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

ABSTRACT

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) approximately 5-7 s when expressed from low to high amounts, respectively. Biochemically, Pit-1 is approximately 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. 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.