We have constructed c-Fos mutants with these serines changed to aspartic acid residues (FosD) to mimic phosphorylation or to alanine residues (FosA) to prevent phosphorylation. Cells expressing FosD exhibited a more extensive transformed phenotype than those expressing either FosA or wild type c-Fos (FosWT). We also observed that FosA has a reduced half-life in comparison with FosD in G1. Furthermore, we observed enhanced AP-1 transactivation activity in cells expressing FosD. These results indicate that phosphorylation of c-Fos at its extreme carboxyterminus, possibly by MAP kinase and RSK, supports the proliferative response by increasing c-Fos stability and/or by increasing its transactivation activity. Under conditions in which the MAP kinase pathway is constitutively activated, c-Fos phosphorylation probably contributes to cellular transformation. The highly conserved nature of these phosphorylation sites in other c-fos family members suggests that these may also be targets of MAP kinase and RSK.

3T3 Cells↗

PubMed1996

A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1.

We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the +1 and -3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.

Amino Acid Sequence↗