Evidence for a phosphorylation-independent role for Ser 32 and 36 in proteasome inhibitor-resistant (PIR) IkappaBalpha degradation in B cells.
Constitutive NF-kappaB activity has emerged as an important cell survival regulator. Canonical inducible NF-kappaB activation involves IkappaB kinase (IKK)-dependent dual phosphorylation of Ser 32 and 36 of IkappaBalpha to cause its beta-TrCP-dependent ubiquitylation and proteasomal degradation. We recently reported that constitutive NF-kappaB (p50/c-Rel) activity in WEHI231 B cells is maintained through proteasome inhibitor-resistant (PIR) IkappaBalpha degradation in a manner that requires Ser 32 and 36, without the requirement of a direct interaction with beta-TrCP. Here we specifically examined whether dual phosphorylation of Ser 32 and 36 was required for PIR degradation. Through mutagenesis studies, we found that dual replacement of Ser 32 and 36 with Glu permitted beta-TrCP and proteasome-dependent, but not PIR, degradation. Moreover, single replacement of either Ser residue with Leu permitted PIR degradation in WEHI231 B cells. These results indicate that PIR degradation occurs in the absence of dual phosphorylation, thereby explaining the beta-TrCP-independent nature of the PIR pathway. Additionally, we found evidence that PIR IkappaBalpha degradation controls constitutive NF-kappaB activation in certain multiple myeloma cells. These results suggest that B lineage cells can differentiate between PIR and canonical IkappaBalpha degradation through the absence or presence of dually phosphorylated IkappaBalpha.
O'Connor
Shelby
S
Program in Cellular and Molecular Biology, Department of Pharmacology, University of Wisconsin, 3795 Medical Sciences Center, 1300 University Avenue, Madison, WI 53706, USA.
Markovina
Stephanie
S
Miyamoto
Shigeki
S
D019943:Amino Acid Substitution; D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D005973:Glutamine / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism*; D007930:Leucine / Q000378:metabolism; D016393:Lymphoma, B-Cell / Q000473:pathology; D051379:Mice; D008954:Models, Biological; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D011233:Precipitin Tests; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D044844:beta-Transducin Repeat-Containing Proteins / Q000378:metabolism
D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D044844:beta-Transducin Repeat-Containing Proteins; D005973:Glutamine; D000072000:NF-KappaB Inhibitor alpha; D012694:Serine; D007930:Leucine