Aberrant expression of the NF-kappaB and IkappaB proteins in B cells from viable motheaten mice.
In viable motheaten mice, a mutation in the gene encoding the phosphatase, SHP1, causes severe immunodeficiency and autoimmunity. A defective phosphatase may result in modified phosphorylation of proteins involved in gene regulation. Since the NFkappaB/IkappaB proteins are regulated through phosphorylation, we wished to understand if the expression of these proteins was altered by the SHP1 defect. Splenic B cells from viable motheaten mice were isolated and assessed for purity by flow cytometry. Levels of each protein in isolated B cells were examined by Western blot analyses. Measurement of RNA levels for each protein was assessed by semi-quantitative RT-PCR. Western blots revealed that, in me(v) whole cell lysates, there were reduced levels of RelA and RelB proteins and increased levels of p50 and c-Rel. Furthermore, we analyzed the protein levels of IkappaBalpha and found that, in me(v), this inhibitor was significantly reduced, while the level of another member of the IkappaB family, IkappaBbeta, was not. To determine if these findings in me(v) were secondary to the autoimmune process, we evaluated NF-kappaB/IkappaB expression in the BXSB murine model of autoimmunity. Unlike me(v), B cells from BXSB/Yaa mice had NF-kappaB complexes composed of the RelA submit, and IkappaBalpha was readily detected. In addition, RNA for the RelA and IkappaBalpha proteins in me(v) and control littermates was detected by RT-PCR, indicating that the reduced amounts of these proteins was not exclusively due to transcriptional defects. We conclude that the differences in NF-kappaB/IkappaB proteins that we have described in me(v) are likely a consequences of the SHP1 defect and could contribute to the clinical disorder that characterizes me(v) mice.
Khaled
A R
AR
Department of Molecular Genetics and Microbiology, University of Florida, Gainesville 32610, USA.
Butfiloski
E J
EJ
Villas
B
B
Sobel
E S
ES
Schiffenbauer
J
J
D000818:Animals; D001327:Autoimmune Diseases / Q000201:enzymology / Q000235:genetics* / Q000276:immunology; D001402:B-Lymphocytes / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000201:enzymology / Q000473:pathology; D020672:I-kappa B Proteins; D007153:Immunologic Deficiency Syndromes / Q000201:enzymology / Q000235:genetics* / Q000276:immunology; D047908:Intracellular Signaling Peptides and Proteins; D008025:Ligases / Q000032:analysis; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D008954:Models, Biological; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D052002:NF-kappa B p50 Subunit; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000172:deficiency / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000032:analysis; D020569:Proto-Oncogene Proteins c-rel; D012333:RNA, Messenger / Q000032:analysis; D013154:Spleen / Q000276:immunology; D051841:Transcription Factor RelB; D014157:Transcription Factors / Q000032:analysis; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; C000606205:Nfkbia protein, mouse; D011518:Proto-Oncogene Proteins; D020569:Proto-Oncogene Proteins c-rel; D012333:RNA, Messenger; C497594:Relb protein, mouse; D014157:Transcription Factors; D000072000:NF-KappaB Inhibitor alpha; D051841:Transcription Factor RelB; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; C516725:Ptpn11 protein, mouse; C509248:Ptpn6 protein, mouse; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases