I k B a Physically Interacts with a Cytoskeleton-Associated Protein through Its Signal Response Domain

The I k B a protein is a key molecular target involved in the control of NF- k B/Rel transcription factors during viral infection or inflammatory reactions. This NF- k B-inhibitory factor is regulated by posttranslational phosphorylation and ubiquitination of its amino-terminal signal response domain that targets I k B a for rapid proteolysis by the 26S proteasome. In an attempt to identify regulators of the I k B a inhibitory activity, we undertook a yeast two-hybrid genetic screen, using the amino-terminal end of I k B a as bait, and identified 12 independent interacting clones. Sequence analysis identified some of these cDNA clones as Dlc-1, a sequence encoding a small, 9-kDa human homolog of the outer-arm dynein light-chain protein. In the two-hybrid assay, Dlc-1 also interacted with full-length I k B a protein but not with N-terminal-deletion-containing versions of I k B a . I k B a interacted in vitro with a glutathione S -transferase–Dlc-1 fusion protein, and RelA(p65) did not displace this association, demonstrating that p65 and Dlc-1 contact different protein motifs of I k B a . Impor-tantly, in HeLa and 293 cells, endogenous and transfected I k B a coimmunoprecipitated with Myc-tagged or endogenous Dlc-1. Indirect immunofluorescence analyzed by confocal microscopy indicated that Dlc-1 and I k B a colocalized with both nuclear and cytoplasmic distribution. Furthermore, Dlc-1 and I k B a were found to associate with the microtubule organizing center, a perinuclear region from which microtubules radiate. Likewise, I k B a colocalized with a -tubulin filaments. Taken together, these results highlight an intriguing interaction between the I k B a protein and the human homolog of a member of the dynein family of motor proteins and provide a potential link between cytoskeleton dynamics and gene regulation.

[1]  T. Maniatis,et al.  Activation of the IκBα Kinase Complex by MEKK1, a Kinase of the JNK Pathway , 1997, Cell.

[2]  V. Ferrans,et al.  rac1 regulates a cytokine-stimulated, redox-dependent pathway necessary for NF-kappaB activation , 1996, Molecular and cellular biology.

[3]  J. Carson,et al.  Brain Cytoplasmic and Flagellar Outer Arm Dyneins Share a Highly Conserved Mr 8,000 Light Chain* , 1996, The Journal of Biological Chemistry.

[4]  J. Frost,et al.  Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members , 1996, Molecular and cellular biology.

[5]  Michael R. Green,et al.  HIV Rev uses a conserved cellular protein export pathway for the nucleocytoplasmic transport of viral RNAs , 1996, Current Biology.

[6]  K. Ray,et al.  Cytoplasmic dynein (ddlc1) mutations cause morphogenetic defects and apoptotic cell death in Drosophila melanogaster , 1996, Molecular and cellular biology.

[7]  R. Vale,et al.  Cell cycle regulation of dynein association with membranes modulates microtubule-based organelle transport , 1996, The Journal of cell biology.

[8]  T. Maniatis,et al.  Site-Specific Phosphorylation of IκBα by a Novel Ubiquitination-Dependent Protein Kinase Activity , 1996, Cell.

[9]  S. Craig,et al.  Assembly of focal adhesions: progress, paradigms, and portents. , 1996, Current opinion in cell biology.

[10]  K. Pfister,et al.  Cytoplasmic dynein is associated with slow axonal transport. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[11]  E. Krebs,et al.  Association of mitogen-activated protein kinase with the microtubule cytoskeleton. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[12]  A. Strasser,et al.  Mice lacking the c-rel proto-oncogene exhibit defects in lymphocyte proliferation, humoral immunity, and interleukin-2 expression. , 1995, Genes & development.

[13]  David Baltimore,et al.  Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-κB , 1995, Nature.

[14]  R. Sternglanz,et al.  Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system , 1995, Molecular and cellular biology.

[15]  SM King,et al.  The M(r) = 8,000 and 11,000 outer arm dynein light chains from Chlamydomonas flagella have cytoplasmic homologues , 1995, The Journal of Biological Chemistry.

[16]  K. Blumer,et al.  Association of p62, a multifunctional SH2- and SH3-domain-binding protein, with src family tyrosine kinases, Grb2, and phospholipase C gamma-1 , 1995, Molecular and cellular biology.

[17]  J. Hiscott,et al.  Subcellular Redistribution of HTLV-I Tax Protein by NF-κB/Rel Transcription Factors , 1994 .

[18]  Donald E. Ingber,et al.  The riddle of morphogenesis: A question of solution chemistry or molecular cell engineering? , 1993, Cell.

[19]  B. Vandenbunder,et al.  High levels of c-rel expression are associated with programmed cell death in the developing avian embryo and in bone marrow cells in vitro , 1993, Cell.

[20]  P. Morin,et al.  The IκB proteins: members of a multifunctional family , 1993 .

[21]  J. McIntosh,et al.  Cytoplasmic dynein plays a role in mammalian mitotic spindle formation , 1993, The Journal of cell biology.

[22]  R. Vallee.  Molecular analysis of the microtubule motor dynein. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[23]  D. von der Ahe,et al.  Cytoskeletal reorganization and TPA differently modify AP-1 to induce the urokinase-type plasminogen activator gene in LLC-PK1 cells. , 1993, Nucleic acids research.

[24]  P. Morin,et al.  GAL4-lϰBα and GAL4-lϰBγ activate transcription by different mechanisms , 1993 .

[25]  K. Katagiri,et al.  Tyrosine-phosphorylation of tubulin during monocytic differentiation of HL-60 cells. , 1993, Journal of immunology.

[26]  P. Morin,et al.  The C terminus of the NF-χB p50 precursor and an IχB isoform contain transcription activation domains , 1992 .

[27]  M. Deutscher,et al.  A sequestered pool of aminoacyl-tRNA in mammalian cells. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[28]  Samuel E. Lux,et al.  Analysis of cDNA for human erythrocyte ankyrin indicates a repeated structure with homology to tissue-differentiation and cell-cycle control proteins , 1990, Nature.

[29]  D. Stern,et al.  The Ick tyrosine protein kinase interacts with the cytoplasmic tail of the CD4 glycoprotein through its unique amino-terminal domain , 1989, Cell.

[30]  V. Fried,et al.  Ubiquitin is a component of the microtubule network. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[31]  D. Baltimore,et al.  Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-κB transcription factor , 1988, Cell.

[32]  G K Lewis,et al.  Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product , 1985, Molecular and cellular biology.

[33]  Justin Schwartz.  Engineering , 1929, Nature.

[34]  Ronald T. Hay,et al.  Nuclear localization of IκBα promotes active transport of NF-κB from the nucleus to the cytoplasm , 1997 .

[35]  C. Mueller-Dieckmann,et al.  Tyrosine Phosphorylation of IkB-a Activates NF-kB without Proteolytic Degradation of IkB-a , 1996 .

[36]  H. Suyang,et al.  Role of unphosphorylated, newly synthesized IκBβ in persistent activation of NF-κB , 1996 .

[37]  J. Hiscott,et al.  Phosphorylation of I k B a in the C-Terminal PEST Domain by Casein Kinase II Affects Intrinsic Protein Stability , 1996 .

[38]  Inder M. Verma,et al.  R~~/NF-KB /IKB familv: intimate tales of association and dissociation , 1995 .

[39]  T. Maniatis,et al.  Signal induced degradation of IkBa requires site-specific ubiquitina-tion , 1995 .

[40]  Steffen Jung,et al.  In Vivo Stimulation of IkB Phosphorylation Is Not Sufficient To Activate NF-kB , 1995 .

[41]  P. Hawkins,et al.  Activation of the small GTP-binding proteins rho and rac by growth factor receptors. , 1995, Journal of cell science.

[42]  K. Wood,et al.  Domain Organization of IkBaand Sites of Interaction with NF-kB p65 , 1995 .

[43]  T. McKinsey,et al.  Coupling of a Signal Response Domain in IkBato Multiple Pathways for NF-kB Activation , 1995 .

[44]  Annie Schmid-AllianaS,et al.  Disruption of Microtubule Network in Human Monocytes Induces Expression of Interleukin-1 but Not That of Interleukin-6 Nor Tumor Necrosis Factor-a , 1993 .

[45]  A. Ciechanover,et al.  Immunofluorescent localization of the ubiquitin-activating enzyme, E1, to the nucleus and cytoskeleton. , 1993, The American journal of physiology.

[46]  Nathan,et al.  Direct association of pp40/IKcBB with rel/NF-KcB transcription factors: Role of ankyrin repeats in the inhibition of DNA binding activity , 2022 .

[47]  Mutual regulation of the transcriptional activator NF-cB and its inhibitor, I1cB-a , 2022 .

[48]  P. Morin,et al.  GAL4-IxBa and GAL4-IxB-y activate transcription by different mechanisms , 2022 .