Characterization of the bovine IkappaB kinases (IKK)alpha and IKKbeta, the regulatory subunit NEMO and their substrate IkappaBalpha.
The Nuclear factor (NF)-kappaB signalling pathway plays a critical role in the regulation and coordination of a wide range of cellular events such as cell growth, apoptosis and cell differentiation. Activation of the IKK (inhibitor of NF-kappaB kinase) complex is a crucial step and a point of convergence of all known NF-kappaB signalling pathways. To analyse bovine IKKalpha (IKK1), IKKbeta (IKK2) and IKKgamma (or NF-kappaB Essential MOdulator, NEMO) and their substrate IkappaBalpha (Inhibitor of NF-kappaB), the corresponding cDNAs of these molecules were isolated, sequenced and characterized. A comparison of the amino acid sequences with those of their orthologues in other species showed a very high degree of identity, suggesting that the IKK complex and its substrate IkappaBalpha are evolutionarily highly conserved components of the NF-kappaB pathway. Bovine IKKalpha and IKKbeta are related protein kinases showing 50% identity which is especially prominent in the kinase and leucine zipper domains. Co-immunoprecipitation assays and GST-pull-down experiments were carried out to determine the composition of bovine IKK complexes compared to that in human Jurkat T cells. Using these approaches, the presence of bovine IKK complexes harbouring IKKalpha, IKKbeta, NEMO and the interaction of IKK with its substrate IkappaBalpha could be demonstrated. Parallel experiments using human Jurkat T cells confirmed the high degree of conservation also at the level of protein-protein interactions. Finally, a yeast two-hybrid analysis showed that bovine NEMO molecules, in addition to the binding to IKKalpha and IKKbeta, also strongly interact with each other.
Rottenberg
Sven
S
Institute of Animal Pathology, Molecular Pathology, University of Berne, Länggassstrasse 122, CH-3012 Berne, Switzerland.
Schmuckli-Maurer
Jacqueline
J
Grimm
Stephan
S
Heussler
Volker T
VT
Dobbelaere
Dirk A E
DA
D000818:Animals; D015152:Blotting, Northern; D002417:Cattle / Q000235:genetics*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D006801:Humans; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins / Q000235:genetics* / Q000378:metabolism; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D000072000:NF-KappaB Inhibitor alpha; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017422:Sequence Analysis, DNA; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; D021122:Protein Subunits; D012333:RNA, Messenger; D000072000:NF-KappaB Inhibitor alpha; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human