Inactivating Pit-1 mutations alter subnuclear dynamics suggesting a protein misfolding and nuclear stress response

Pit-1, a POU-class nuclear DNA-binding transcription factor, specifies three of the parenchymal cell types in anterior pituitary ontogeny. Using fluorescent fusions and live cell imaging, we have compared the dynamic behavior of wild-type and inactivating Pit-1 point mutations. Fluorescence recovery after photobleaching (FRAP) and real-time extraction data indicate that wild-type Pit-1 has a dynamic mobility profile, with t_1/2s ∼5-7 s when expressed from low to high amounts, respectively. Biochemically, Pit-1 is ∼50% retained according to direct observation during extraction, indicating a dynamic interaction with nuclear structure. An analysis of transiently expressed Pit-1 carrying two different debilitating mutations reveals that they translocate normally to the nucleus, but exhibit two different levels of mobility, both clearly distinguishable from wild-type Pit-1. At low expression levels, the t_1/2s of Pit_W261C and Pit_A158P are extremely rapid (0.3 and 0.6 s t_1/2s, respectively). At higher expression levels, unlike wild-type Pit-1, both mutant proteins become immobilized and insoluble, and fractionate completely with the insoluble nuclear matrix. Relative to wild-type, over expression of mutated Pit-1 elicits a nuclear stress response indicated by increased levels of heat shock inducible heat shock protein 70 (Hsp70), and reorganization of heat shock factor-1. The decreased mobility of Pit_A158P relative to Pit_W261C at low expression levels correlates with its ability to partially activate when expressed at low levels and its ability to bind cognate DNA. At high expression levels, lower Pit_A158P activation correlates with its immobilization and insolubility. These data suggest a link between specific rates of intranuclear mobility and Pit-1 transcription function, perhaps to insure sufficient interactions with chromatin, or in the case of non-DNA binding Pit-1, interaction as a repressor (Scully and Rosenfeld [2002]: Science 295:2231-2235). These data imply inactivating mutations can lead to an intranuclear sorting away from transcription related pathways, and at least in part to a misfolded protein pathway. Taken together, caution is suggested when interpreting point (or other) mutational analyses of transactivator function, as new compartmentation, especially in the context of expression levels, may cloud the distinction between defining functional molecular domains and intranuclear processing of misfolded proteins.