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J Blenis

Publications and source records attributed to J Blenis.

At least 73 records · Page 4Linked to original sources
PubMed1994

Activation of early growth response 1 gene transcription and pp90rsk during induction of monocytic differentiation.

The present work has studied mechanisms responsible for induction of early growth response 1 (EGR-1) gene expression during monocytic differentiation of U-937 myeloid leukemia cells. Differentiation of U-937 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of the serine/threonine protein kinase C, was associated with transcriptional activation of EGR-1 promoter-reporter constructs. The EGR-1 promoter contains six CC(A/T)6GG (CArG) motifs. The two 5'-most distal CArG sequences conferred TPA inducibility. In contrast, there was little effect of TPA on EGR-1 transcription in a TPA-resistant U-937 cell variant, designated TUR. Treatment of both U-937 and TUR cells with okadaic acid, an inhibitor of serine/threonine protein phosphatases 1 and 2A, was associated with induction of monocytic differentiation and EGR-1 transcription through the 5'-most CArG element. Since these findings supported the involvement of serine/threonine protein phosphorylation in the regulation of EGR-1 expression, we studied activation of the 40S ribosomal protein S6 serine/threonine kinases, pp70S6K and pp90rsk. Although both kinases participate in regulating cell growth, there was no detectable activation of pp70S6K during TPA- or okadaic acid-induced monocytic differentiation. Moreover, rapamycin, an inhibitor of pp70S6K activation, had no effect on induction of EGR-1 expression. In contrast, analysis of pp90rsk activity by phosphorylation of a peptide derived from S6 protein demonstrated stimulation of this kinase in TPA-treated U-937, and not TUR, cells. Okadaic acid treatment of both cell types was associated with activation of pp90rsk.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Differentiation↗
PubMed1993

Phosphorylation of the c-Fos transrepression domain by mitogen-activated protein kinase and 90-kDa ribosomal S6 kinase.

Phosphorylation of the C terminus of c-Fos has been implicated in serum response element-mediated repression of c-fos transcription after its induction by serum growth factors. The growth-regulated enzymes responsible for this phosphorylation in early G1 phase of the cell cycle and the sites of phosphorylation have not been identified. We now provide evidence that two growth-regulated, nucleus- and cytoplasm-localized protein kinases, 90-kDa ribosomal S6 kinase (RSK) and mitogen-activated protein kinase (MAP kinase), contribute to the serum-induced phosphorylation of c-Fos. The major phosphopeptides derived from biosynthetically labeled c-Fos correspond to phosphopeptides generated after phosphorylation of c-Fos in vitro with both RSK and MAP kinase. The phosphorylation sites identified for RSK (Ser-362) and MAP kinase (Ser-374) are in the transrepression domain. Cooperative phosphorylation at these sites by both enzymes was observed in vitro and reflected in vivo by the predominance of the peptide phosphorylated on both sites, as opposed to singly phosphorylated peptides. This study suggests a role for nuclear RSK and MAP kinase in modulating newly synthesized c-Fos phosphorylation and downstream signaling.

3T3 Cells↗
PubMed1993

Activation of the early growth response 1 gene and nuclear pp90rsk in human myeloid leukemia cells by 1-(beta-D-arabinofuranosyl)cytosine.

The early growth response 1 (EGR-1) gene is induced by mitogens, differentiating stimuli, and certain genotoxic agents in diverse cell types. The present work has examined the effects of 1-(beta-D-arabinofuranosyl)cytosine (ara-C), an antileukemia agent that misincorporates into DNA, on EGR-1 expression. Treatment of HL-525 myeloid leukemia cells with ara-C was associated with transient increases in EGR-1 mRNA levels. Nuclear run-on assays showed that this effect is related at least in part to activation of EGR-1 gene transcription. Sequences responsive to ara-C-induced signals were determined by deletion analysis of the EGR-1 promoter. The results demonstrate that ara-C inducibility of the EGR-1 gene is conferred by a region containing six serum response or CC(A/T)6GG (CArG) motifs. Further analysis demonstrated that the first two distal or 5'-most CArG elements are functional in the ara-C response. An oligomer corresponding to the first CArG element also conferred ara-C inducibility of the minimal thymdine kinase gene promoter, while no inducibility was detectable using a similar oligomer containing a mutated CArG box. Other work has demonstrated that the nuclear serum response factor (SRF) interacts with the CArG box in the EGR-1 promoter and that the serine/threonine pp90rsk protein kinase phosphorylates SRF in vitro at sites phosphorylated in vivo. The present studies demonstrate that ara-C has little if any effect on cytosolic pp90rsk as determined by immunoblotting to assess electrophoretic mobility and by immune-complex kinase assays using S6 peptide as substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1993

Signal transduction via the MAP kinases: proceed at your own RSK.

An explosion of new information linking activation of cell surface signal initiators to changes in gene expression has recently emerged. The focus of much of this information has centered around the agonist-dependent activation of the mitogen-activated protein (MAP) kinases. Although this intracellular signal transduction pathway is extremely complex, conservation of many of its components has been observed in yeast, nematodes, Drosophila, and mammals. Thus, these signaling proteins may participate in the regulation of a variety of cellular processes.

Animals↗
PubMed1993

Failure of rapamycin to block proliferation once resting cells have entered the cell cycle despite inactivation of p70 S6 kinase.

Rapamycin (RAP) has recently been shown to inhibit the phosphorylation and activity of p70 S6 kinase (p70s6k). In interleukin (IL)-2-induced cell activation of the human IL-2-dependent T-cell line, Kit225, RAP inhibited p70s6k phosphorylation and activation, but not the activation of MAP kinase, p90 S6 kinase (p90rsk), early tyrosine kinases, or the transcription of the c-fos and c-myc genes. Cell cycle progression induced by IL-2 was arrested by RAP prior to p110Rb phosphorylation and the major increase in total RNA synthesis, both of which were initiated around 6 h after addition of IL-2 and 9 h before the beginning of DNA synthesis. Interestingly, RAP could not inhibit DNA synthesis if addition of the drug was delayed for 6 h after addition of IL-2, despite the fact that even at this time, RAP rapidly induced the accumulation of the dephosphorylated form of p70s6k and that p70s6k was inactivated within 1 h of RAP addition. Furthermore, when RAP was added to continuously growing Kit225 cells, cell proliferation was maintained for at least two additional cell cycles, in the absence of apparent p70s6k activity. These results indicate that 1) among the earliest detectable signals after IL-2 treatment, RAP selectively inhibits p70s6k activation, 2) RAP inactivates p70s6k regardless of the stage of the cell cycle in which the drug is added, 3) RAP blocks resting T-cells from entering the cell cycle, but does not directly arrest cell cycle progression once cells have entered the cycle, and 4) inactivation of p70s6k does not cause immediate arrest of cell cycle progression once cells have entered the cycle.

Amino Acid Sequence↗
PubMed1993

Dissociation of pp70 ribosomal protein S6 kinase from insulin-stimulated glucose transport in 3T3-L1 adipocytes.

The metabolic and mitogenic actions of insulin have been proposed to be mediated by cellular serine/threonine kinases such as the ribosomal protein S6 kinases pp70-S6 (pp70-S6 kinase) and pp90rsk and the erk-encoded mitogen-activated protein kinases (pp42mapk and pp44mapk). Rapamycin completely blocked activation of pp70-S6 kinase by insulin in 3T3-L1 adipocytes, but did not inhibit insulin-stimulated glucose transport, translocation of GLUT4 to the cell surface, or activation of pp90rsk or pp44mapk by insulin. Concordant with the inhibition of kinase activity, rapamycin prevented the insulin-induced decrease in mobility of pp70-S6 kinase visualized by SDS-polyacrylamide gel electrophoresis, reflecting a reduction in the hormone-stimulated phosphorylation of the enzyme. The structurally related macrolide, FK506, had no effect on pp70-S6 kinase or hexose uptake. These data demonstrate that rapamycin blocks insulin activation of pp70-S6 kinase in 3T3-L1 adipocytes and that pp70-S6 kinase is not required in the signaling pathway leading to insulin-stimulated glucose transport.

3T3 Cells↗
PubMed1993

Activation of MAP kinases, pp90rsk and pp70-S6 kinases in mouse mast cells by signaling through the c-kit receptor tyrosine kinase or Fc epsilon RI: rapamycin inhibits activation of pp70-S6 kinase and proliferation in mouse mast cells.

The high-affinity receptor for IgE, Fc epsilon RI, represents the major cell surface structure through which mast cells express immunologically specific secretory function. By contrast, the stem cell factor receptor (SCFR), which is encoded by c-kit, is essential for normal mast cell development. The signaling pathways initiated by the stimulation of mast cells through the Fc epsilon RI, which lacks intrinsic kinase activity, and the SCFR, a member of the receptor tyrosine kinase family, generally have been regarded to be distinct. We report here that mouse mast cells stimulated either with SCF or with IgE and specific antigen exhibit a remarkably similar pattern of activation of mitogen-activated protein kinases (MAPK), 90 kDa-S6 kinases (pp90rsk), and pp70-S6 kinases (pp70-S6K). These results indicate that all three families of protein kinases are associated with the cell surface receptor-dependent activation of secretion, as well as proliferation, in mast cells. We also show that the immunosuppressant rapamycin, but not FK506, can inhibit both SCF-dependent pp70-S6 kinase activation and SCF-dependent proliferation in mouse mast cells, without suppressing IgE- and antigen-dependent mediator release. These findings suggest that the activation of pp70-S6 kinase represents an important link in the stimulation of cell proliferation by SCF. Our results also indicate that the intracellular signaling pathways initiated by stimulation of mast cells through the Fc epsilon RI or the SCFR exhibit more overlap than has previously been appreciated.

Animals↗
PubMed1993

A growth factor-induced kinase phosphorylates the serum response factor at a site that regulates its DNA-binding activity.

A signaling pathway by which growth factors may induce transcription of the c-fos proto-oncogene has been characterized. Growth factor stimulation of quiescent fibroblasts activates a protein kinase cascade that leads to the rapid and transient phosphorylation of the serum response factor (SRF), a regulator of c-fos transcription. The in vivo kinetics of SRF phosphorylation and dephosphorylation parallel the activation and subsequent repression of c-fos transcription, suggesting that this phosphorylation event plays a critical role in the control of c-fos expression. The ribosomal S6 kinase pp90rsk, a growth factor-inducible kinase, phosphorylates SRF in vitro at serine 103, the site that becomes newly phosphorylated upon growth factor stimulation in vivo. Phosphorylation of serine 103 significantly enhances the affinity and rate with which SRF associates with its binding site, the serum response element, within the c-fos promoter. These results suggest a model in which the growth factor-induced phosphorylation of SRF at serine 103 contributes to the activation of c-fos transcription by facilitating the formation of an active transcription complex at the serum response element.

3T3 Cells↗
PubMed1993

Functional domains and phosphorylation of the orphan receptor Nur77.

Nur77 represents a unique class within the steroid receptor superfamily since its synthesis is tightly regulated by extracellular signals and it is capable of potent transactivation activity in the absence of an exogenously added ligand. In this study, we sought to dissect the functional domains regulating the activities of Nur77 by deletion mapping. We demonstrate that whereas the transactivation activity of Nur77 resides in the amino-terminal domain, the carboxy-terminal domain regulates this activity. A short deletion from the carboxy terminus eliminates transactivation activity while a further deletion restores the activity. Deletion of the domain immediately carboxyl to the zinc fingers motif eliminates both DNA binding activity and nuclear localization, thus abolishing transactivation. Nur77 is posttranslationally modified predominantly by phosphorylation, which occurs primarily at the N-terminal domain. The growth-related kinase pp90rsk, but neither the pp44mapk nor the pp70s6k, can phosphorylate recombinant Nur77 in vitro. Furthermore, we have identified a site within the region required for sequence-specific DNA binding, Ser-354, that is phosphorylated by pp90rsk in vitro; this site is also phosphorylated in vivo. The possibility that phosphorylation might affect DNA binding is discussed.

3T3 Cells↗
PubMed1992

Rapamycin selectively inhibits interleukin-2 activation of p70 S6 kinase.

The macrolide rapamycin induces cell cycle G1 arrest in yeast and in mammalian cells, which suggests that an evolutionarily conserved, rapamycin-sensitive pathway may regulate entry into S phase. In mammals, rapamycin inhibits interleukin-2 receptor-induced S phase entry and subsequent T-cell proliferation, resulting in immunosuppression. Here we show that interleukin-2 selectively stimulates the phosphorylation and activation of p70 S6 kinase but not the erk-encoded MAP kinases and rsk-encoded S6 kinases. Rapamycin completely and rapidly inhibits interleukin-2-induced phosphorylation and activation of p70 S6 kinase at concentrations comparable to those blocking S phase entry of T cells (0.05-0.2 nM). The structurally related macrolide FK506 competitively antagonizes the actions of rapamycin, indicating that these effects are mediated by FKBP, which binds the transition-state mimic structure common to both rapamycin and FK506 (refs 4, 6, 9-11). The selective blockade of the p70 S6 kinase activation cascade by the rapamycin-FKBP complex implicates this signalling pathway in the regulation of T cell entry into S phase.

Carrier Proteins↗
PubMed1992

Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases.

The macrolide rapamycin blocks cell cycle progression in yeast and various animal cells by an unknown mechanism. We demonstrate that rapamycin blocks the phosphorylation and activation of the 70 kd S6 protein kinases (pp70S6K) in a variety of animal cells. The structurally related drug FK506 had no effect on pp70S6K activation but at high concentrations reversed the rapamycin-induced block, confirming the requirement for the rapamycin and FK506 receptor, FKBP. Rapamycin also interfered with signaling by these S6 kinases, blocking serum-stimulated S6 phosphorylation and delaying entry of Swiss 3T3 cells into S phase. Neither rapamycin nor FK506 blocked activation of a distinct family of S6 kinases (RSKs) or the MAP kinases. These studies identify a rapamycin-sensitive signaling pathway, argue for a ubiquitous role for FKBPs in signal transduction, indicate that FK506-FKBP-calcineurin complexes do not interfere with pp70S6K signaling, and show that in fibroblasts pp70S6K, not RSK, is the physiological S6 kinase.

3T3 Cells↗
PubMed1992

ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf-1, and RSK.

p21c-ras plays a critical role in mediating tyrosine kinase-stimulated cell growth and differentiation. However, the pathways through which p21c-ras propagates these signals remain unknown. We report that in PC12 cells, expression of a dominant inhibitory mutant of ras, c-Ha-ras(Asn-17), antagonizes growth factor- and phorbol ester-induced activation of the erk-encoded family of MAP kinases, the 85-92 kd RSKs, and the kinase(s) responsible for hyperphosphorylation of the proto-oncogene product Raf-1. In addition, we find that expression of the activated ras oncogene is sufficient to stimulate these events. These data indicate that ras mediates nerve growth factor receptor and protein kinase C modulation of MAP kinases, RSKs, and Raf-1.

Animals↗
PubMed1992

Nuclear localization and regulation of erk- and rsk-encoded protein kinases.

We demonstrate that members of the erk-encoded family of mitogen-activated protein (MAP) kinases (pp44/42mapk/erk) and members of the rsk-encoded protein kinases (RSKs or pp90rsk) are present in the cytoplasm and nucleus of HeLa cells. Addition of growth factors to serum-deprived cells results in increased tyrosine and threonine phosphorylation and in the activation of cytosolic and nuclear MAP kinases. Activated MAP kinases then phosphorylate (serine/threonine) and activate RSKs. Concurrently, a fraction of the activated MAP kinases and RSKs enter the nucleus. In addition, a distinct growth-regulated RSK-kinase activity (an enzyme[s] that phosphorylates recombinant RSK in vitro and that may be another member of the erk-encoded family of MAP kinases) was found associated with a postnuclear membrane fraction. Regulation of nuclear MAP kinase and RSK activities by growth factors and phorbol ester is coordinate with immediate-early gene expression. Indeed, in vitro, MAP kinase and/or RSK phosphorylates histone H3 and the recombinant c-Fos and c-Jun polypeptides, transcription factors phosphorylated in a variety of cells in response to growth stimuli. These in vitro studies raise the possibility that the MAP kinase/RSK signal transduction pathway represents a protein-Tyr/Ser/Thr phosphorylation cascade with the spatial distribution and temporal regulation that can account for the rapid transmission of growth-regulating information from the membrane, through the cytoplasm, and to the nucleus.

Amino Acid Sequence↗
PubMed1991

Mitogen-activated Swiss mouse 3T3 RSK kinases I and II are related to pp44mpk from sea star oocytes and participate in the regulation of pp90rsk activity.

Using a recombinant rsk gene product as a substrate for in vitro kinase assays, we have identified two mitogen-activated Swiss 3T3 RSK protein kinase activities (referred to as RSK kinase I and RSK kinase II, based on their order of elution from phenyl-Sepharose). Polyclonal antisera prepared against maturation-regulated 44-kDa myelin basic protein (MBP) kinase (pp44mpk) purified from sea star oocytes demonstrated immunocrossreactivity with polypeptides of approximately 44 kDa in the RSK kinase I preparation and approximately 42 kDa in the RSK kinase II preparation, respectively. These polypeptides were also recognized by anti-phosphotyrosine antibodies, and either phosphatase 1B or 2A (tyrosine- and serine/threonine-specific phosphatases, respectively) separately inactivated RSK phosphotransferase activity supporting the notion that tyrosine and serine/threonine phosphorylation are required for activity. In vitro, both RSK kinases and MBP kinase phosphorylated recombinant RSK and generated nearly identical two-dimensional tryptic phosphopeptide maps. They also phosphorylated MBP and microtubule-associated protein 2 but not 40S ribosomal protein S6. Furthermore, these protein kinases phosphorylated and partially activated pp90rsk in immune complexes obtained from quiescent cells.

Animals↗
PubMed1991

Heat shock induces two distinct S6 protein kinase activities in quiescent mammalian fibroblasts.

The regulation of S6 kinase activity was used to monitor perturbations of intracellular signaling activity during heat shock of quiescent murine and human fibroblasts. Previous reports on exponentially growing insect and plant cells had indicated that 40S ribosomal protein S6 is dephosphorylated during heat shock; thus inhibition of S6 kinase activity by heat shock was anticipated in NIH 3T3 fibroblasts and human cells (HeLa, diploid embryonic fibroblasts MRC-5, and skin-derived fibroblasts). Unexpectedly, two distinct S6 protein kinases were activated in quiescent fibroblasts after heat exposure. One of the enzymes was partially purified by sequential column chromatography and was determined to be equivalent to the enzyme activated by serum and other growth factors, referred to here as pp70-S6 protein kinase. The other protein S6 kinase, pp90rsk, was identified by a specific immunoprecipitation assay. Monitoring both enzymatic activities during heat shock revealed a temporal pattern of activation that was reversed when compared to non-stressed, mitogen-stimulated cells. Finally, heat shock stimulated protein S6 phosphorylation in cultured, quiescent mammalian cells. These data demonstrate that specific protein kinases can be activated during heat shock, and that some early mitogenic signals may also participate in the response of cells to physiologic stress.

Animals↗
PubMed1991

Regulation of pp90rsk phosphorylation and S6 phosphotransferase activity in Swiss 3T3 cells by growth factor-, phorbol ester-, and cyclic AMP-mediated signal transduction.

Somatic cell homologs to the Xenopus laevis S6 protein kinases (referred to collectively as pp90rsk) have recently been identified and partially characterized. Here we examine alterations in pp90rsk phosphorylation and S6 phosphotransferase activity in response to regulators of multiple signal transduction systems: purified growth factors, phorbol ester, changes in cyclic AMP (cAMP) levels, and sodium vanadate. All reagents tested increased pp90rsk serine and threonine phosphorylation, but only those agents that regulate cell proliferation and sodium vanadate activated its S6 kinase activity. In addition to the cAMP-stimulated phosphorylation of pp90rsk, a simple correlation between the extent of growth-regulated pp90rsk phosphorylation and S6 phosphotransferase activity was not observed. Quantitative phosphorylation of pp90rsk continued to increase after its S6 kinase activity began its return towards basal levels. However, a close correlation between the appearance and disappearance of a slow-mobility form of phosphorylated pp90rsk (by electrophoresis) and pp90rsk activity was observed. In addition, pp90rsk was regulated by both protein kinase C-independent and -dependent signaling mechanisms. The extent of protein kinase C participation, however, varied depending on which growth factor receptor was activated. Furthermore, growth factor-specific differences in the temporal regulation of pp90rsk S6 phosphotransferase activity were also observed. These results support the notion that the complex regulation of the rsk gene product constitutes one of the primary responses of animal cells to mitogenic signals.

Animals↗