Abstract
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 NFκB/IκB 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 mev whole cell lysates, there were reduced levels of Re1A and Re1B proteins and increased levels of p50 and c-Re1. Furthermore, we analyzed the protein levels of IκBU and found that, in mev, this inhibitor was significantly reduced, while the level of another member of the IκB family, IκBβ, was not. To determine if these findings in mev were secondary to the autoimmune process, we evaluated NF-KB/IKB expression in the BXSB murine model of autoimmunity. Unlike mev, B cells from BXSB/Yaa mice had NF-κB complexes composed of the Re1A submit, and IκBα was readily detected. In addition, RNA for the Re1A and IκBα proteins in mev 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-κB/IκB proteins that we have described in mev are likely a consequences of the SHP1 defect and could contribute to the clinical disorder that characterizes mev mice.
Key Words: