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Signal transducers and activators of transcription (STATs) regulate a variety of cellular functions, including differentiation and proliferation. STAT3 and STAT5 are known to play important roles in brain processes, such as energy homeostasis and neuronal development. We isolated a novel splicing variant of STAT5A from a cDNA library of the mouse brainstem. This variant, STAT5A_ΔE18, lacked exon 18 and caused a frameshift in the C-terminus, resulting in deletion of a tyrosine phosphorylation site and a transactivation domain. Although the frameshift region had no characteristic motifs, it was highly serine/threonine-rich and contained a short proline-rich sequence. Expression of STAT5A_ΔE18 was detected in the mouse brainstem, lung and thymus, but not in the mouse cerebrum or cerebellum. We developed a specific antibody against STAT5A_ΔE18 and investigated the intracellular localization of this variant. STAT5A_ΔE18 showed dot-like structures in the cytoplasm and could not translocate into the nucleus after prolactin treatment. STAT5A_ΔE18 showed a strong tendency to aggregate, which led to coaggregation with STAT5A_full-length. This coaggregation inhibited the nuclear transport of STAT5A and suppressed prolactin-induced activation of STAT5A.