A Mutant Stat5b with Weaker DNA Binding Affinity Defines a Key Defective Pathway in Nonobese Diabetic Mice

A number of cytokines that finely regulate immune response have been implicated in the pathogenesis or protection of type 1 diabetes and other autoimmune diseases. It is, therefore, of pivotal importance to examine a family of proteins that serve as signal transducers and activators of transcription (STATs), which regulate the transcription of a variety of cytokines. We report here a defective gene (Stat5b) located on chromosome 11 within a previously mapped T1D susceptibility interval (Idd4) in the nonobese diabetic (NOD) mice. Our sequencing analysis revealed a unique mutation C1462A that results in a leucine to methionine (L327M) in Stat5b of NOD mice. Leu ³²⁷, the first residue in the DNA binding domain of STAT proteins, is conserved in all identified mammalian STAT proteins. Homology modeling predicted that the mutant Stat5b has a weaker DNA binding, which was confirmed by DNA-protein binding assays. The inapt transcriptional regulation ability of the mutated Stat5b is proved by decreased levels of RNA of Stat5b-regulated genes (IL-2Rβ and Pim1). Consequently, IL-2Rβ and Pim1 proteins were shown by Western blotting to have lower levels in NOD compared with normal B6 mice. These proteins have been implicated in immune regulation, apoptosis, activation-induced cell death, and control of autoimmunity. Therefore, the Stat5b pathway is a key molecular defect in NOD mice.