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.

Dynamic Interactions between Pit-1 and C/EBPα in the Pituitary Cell Nucleus

“…Furthermore, the FRAP analysis showed that the Pit-1 W261C mutation had significantly higher intranuclear mobility than the wild-type protein. This result generally agreed with an earlier study in nonpituitary cells (39), although the mobilities we observed here in pituitary cells were much slower than those reported for the HeLa cells. Since the FRAP results represent the averages of many different interactions, including specific interactions with chromatin and the cooperative associations with protein partners, it seems likely that the slower mobilities observed here could reflect these pituitary-specific interactions.…”

“…Pituitary GHFT1 cells expressing the different CFP fusion proteins were identified using low-intensity illumination with the 405-nm laser line. Sharp and colleagues (39) showed that the mobility of GFP-Pit-1 in HeLa cells was not significantly different over a broad range of expression levels, but it is recognized that the level of protein expression can influence protein mobilities. We compared the relative expression levels of CFP-Pit-1 to the endogenous Pit-1 in GHFT1 cells (Fig.…”

Pax6 localizes to chromatin-rich territories and displays a slow nuclear mobility altered by disease mutations

“…It is striking that the three homeodomain proteins, Pax6, Pax3 and HoxB7, all have a localization pattern that overlaps with the Hoechst dye. We also found the subnuclear distribution pattern of Pax6 to be very similar to what is reported for other transcription factors like Pax3 [48] and Pit-1 [64].…”

“…They further suggest that this is a general characteristic of diseasecausing missense alleles, something our FRAP results on disease-causing Pax6 mutations support. Several studies of transcription factor mutations show a correlation between increased mobility and decreased DNA-binding affinity [35,64,74]. However, the opposite was found for disease-causing mutations in Pax3 where intact DNA binding correlated with increased nuclear mobility [48].…”

Aberrant Folding of a Mutant Stat5b Causes Growth Hormone Insensitivity and Proteasomal Dysfunction

“…Except for this disorder, no natural variants of transcription factors have been shown to be prone to protein aggregation. A substitution mutation in the pituitary transcriptional activator Pit-1 has been found to become mislocalized within the nucleus when overexpressed, possibly because of changes in its structure, although no structural determinations have been performed (40).…”