Regulation of Stress-Inducible Phosphoprotein 1 Nuclear Retention by Protein Inhibitor of Activated STAT PIAS1

Stress-inducible phosphoprotein 1 (STI1), a cochaperone for Hsp90, has been shown to regulate multiple pathways in astrocytes, but its contributions to cellular stress responses are not fully understood. We show that in response to irradiation-mediated DNA damage stress STI1 accumulates in the nucleus of astrocytes. Also, STI1 haploinsufficiency decreases astrocyte survival after irradiation. Using yeast two-hybrid screenings we identified several nuclear proteins as STI1 interactors. Overexpression of one of these interactors, PIAS1, seems to be specifically involved in STI1 nuclear retention and in directing STI1 and Hsp90 to specific sub-nuclear regions. PIAS1 and STI1 co-immunoprecipitate and PIAS1 can function as an E3 SUMO ligase for STI. Using mass spectrometry we identified five SUMOylation sites in STI1. A STI1 mutant lacking these five sites is not SUMOylated, but still accumulates in the nucleus in response to increased expression of PIAS1, suggesting the possibility that a direct interaction with PIAS1 could be responsible for STI1 nuclear retention. To test this possibility, we mapped the interaction sites between PIAS1 and STI1 using yeast-two hybrid assays and surface plasmon resonance and found that a large domain in the N-terminal region of STI1 interacts with high affinity with amino acids 450–480 of PIAS1. Knockdown of PIAS1 in astrocytes impairs the accumulation of nuclear STI1 in response to irradiation. Moreover, a PIAS1 mutant lacking the STI1 binding site is unable to increase STI1 nuclear retention. Interestingly, in human glioblastoma multiforme PIAS1 expression is increased and we found a significant correlation between increased PIAS1 expression and STI1 nuclear localization. These experiments provide evidence that direct interaction between STI1 and PIAS1 is involved in the accumulation of nuclear STI1. This retention mechanism could facilitate nuclear chaperone activity.Stress-inducible phosphoprotein I (STI1)¹ is a conserved cochaperone protein that assists Hsp90 in managing client proteins, by mediating the transfer of proteins between Hsp70 and Hsp90 (1–3). STI1 contains several tetratricopeptide-repeat domains (TRP) that can serve as interaction modules with Hsp90 and Hsp70 (4). STI1 helps to drive the sequential steps involved in the Hsp90 chaperone machinery (5) and regulates the ATPase activity of Hsp90 (6, 7). STI1 is also secreted by distinct cells (8–12), using a noncanonical mechanism involving extracellular vesicles (11). Secreted STI1 can activate multiple signaling pathways in distinct cell types (8–10, 13–18).Elimination of STI1 in yeast sensitizes cells to Hsp90 inhibitors, but it is not by itself lethal (19). STI1 can also be eliminated in C. elegans, although it results in decreased life span (20). In contrast, STI1 mutant mice do not survive E10.5 and present several morphological defects, owing to decreased levels of several Hsp90-client proteins (21). Mouse embryonic fibroblasts obtained from STI1-deficient embryos also fail to thrive and present increased levels of the DNA damage marker γ-H2AX, suggestive of increased cellular stress (21). Hence, in mammals STI1 seems to play additional roles in cellular survival that are not yet fully understood.STI1 is abundantly expressed in the cytoplasm of cells, but can also be found in the Golgi (22), in vesicles and in multivesicular bodies (11). Moreover, this cochaperone has been shown to shuttle between the cytoplasm and the nucleus in cell lines (23). Cellular stress, arrest in G1/S phase of the cell cycle and phosphorylation are factors that seem to regulate STI1 nuclear localization (23, 24). Presumably nuclear STI1 can regulate chaperone activity, but whether it can interact with nuclear proteins is unknown.Previous experiments using cell lines have shown that knockdown of STI1 increases susceptibility of cells to irradiation (25). Whether changes in STI1 levels in primary differentiated cells, such as astrocytes, may affect their response to irradiation stress is unknown. This is of interest, as astrocytes, which can give rise to distinct tumor cells, are highly radioresistant (26). Indeed, astrocytes have a noncanonical DNA damage response (DDR) to irradiation (26). Here we show that STI1 undergoes nuclear translocation in astrocytes after γ-radiation-induced DNA damage. Moreover, astrocytes haploinsufficient for STI1 are more susceptible to cell death induced by irradiation. To understand potential mechanisms involved with STI1 nuclear retention, we have performed yeast-two hybrid screenings to identify STI1 nuclear partners. We identified protein inhibitor of activated STAT (PIAS1) as a direct interactor of STI1 and provide evidence that it acts as a small ubiquitin-like modifier (SUMO) E3 ligase for STI1. We show this interaction is involved with STI1 nuclear retention after irradiation. Interestingly, tissue microarray analysis demonstrated that higher PIAS1 levels are found in glioblastoma multiforme (GBM) when compared with non-neoplastic tissue. Furthermore, we uncovered a positive relationship between increased PIAS1 expression in GBMs and augmented STI1 nuclear localization. Our results reveal a novel mechanism by which increased expression of PIAS1, as observed in GBM, can increase the retention of nuclear STI1, a critical regulator of the chaperone machinery.