CoChat
Sign Up
Cited by 83Genomics and Chromatin DynamicsCytokine Signaling Pathways and InteractionsCell death mechanisms and regulation

Interleukin-3 Stimulation of mcl-1 Gene Transcription Involves Activation of the PU.1 Transcription Factor through a p38 Mitogen-Activated Protein Kinase-Dependent Pathway

Open access
Wang, Ju-Ming, Lai, Ming-Zong, Yang-Yen, Hsin-Fang·2003·Molecular and Cellular Biology
DOI 10.1128/mcb.23.6.1896-1909.2003PMC PMC149468
Open this paper in CoChat — free account

Abstract

We have previously demonstrated that the antiapoptotic gene mcl-1 is activated by interleukin-3 (IL-3) in Ba/F3 pro-B cells through two promoter elements designated the CRE-2 and SIE motifs. While the CRE-2-binding complex contains the CREB protein and is activated by IL-3 through the phosphatidylinositol 3-kinase/Akt-dependent pathway, the identity and cytokine activation pathway of the SIE-binding complex remains unclear. In this report, we demonstrated that PU.1 is one component of the SIE-binding complex. A chromatin immunoprecipitation assay further confirmed that PU.1 binds to the mcl-1 promoter region containing the SIE motif in vivo. While IL-3 stimulation does not significantly alter the SIE-binding activity of PU.1, it markedly increases PU.1's transactivation activity. The latter effect coincides with the increased phosphorylation of PU.1 following IL-3 activation of a p38 mitogen-activated protein kinase (p38(MAPK))-dependent pathway. A serine-to-alanine substitution at position 142 significantly weakens PU.1's ability to be phosphorylated by the p38(MAPK) immunocomplex. Furthermore, this S142A mutant is impaired in the ability to be further stimulated by IL-3 to transactivate the mcl-1 reporter through the SIE motif. Taken together, our results demonstrate that IL-3 stimulation of mcl-1 gene transcription through the SIE motif involves phosphorylation of PU.1 at serine 142 by a p38(MAPK)-dependent pathway.

AI snapshot

Written from the paper’s full text.

TL;DR

IL-3 stimulation of the antiapoptotic gene *mcl-1* through its SIE promoter motif involves PU.1, whose transactivation activity is increased by p38MAPK-dependent phosphorylation at serine 142. This defines a p38MAPK/PU.1 pathway that acts alongside the previously described PI3-K/Akt/CREB pathway at the *mcl-1* locus.

Key findings

  • PU.1 was identified as a component of the SIE-binding complex on the *mcl-1* promoter; in a screen of 5 × 10⁵ clones, 23 positives represented 11 distinct cDNAs, and PU.1 cDNA was pulled out four times.
  • IL-3 did not significantly change PU.1 DNA-binding activity, but it markedly increased PU.1 transactivation activity; ChIP confirmed PU.1 bound the *mcl-1* promoter region containing the SIE motif in vivo.
  • IL-3 stimulated the p(−203/+10)mcl-luc reporter about three- to fourfold; mutation of CRE-2 or SIE reduced this to about twofold. SB203580 and wortmannin each inhibited IL-3 stimulation of *mcl-1* mRNA by approximately 50%, and together nearly completely blocked it.
  • A serine-to-alanine substitution at PU.1 position 142 (S142A) markedly weakened phosphorylation by the p38MAPK immunocomplex and impaired IL-3-stimulated transactivation through the SIE motif; S148A did not significantly affect this phosphorylation.

Methods

  • Used IL-3-dependent mouse Ba/F3 pro-B cells, including a Ba/F3 derivative stably overexpressing HA-tagged PU.1.
  • Combined cDNA expression library screening, oligonucleotide pulldown, gel shift/supershift, chromatin immunoprecipitation, reporter luciferase assays, Northern/Western blotting, and in vitro kinase assays.
  • Tested wild-type and mutant PU.1 constructs, dominant negative p38α/p38β and PI3-K constructs, and chemical inhibitors SB203580 and wortmannin.

Limitations

  • Stated in the paper: the identity of the putative p38α-associated kinase that phosphorylates PU.1 at Ser-142 remains unknown; the data cannot distinguish whether PU.1 is directly phosphorylated by p38MAPK or by another associated kinase in the p38MAPK immunocomplex.
  • Stated in the paper: the role of the B1 SIE-binding complex is unclear, and it is not known whether B1 contains PU.1 or contributes to SIE-mediated IL-3 induction.
  • Evident from the text: the mechanistic conclusions rely mainly on Ba/F3 cells and reporter assays, with no PU.1-null or in vivo rescue experiments to establish the pathway’s developmental or physiological role.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1128/mcb.23.6.1896-1909.2003

Cites

63 of 63 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

19 of 39 cited sources checked · 12 supported · 0 flagged · accuracy not yet measured

  • Premisecitation
    § RESULTS

    HA/PU.1dlN133 has a dominant negative effect.

    “a vector expressing mutant PU.1 (HA/PU.1dlN133) that was previously shown to have a dominant negative effect (43).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [43] Shin, M. K., and M. E. Koshland. 1993. Ets-related protein PU.1 regulates expression of the immunoglobulin J-chain gene through a novel Ets-binding element. Genes Dev. 7:2006-2015. [DOI] [PubMed] [Google Scholar] source
  • Findingcitation
    § RESULTS

    Mutation of the CRE-2 or SIE motif resulted in a reporter that still responded to IL-3 stimulation but to a lesser extent (about twofold).

    “Mutation of the CRE-2 or SIE motif resulted in a reporter that still responded to IL-3 stimulation but to a lesser extent (about twofold)”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [49] Wang, J.-M., J.-R. Chao, W. Chen, M.-L. Kuo, J. J.-Y. Yen, and H.-F. Yang-Yen. 1999. The antiapoptotic gene mcl-1 is up-regulated by the phosphatidylinositol 3-kinase/Akt signaling pathway through a transcription factor complex containing CREB. Mol. Cell. Biol. 19:6195-6206. [DOI] [PMC free article] [PubMed] [Google Scholar] source

      The Results (Fig. 7B; page unavailable) report that mutating either CRE-2 or SIE reduced IL-3 induction by approximately 40%, so the reporters remained responsive. The copy does not give an approximately twofold response for those mutant reporters; its ≤twofold figure refers to different reporters containing a single motif inserted upstream of an SV40 promoter (Fig. 8). (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • Premisecitation
    § Abstract

    IL-3 inducibility of mcl-1 in Ba/F3 pro-B cells is mediated mainly through two upstream DNA motifs located at positions −70 (CRE-2) and −87 (SIE).

    “Through cloning and extensive characterization of the mcl-1 promoter, we have found that the IL-3 inducibility of this gene in Ba/F3 pro-B cells is mediated mainly through two upstream DNA motifs located at positions −70 (the CRE-2 site) and −87 (the SIE site) (49).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [49] Wang, J.-M., J.-R. Chao, W. Chen, M.-L. Kuo, J. J.-Y. Yen, and H.-F. Yang-Yen. 1999. The antiapoptotic gene mcl-1 is up-regulated by the phosphatidylinositol 3-kinase/Akt signaling pathway through a transcription factor complex containing CREB. Mol. Cell. Biol. 19:6195-6206. [DOI] [PMC free article] [PubMed] [Google Scholar] source
  • Findingcitation
    § RESULTS

    IL-3 stimulated (about three- to fourfold) the luciferase activity of cells transfected with p(−203/+10)mcl-luc.

    “IL-3 stimulated (about three- to fourfold) the luciferase activity of cells transfected with a luciferase reporter driven by the mcl-1 promoter region between −203 and +10, i.e., the p(−203/+10)mcl-luc construct”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [49] Wang, J.-M., J.-R. Chao, W. Chen, M.-L. Kuo, J. J.-Y. Yen, and H.-F. Yang-Yen. 1999. The antiapoptotic gene mcl-1 is up-regulated by the phosphatidylinositol 3-kinase/Akt signaling pathway through a transcription factor complex containing CREB. Mol. Cell. Biol. 19:6195-6206. [DOI] [PMC free article] [PubMed] [Google Scholar] source

      Materials and Methods identifies p(−203/+10)mcl-luc as an mcl-1 promoter luciferase reporter tested in Ba/F3 cells with and without IL-3. The Results report IL-3 induction of that reporter (Fig. 7B), but the passages in our copy do not state the claimed three- to fourfold magnitude. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Premisecitation
    § RESULTS

    IL-3 activation of mcl-1 gene transcription through the SIE motif occurs via an unknown but PI3-K/Akt-independent pathway.

    “We have previously demonstrated that IL-3 activation of mcl-1 gene transcription through the SIE motif occurs via an unknown but PI3-K/Akt-independent pathway (49).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [49] Wang, J.-M., J.-R. Chao, W. Chen, M.-L. Kuo, J. J.-Y. Yen, and H.-F. Yang-Yen. 1999. The antiapoptotic gene mcl-1 is up-regulated by the phosphatidylinositol 3-kinase/Akt signaling pathway through a transcription factor complex containing CREB. Mol. Cell. Biol. 19:6195-6206. [DOI] [PMC free article] [PubMed] [Google Scholar] source
  • May combine two claims. 23
  • Classified as probably not a substantial claim. 23

  • May combine two claims.

    Premisecitation
    § Abstract

    Cytokine activation of the mcl-1 gene is regulated at the transcriptional level and requires the membrane-distal region between amino acids 573 and 755 of the common β chain of the GM-CSF and IL-3 receptors.

    “Cytokine activation of the mcl-1 gene is regulated at the transcriptional level and requires the membrane-distal region between amino acids 573 and 755 of the common β chain of the GM-CSF and IL-3 receptors (5).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [5] Chao, J.-R., J.-M. Wang, S.-F. Lee, H.-W. Peng, Y.-H. Lin, C.-H. Chou, J.-C. Li, H.-M. Huang, C.-K. Chou, M.-L. Kuo, J. J.-Y. Yen, and H.-F. Yang-Yen. 1998. mcl-1 is an immediate-early gene activated by the granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling pathway and is one component of the GM-CSF viability response. Mol. Cell. Biol. 18:4883-4898. [DOI] [PMC free article] [PubMed] [Google Scholar] source

  • May combine two claims.

    Premisecitation
    § Abstract

    mcl-1 is an immediate-early gene activated by the GM-CSF and IL-3 signaling pathways, and the mcl-1 gene product is one component of the viability response of these two cytokines.

    “We have previously demonstrated that mcl-1 is an immediate-early gene activated by the GM-CSF and IL-3 signaling pathways and that the mcl-1 gene product is one component of the viability response of these two cytokines (5).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [5] Chao, J.-R., J.-M. Wang, S.-F. Lee, H.-W. Peng, Y.-H. Lin, C.-H. Chou, J.-C. Li, H.-M. Huang, C.-K. Chou, M.-L. Kuo, J. J.-Y. Yen, and H.-F. Yang-Yen. 1998. mcl-1 is an immediate-early gene activated by the granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling pathway and is one component of the GM-CSF viability response. Mol. Cell. Biol. 18:4883-4898. [DOI] [PMC free article] [PubMed] [Google Scholar] source

  • May combine two claims.

    Premisecitation
    § Abstract

    The CRE-2 and SIE promoter elements can each confer IL-3 inducibility on a heterologous promoter but work additively in mediating IL-3 response via two different signaling pathways.

    “Interestingly, these two promoter elements can each confer IL-3 inducibility on a heterologous promoter but work additively in mediating IL-3 response via two different signaling pathways.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [49] Wang, J.-M., J.-R. Chao, W. Chen, M.-L. Kuo, J. J.-Y. Yen, and H.-F. Yang-Yen. 1999. The antiapoptotic gene mcl-1 is up-regulated by the phosphatidylinositol 3-kinase/Akt signaling pathway through a transcription factor complex containing CREB. Mol. Cell. Biol. 19:6195-6206. [DOI] [PMC free article] [PubMed] [Google Scholar] source

  • May combine two claims.

    Premisecitation
    § Abstract

    The CRE-2-binding complex contains the CREB protein and is induced and activated by IL-3 via activation of the PI3-K/Akt-dependent pathway.

    “While the CRE-2-binding complex (which contains the CREB protein) is induced and activated by IL-3 via activation of the phosphatidylinositol 3-kinase (PI3-K)/Akt-dependent pathway”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [49] Wang, J.-M., J.-R. Chao, W. Chen, M.-L. Kuo, J. J.-Y. Yen, and H.-F. Yang-Yen. 1999. The antiapoptotic gene mcl-1 is up-regulated by the phosphatidylinositol 3-kinase/Akt signaling pathway through a transcription factor complex containing CREB. Mol. Cell. Biol. 19:6195-6206. [DOI] [PMC free article] [PubMed] [Google Scholar] source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § Abstract

    The identity and the IL-3 activation pathway of the SIE-binding complex remain to be determined.

    “the identity and the IL-3 activation pathway of the SIE-binding complex remain to be determined (49).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [49] Wang, J.-M., J.-R. Chao, W. Chen, M.-L. Kuo, J. J.-Y. Yen, and H.-F. Yang-Yen. 1999. The antiapoptotic gene mcl-1 is up-regulated by the phosphatidylinositol 3-kinase/Akt signaling pathway through a transcription factor complex containing CREB. Mol. Cell. Biol. 19:6195-6206. [DOI] [PMC free article] [PubMed] [Google Scholar] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Abstract

    PU.1 expression is restricted specifically to cells of the hematopoietic lineage, including B cells, macrophages, mast cells, neutrophils, and early erythroblasts.

    “The expression of PU.1 is restricted specifically to cells of the hematopoietic lineage. These include B cells, macrophages, mast cells, neutrophils, and early erythroblasts (6, 10, 12, 20, 32, 37).”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [6] Chen, H. M., P. Zhang, M. T. Voso, S. Hohaus, D. A. Gonzalez, C. K. Glass, D. E. Zhang, and D. G. Tenen. 1995. Neutrophils and monocytes express high levels of PU.1 (Spi-1) but not Spi-B. Blood 85:2918-2928. [PubMed] [Google Scholar] source
    • cites [10] Galson, D. L., J. O. Hensold, T. R. Bishop, M. Schalling, A. D. D'Andrea, C. Jones, P. E. Auron, and D. E. Housman. 1993. Mouse β-globin DNA-binding protein B1 is identical to a proto-oncogene, the transcription factor Spi-1/PU.1, and is restricted in expression to hematopoietic cells and the testis. Mol. Cell. Biol. 13:2929-2941. [DOI] [PMC free article] [PubMed] [Google Scholar] source

      The Results and Discussion (page unavailable) report PU.1/Spi-1 expression in B cells, macrophages, mast cells, and developing erythroid cells. The paper does not establish expression in neutrophils, and its finding of expression in the testis—possibly in Leydig cells—does not support an unqualified restriction to hematopoietic cells. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [12] Hromas, R., A. Orazi, R. S. Neiman, R. Maki, C. Van Beveran, J. Moore, and M. Klemsz. 1993. Hematopoietic lineage- and stage-restricted expression of the ETS oncogene family member PU.1. Blood 82:2998-3004. [PubMed] [Google Scholar] source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § Abstract

    PU.1 is a member of the Ets family of transcription factors, characterized by a DNA-binding domain that recognizes a core DNA element containing 5′-GGAA/T-3′.

    “PU.1 is a member of the Ets family of transcription factors, and this family of proteins is characterized by the presence of a DNA-binding domain that recognizes a core DNA element containing the 5′-GGAA/T-3′ motif (16, 28, 31).”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [16] Karim, F. D., L. D. Urness, C. S. Thummel, M. J. Klemsz, S. R. McKercher, A. Celada, C. Van Beveren, R. A. Maki, C. V. Gunther, and J. A. Nye. 1990. The ETS-domain: a new DNA-binding motif that recognizes a purine-rich core DNA sequence. Genes Dev. 4:1451-1453. [DOI] [PubMed] [Google Scholar] source

      The paper identifies PU.1 as an ETS-domain protein and describes the ETS-domain as a DNA-binding motif. Its discussion of binding sites identifies a common GGAA motif, but our copy does not establish the alternative GGAT sequence represented by “GGAA/T.” (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [28] Macleod, K., D. Leprince, and D. Stehelin. 1992. The ets gene family. Trends Biochem. Sci. 17:251-256. [DOI] [PubMed] [Google Scholar] source
    • cites [31] Nye, J. A., J. M. Petersen, C. V. Gunther, M. D. Jonsen, and B. J. Graves. 1992. Interaction of murine ets-1 with GGA-binding sites establishes the ETS domain as a new DNA-binding motif. Genes Dev. 6:975-990. [DOI] [PubMed] [Google Scholar] source

      The paper identifies PU.1 as an Ets-family member and describes the conserved ETS domain as a DNA-binding domain. In “Sequence specificity of DNA binding of the ETS domain proteins” (page not provided), it says all Ets-family members bind sites with a central GGA triplet; the more specific GGAA/T sequence appears in the Ets-1 consensus, but is not established as a family-wide motif. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Abstract

    Many B-cell- and myeloid-specific genes, including those encoding immunoglobulins, receptors, and enzymes, have been reported to be directly regulated by PU.1 or have a potential PU.1-binding site in their promoters.

    “Consistent with this finding, many B-cell- and myeloid-specific genes, including those encoding immunoglobulins, receptors, and enzymes, have been reported to be directly regulated by PU.1 or have a potential PU.1-binding site in their promoters (7, 26, 53).”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [7] DeKoter, R. P., H. J. Lee, and H. Singh. 2002. PU.1 regulates expression of the interleukin-7 receptor in lymphoid progenitors. Immunity 16:297-309. [DOI] [PubMed] [Google Scholar] source
    • cites [26] Lloberas, J., C. Soler, and A. Celada. 1999. The key role of PU.1/SPI-1 in B cells, myeloid cells and macrophages. Immunol. Today 20:184-189. [DOI] [PubMed] [Google Scholar] source
    • cites [53] Yordy, J. S., and R. C. Muise-Helmericks. 2000. Signal transduction and the Ets family of transcription factors. Oncogene 19:6503-6513. [DOI] [PubMed] [Google Scholar] source

      The indexed paper’s Abstract/intro passage says Spi-1 (PU.1) and C/EBP factors regulate many immunity- and hematopoiesis-related genes, naming macrophage receptors and neutrophil elastase; it also identifies a Spi-1-binding site in the il1b promoter. The cited passages support the myeloid receptor and enzyme examples, but do not establish the claim about B-cell-specific genes or immunoglobulins. Page numbers are unavailable in our copy. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation
    § DISCUSSION

    The CRE-2-binding complex contains the CREB protein and is activated by IL-3 through a PI3-K/Akt-dependent pathway.

    “the CRE-2-binding complex contains the CREB protein and is activated by IL-3 through a PI3-K/Akt-dependent pathway (49).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [49] Wang, J.-M., J.-R. Chao, W. Chen, M.-L. Kuo, J. J.-Y. Yen, and H.-F. Yang-Yen. 1999. The antiapoptotic gene mcl-1 is up-regulated by the phosphatidylinositol 3-kinase/Akt signaling pathway through a transcription factor complex containing CREB. Mol. Cell. Biol. 19:6195-6206. [DOI] [PMC free article] [PubMed] [Google Scholar] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    Phosphorylation of two serine residues (positions 41 and 45) located at one of the acidic transactivation domains is necessary for macrophage colony-stimulating factor-dependent proliferation of bone marrow macrophages.

    “phosphorylation of two serine residues (positions 41 and 45) located at one of the acidic transactivation domains is necessary for macrophage colony-stimulating factor-dependent proliferation of bone marrow macrophages (4)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [4] Celada, A., F. E. Borras, C. Soler, J. Lloberas, M. Klemsz, C. van Beveren, S. McKercher, and R. A. Maki. 1996. The transcription factor PU.1 is involved in macrophage proliferation. J. Exp. Med. 184:61-69. [DOI] [PMC free article] [PubMed] [Google Scholar] source

      The Results (Fig. 7B; page not supplied) report that substituting alanine for serine 41 or 45 prevented PU.1 from enhancing proliferation of bone marrow-derived macrophages and lowered basal proliferation. The paper infers that phosphorylation at one or both positions likely activates an important domain, but does not establish that phosphorylation of both is necessary or identify that domain as acidic. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    STAT3 protein in peripheral blood mononuclear cell extracts isolated from patients with large granular lymphocyte leukemia has been reported to bind to the murine SIE motif.

    “the STAT3 protein in the peripheral blood mononuclear cell extracts isolated from patients with large granular lymphocyte leukemia has been reported to bind to the murine SIE motif (9).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [9] Epling-Burnette, P. K., J. H. Liu, R. Catlett-Falcone, J. Turkson, M. Oshiro, R. Kothapalli, Y. Li, J.-M. Wang, H.-F. Yang-Yen, J. Karras, R. Jove, and T. P. Loughran, Jr. 2001. Inhibition of STAT3 signaling leads to apoptosis of leukemic large granular lymphocytes and decreased Mcl-1 expression. J. Clin. Investig. 107:351-362. [DOI] [PMC free article] [PubMed] [Google Scholar] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    Mcl-1 has been found to be expressed in many other cell types of nonhematopoietic origin.

    “Mcl-1 has been found to be expressed in many other cell types of nonhematopoietic origin (23, 39).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [23] Krajewski, S., S. Bodrug, M. Krajewska, A. Shabaik, R. Gascoyne, K. Berean, and J. C. Reed. 1995. Immunohistochemical analysis of Mcl-1 protein in human tissues: differential regulation of Mcl-1 and Bcl-2 protein production suggests a unique role for Mcl-1 in control of programmed cell death in vivo. Am. J. Pathol. 146:1309-1319. [PMC free article] [PubMed] [Google Scholar]
    • cites [39] Rinkenberger, J. L., S. Horning, B. Klocke, K. Roth, and S. J. Korsmeyer. 2000. Mcl-1 deficiency results in peri-implantation embryonic lethality. Genes Dev. 14:23-27. [PMC free article] [PubMed] [Google Scholar] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § RESULTS

    PU.1 cDNA was pulled out four times (all with different sizes) in the library screen.

    “PU.1 cDNA was pulled out four times (all with different sizes) in our library screen”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    Neither isotype-matched rabbit IgG nor an antibody recognizing p53 precipitated the C fragment.

    “As shown in Fig. 2C, neither an isotype-matched rabbit IgG (lanes 2 and 3) nor an antibody recognizing an irrelevant transcription factor (p53, lanes 6 and 7) precipitated this C fragment.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    The pulldown of PU.1 was specific to the SIE element, inhibited by wild-type SIE but not mSIE.

    “The pulldown of PU.1 in this experiment was specific to the SIE element, as it was specifically inhibited by oligonucleotides containing the wild-type SIE site but not by oligonucleotides containing the mSIE site (data not shown).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    Of 5 × 105 clones screened, 23 were positive and sequence analysis revealed they represented clones derived from 11 distinct cDNAs.

    “Of 5 × 105 clones screened, 23 were positive and sequence analysis revealed that they represented clones derived from 11 distinct cDNAs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    In the antibody supershift assay, the B2 complex, but not the B1 complex, was specifically supershifted by the PU.1 antibody.

    “Unexpectedly, it was the B2 but not the B1 complex that was specifically supershifted by the PU.1 antibody (compare lanes 4 and 5 in Fig. 1C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    The identity of the protein-DNA complex formed with HA/PU.1 was confirmed by supershift with PU.1 antibody or competition with wild-type SIE but not mSIE.

    “The identity of the protein-DNA complex formed with HA/PU.1 in this assay was further confirmed either by its ability to be supershifted by a PU.1 antibody (lane 4) or by its ability to be competed out by a 100-fold molar excess of unlabeled wild-type SIE but not mSIE oligonucleotide (data not shown).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § DISCUSSION

    PU.1 binds to the SIE element constitutively but its transactivation activity is increased upon IL-3 stimulation of cells.

    “PU.1 binds to the SIE element constitutively but its transactivation activity is increased upon IL-3 stimulation of cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    The inhibitory effect of DN-p38α was specifically mediated through the SIE motif, as IL-3 inducibility of the CRE-2 reporter was not affected by DN-p38α.

    “This inhibitory effect was specifically mediated through the SIE motif, as under the same conditions, the IL-3 inducibility of the CRE-2 reporter (pGL-2XCRE-2) was not affected by DN-p38α (compare lanes 9 and 10 in Fig. 5B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    Either inhibitor had an approximately 50% inhibitory effect, and both together nearly completely blocked IL-3 stimulation of mcl-1 expression.

    “While either inhibitor had an approximately 50% inhibitory effect, both inhibitors, when added together to cells, nearly completely blocked IL-3 stimulation of mcl-1 expression.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: SB203580 specifically inhibits the α and β isoforms of p38MAPK.

  • Classified as probably not a substantial claim.

    Findingnone given
    § DISCUSSION

    The core sequences of the CRE-2 and SIE motifs are separated by 9 bp.

    “the core sequences of the CRE-2 and SIE motifs are only separated by 9 bp”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § DISCUSSION

    In the gel shift assay with the SIE probe, in addition to the B2 complex that contains PU.1, another specific B1 complex was consistently detected in the Ba/F3 cell extracts.

    “in addition to the B2 complex that contains PU.1, another specific B1 complex was consistently detected in the Ba/F3 cell extracts (Fig. 1).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § DISCUSSION

    The formation of the B1 complex, like that of B2, is not influenced by the presence or absence of IL-3.

    “The formation of this B1 complex, like that of B2, is not influenced by the presence or absence of IL-3.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § DISCUSSION

    Although STAT3 is activated by IL-3 in Ba/F3 cells, no evidence suggests that it binds to SIE or plays a role in the IL-3 regulation of murine mcl-1 gene transcription.

    “although STAT3 is activated by IL-3 in Ba/F3 cells (30; our unpublished data), no evidence suggests that it binds to SIE or plays a role in the IL-3 regulation of murine mcl-1 gene transcription (our unpublished results).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [30] Nagata, Y., and K. Todokoro. 1996. Interleukin 3 activates not only JAK2 and STAT5, but also Tyk2, STAT1, and STAT3. Biochem. Biophys. Res. Commun. 221:785-789. [DOI] [PubMed] [Google Scholar] source

  • May combine two claims.

    Findingown data
    § DISCUSSION

    PU.1 binds to the SIE motif of the mcl-1 promoter irrespective of IL-3 stimulation, and IL-3 stimulates its phosphorylation-and-transactivation function.

    “PU.1 binds to the SIE motif of the mcl-1 promoter irrespective of IL-3 stimulation, and IL-3 stimulates its phosphorylation-and-transactivation function.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § Abstract

    PU.1 deficiency results in the absence of morphologically normal B cells and macrophages, disrupted granulopoiesis, and aberrant T lymphopoiesis.

    “Knockout mouse studies have demonstrated that PU.1 deficiency results in the absence of morphologically normal B cells and macrophages, disrupted granulopoiesis, and aberrant T lymphopoiesis (29, 41).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [29] McKercher, S. R., B. E. Torbett, K. L. Anderson, G. W. Henkel, D. J. Vestal, H. Baribault, M. Klemsz, A. J. Feeney, G. E. Wu, C. J. Paige, and R. A. Maki. 1996. Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. EMBO J. 15:5647-5658. [PMC free article] [PubMed] [Google Scholar] source
    • cites [41] Scott, E. W., M. C. Simon, J. Anastasi, and H. Singh. 1994. Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages. Science 265:1573-1577. [DOI] [PubMed] [Google Scholar] source

  • May combine two claims.

    Interpretationown data
    § RESULTS

    The p38MAPK and PI3-K pathways both contribute to the regulation of Mcl-1 expression and to the survival activity of IL-3.

    “suggests that the p38MAPK and PI3-K pathways both contribute to the regulation of Mcl-1 expression, as well as to the survival activity of IL-3.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: SB203580 specifically inhibits p38MAPK activity in the cells used, without affecting other kinases that could influence the measured phosphorylation, apoptosis, or transcription.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    Serine 148 phosphorylation is required for the lipopolysaccharide-induced transactivation function of PU.1.

    “The serine 148 phosphorylation was also found to be required for the lipopolysaccharide-induced transactivation function of PU.1 (27).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [27] Lodie, T. A., R. Savedra, Jr., D. T. Golenbock, C. P. Van Beveren, R. A. Maki, and M. J. Fenton. 1997. Stimulation of macrophages by lipopolysaccharide alters the phosphorylation state, conformation, and function of PU.1 via activation of casein kinase II. J. Immunol. 158:1848-1856. [PubMed] [Google Scholar] source

  • May combine two claims.

    Interpretationown data
    § RESULTS

    PU.1 is involved in IL-3 stimulation of mcl-1 gene transcription and its effect is mediated through binding to the SIE motif.

    “Taken together, these results suggest that PU.1 is involved in IL-3 stimulation of mcl-1 gene transcription and that its effect is mediated through binding to the SIE motif.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § RESULTS

    Phosphorylation of PU.1 at serine 148 is required for its interaction with PIP (NF-EM5) and activates the immunoglobulin κ 3′ enhancer.

    “phosphorylation at serine 148 is required for its interaction with a B-cell-restricted transcription factor, PIP (NF-EM5), and activates the immunoglobulin κ 3′ enhancer (35).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [35] Pongubala, J. M., C. Van Beveren, S. Nagulapalli, M. J. Klemsz, S. R. McKercher, R. A. Maki, and M. L. Atchison. 1993. Effect of PU.1 phosphorylation on interaction with NF-EM5 and transcriptional activation. Science 259:1622-1625. [DOI] [PubMed] [Google Scholar] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    Serine phosphorylation at position 148 in the proline-, glutamic-acid-, serine-, and threonine-rich domain mediates the interaction between PU.1 and the B-cell enhancer factor (NF-EM5).

    “a serine phosphorylation at position 148 in the proline-, glutamic-acid-, serine-, and threonine-rich domain mediates the interaction between PU.1 and the B-cell enhancer factor (NF-EM5).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [35] Pongubala, J. M., C. Van Beveren, S. Nagulapalli, M. J. Klemsz, S. R. McKercher, R. A. Maki, and M. L. Atchison. 1993. Effect of PU.1 phosphorylation on interaction with NF-EM5 and transcriptional activation. Science 259:1622-1625. [DOI] [PubMed] [Google Scholar] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    The activity of PU.1 is primarily regulated posttranscriptionally by phosphorylation.

    “The activity of PU.1 is primarily regulated posttranscriptionally by phosphorylation (reviewed in reference 26).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [26] Lloberas, J., C. Soler, and A. Celada. 1999. The key role of PU.1/SPI-1 in B cells, myeloid cells and macrophages. Immunol. Today 20:184-189. [DOI] [PubMed] [Google Scholar] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    Casein kinase II has been suggested to be responsible for phosphorylation of PU.1 at serine 148.

    “casein kinase II has been suggested to be responsible for phosphorylation of PU.1 at serine 148 (27, 35)”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [27] Lodie, T. A., R. Savedra, Jr., D. T. Golenbock, C. P. Van Beveren, R. A. Maki, and M. J. Fenton. 1997. Stimulation of macrophages by lipopolysaccharide alters the phosphorylation state, conformation, and function of PU.1 via activation of casein kinase II. J. Immunol. 158:1848-1856. [PubMed] [Google Scholar] source
    • cites [35] Pongubala, J. M., C. Van Beveren, S. Nagulapalli, M. J. Klemsz, S. R. McKercher, R. A. Maki, and M. L. Atchison. 1993. Effect of PU.1 phosphorylation on interaction with NF-EM5 and transcriptional activation. Science 259:1622-1625. [DOI] [PubMed] [Google Scholar] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    Unlike the SIE motif reported in this study, the tetradecanoyl phorbol acetate inducibility of SRE in the human mcl-1 promoter is mediated through the extracellular signal-regulated kinase pathway.

    “Unlike the SIE motif reported in this study, the tetradecanoyl phorbol acetate inducibility of SRE in the human mcl-1 promoter was shown to be mediated through the extracellular signal-regulated kinase pathway (47).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [47] Townsend, K. J., P. Zhou, L. Qian, C. K. Bieszczad, C. H. Lowrey, A. Yen, and R. W. Craig. 1999. Regulation of MCL1 through a serum response factor/Elk-1-mediated mechanism links expression of a viability-promoting member of the BCL2 family to the induction of hematopoietic cell differentiation. J. Biol. Chem. 274:1801-1813. [DOI] [PubMed] [Google Scholar] source

  • May combine two claims.

    Interpretationown data
    § DISCUSSION

    IL-3-stimulated transactivation activity of PU.1 is inhibited by the dominant negative mutant form of p38α but not that of p38β, suggesting that serine 142 phosphorylation is induced via a p38α-dependent pathway.

    “the fact that IL-3-stimulated transactivation activity of PU.1 is inhibited by the dominant negative mutant form of p38α but not that of p38β (Fig. 8B) suggests that serine 142 phosphorylation is induced via a p38α-dependent pathway.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The dominant negative p38α mutant specifically inhibits p38α signaling without affecting p38β or PI3-K signaling in the transactivation assays.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    Both serum response factor and Elk-1 act coordinately to affect both the basal activity and tetradecanoyl phorbol acetate inducibility of the human mcl-1 gene in human K-562 cells.

    “Both serum response factor and Elk-1 act coordinately to affect both the basal activity and tetradecanoyl phorbol acetate inducibility of the human mcl-1 gene in human K-562 cells.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [47] Townsend, K. J., P. Zhou, L. Qian, C. K. Bieszczad, C. H. Lowrey, A. Yen, and R. W. Craig. 1999. Regulation of MCL1 through a serum response factor/Elk-1-mediated mechanism links expression of a viability-promoting member of the BCL2 family to the induction of hematopoietic cell differentiation. J. Biol. Chem. 274:1801-1813. [DOI] [PubMed] [Google Scholar] source

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

    Interpretationreasoning
    § DISCUSSION

    Our identification of PU.1 as being involved in IL-3 regulation of mcl-1 gene expression suggests that Mcl-1 may also contribute to the development of myeloid and/or lymphoid cells.

    “Our identification of PU.1 as being involved in IL-3 regulation of mcl-1 gene expression suggests that Mcl-1 may also contribute to the development of myeloid and/or lymphoid cells.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data
    § DISCUSSION

    The results in Fig. 7 cannot distinguish whether PU.1 is directly phosphorylated by p38MAPK or by another associated kinase in the p38MAPK immunocomplex.

    “our results presented in Fig. 7 cannot distinguish whether PU.1 is directly phosphorylated by p38MAPK or by another associated kinase in the p38MAPK immunocomplex”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    An SIE-like element (SRE) is present in the human mcl-1 gene promoter between nucleotides −105 and −92.

    “An SIE-like element (termed SRE) is present in the human mcl-1 gene promoter (between nucleotides −105 and −92) (47).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [47] Townsend, K. J., P. Zhou, L. Qian, C. K. Bieszczad, C. H. Lowrey, A. Yen, and R. W. Craig. 1999. Regulation of MCL1 through a serum response factor/Elk-1-mediated mechanism links expression of a viability-promoting member of the BCL2 family to the induction of hematopoietic cell differentiation. J. Biol. Chem. 274:1801-1813. [DOI] [PubMed] [Google Scholar] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    The SRE DNA element is recognized by a protein complex containing serum response factor and Elk-1.

    “This DNA element was shown to be recognized by a protein complex containing serum response factor and Elk-1.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [47] Townsend, K. J., P. Zhou, L. Qian, C. K. Bieszczad, C. H. Lowrey, A. Yen, and R. W. Craig. 1999. Regulation of MCL1 through a serum response factor/Elk-1-mediated mechanism links expression of a viability-promoting member of the BCL2 family to the induction of hematopoietic cell differentiation. J. Biol. Chem. 274:1801-1813. [DOI] [PubMed] [Google Scholar] source

  • May combine two claims.

    Interpretationreasoning
    § DISCUSSION

    IL-3 stimulates the phosphorylation of PU.1 at serine 142, which either directly increases the transactivation activity of PU.1 or increases PU.1's ability to recruit another transcriptional activator and activates mcl-1 gene transcription in an indirect manner.

    “IL-3 stimulates the phosphorylation of PU.1 at serine 142, which either directly increases the transactivation activity of PU.1 or increases PU.1's ability to recruit another transcriptional activator and activates mcl-1 gene transcription in an indirect manner.”
    Quote in the paperSubstantial claim
42 of 42

  • The paper relies on this without support.

    SB203580 specifically inhibits the α and β isoforms of p38MAPK.

    “SB203580, which specifically inhibits the α and β isoforms of p38MAPK, manifested the best inhibitory effect (data not shown).”

  • The paper relies on this without support.

    SB203580 specifically inhibits p38MAPK activity in the cells used, without affecting other kinases that could influence the measured phosphorylation, apoptosis, or transcription.

    “blocking of the p38 pathway by treatment of cells with SB203580 also induced apoptosis of cells cultivated in IL-3-containing medium.”
    “IL-3-induced phosphorylation of PU.1 was inhibited by pretreatment of cells with SB203580”

  • The paper relies on this without support.

    The dominant negative p38α mutant specifically inhibits p38α signaling without affecting p38β or PI3-K signaling in the transactivation assays.

    “coexpression of DN-p38α but not DN-p38β or DN-p85 significantly attenuated the stimulation effect of IL-3 on the transactivation activity of PU.1”

  • The paper relies on this without support.

    HA-tagged PU.1 overexpressed in Ba/F3 cells behaves like endogenous PU.1 with respect to IL-3-induced phosphorylation.

    “Ba/F3 cells stably overexpressing HA-tagged PU.1 were stimulated with IL-3 in the presence of [32P]orthophosphate”

  • The paper relies on this without support.

    IL-3 activates the p38MAPK pathway in Ba/F3 cells.

    “p38MAPK immunoprecipitated from cells treated with IL-3 or anisomycin (a known p38MAPK activator)”

  • The paper relies on this without support.

    The SIE-containing reporter constructs used in the transient transfection assays reflect the regulation of the endogenous mcl-1 promoter by IL-3 and PU.1.

    “the −203/+10dlC reporter was used because it could eliminate the stimulation effect of IL-3 through the CRE-2 site via the PI3-K/Akt-dependent pathway (49).”

  • The paper relies on this without support.

    The kinase activity of the p38MAPK immunocomplex in vitro reflects p38MAPK-dependent phosphorylation relevant to the in vivo IL-3 response.

    “The p38MAPK immunocomplex purified in both cases phosphorylated the bacterially produced His-tagged PU.1 (His/PU.1) protein (lanes 2 and 4).”
7 of 7

Cites

Flagged first, then by citations

Cited by

Sorted by citations

Related

Authors
Wang, J.-M., Lai, M.-Z., & Yang-Yen, H.-F.
Affiliations
  • Institute of Molecular Biology, Academia Sinica (TW)
  • Institute of Molecular Biology, Academia Sinica
ORCID
Venue
Molecular and Cellular Biology
Publisher
Informa UK Limited
Published
2003-03
Volume
23
Issue
6
Pages
1896-1909
ISSN
  • 0270-7306 (ISSN-L)
  • 1098-5549
DOI
10.1128/mcb.23.6.1896-1909.2003
PMC
PMC149468
OpenAlex
W2172257243
Cited by
83
References
57
Citations by year
2025: 1 · 2024: 1 · 2023: 1 · 2021: 1 · 2020: 2 · 2019: 3 · 2018: 1 · 2017: 2 · 2016: 2 · 2014: 4 · 2013: 4 · 2012: 1
FWCI
3.44
Type
journal article
Language
en
Open access
Licence
journals.asm.org/non-commercial-tdm-license (tdm)
Locations
  • Molecular and Cellular Biology (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Health Sciences › Medicine › Oncology › Cytokine Signaling Pathways and Interactions
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Cell death mechanisms and regulation
Keywords
interleukin-3, Mcl-1, PU.1, p38 MAPK, transcriptional activation, CREB, anti-apoptotic gene expression, chromatin immunoprecipitation
MeSH terms
Wortmannin, Phosphoinositide-3 Kinase Inhibitors, Proto-Oncogene Protein Spi-1, Androstadienes, Animals, Cell Line, Enzyme Inhibitors, Hematopoietic Stem Cells

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-07.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-07.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review