To examine whether FKBP51 regulates cellular response to multiple classes of chemotherapeutic drugs, we used microtubule stabilizers and Topoisomerase I and II inhibitors to treat the pancreatic cancer cell line SU86. Downregulation of FKBP51 with two different siRNAs resulted in increased resistance to these treatments ( and Figure S1A
). Downregulation of FKBP51 also resulted in resistance to these drugs in the lung cancer cell line A549 and the breast cancer cell line MDA-MB-231 (Figure S2
). Furthermore, loss of FKBP51 expression resulted in increased resistance of mouse embryonic fibroblasts (MEFs) to gemcitabine (). In contrast, overexpression of FKBP51 resulted in hypersensitivity to gemcitabine (). Overall, these results have established an important role of FKBP51 in regulating cellular response to a wide range of clinically important anti-neoplastic agents in both transformed and non-transformed cells.
FKBP51 regulates cellular response to genotoxic stress
This raised the question, how does FKBP51 regulate cellular response to these therapeutics? We found that overexpression of FKBP51 resulted in a reduced phosphorylation of Akt at Ser473, but had no effect on the phosphorylation of Thr308 (). There was no visible difference in Akt phosphorylation with or without gemcitabine treatment. On the other hand, downregulation of FKBP51 resulted in increased Ser473 phosphorylation, with no effect on Thr308 phosphorylation ( and Figure S1A
). Furthermore, hyperphosphorylation of Ser473 was observed in FKBP51−/−
MEFs (). These results suggest that FKBP51 negatively regulates Akt phosphorylation, which might account for its effects on cell survival.
FKBP51 regulates Akt phosphorylation at Ser473 by promoting Akt-PHLPP interaction
To further confirm that FKBP51 regulates Akt activity, we examined the phosphorylation of downstream substrates of Akt, such as GSK-3β and FOXO1. We found that overexpression of FKBP51 decreased the phosphorylation of GSK-3β (pSer9GSK-3β) and FOXO1 (pThr24 FoxO1) (, lower panels), which was consistent with decreased Akt phosphorylation. In contrast, downregulation of FKBP51 significantly increased the phosphorylation of GSK-3β and FOXO1 (, lower panels). These results confirmed that FKBP51 inhibits Ser473 phosphorylation and Akt activity.
Since FKBP51 specifically regulates Ser473 phosphorylation, but not Thr308 phosphorylation, it is likely that FKBP51 regulates signaling events that directly control Akt phosphorylation at Ser473. The Ser473 of Akt is specifically phosphorylated by mTORC2 and dephosphorylated by PHLPP phosphatases (PHLPP1 and PHLPP2) (Brognard et al., 2007
; Gao et al., 2005
; Sarbassov et al., 2005
). Another FKBP family member, FKBP38, has previously been shown to bind and inhibit mTORC1 activity (Bai et al., 2007
). However, we could not detect any interaction between FKBP51 and mTOR. Instead, we found that PHLPP1 and Akt were coimmunoprecipitated with FKBP51 (, upper panels). Similarly, FKBP51 and PHLPP1 were coimmunoprecipitated with Akt (, lower panels). These results suggest that FKBP51, Akt and PHLPP could exist as a complex in cells.
The findings that FKBP51 interacts with PHLPP and Akt led us to hypothesize that FKBP51 acts as a scaffolding protein that promotes the interaction between Akt and PHLPP, thereby enhancing the dephosphorylation of Akt. To test this hypothesis, we overexpressed FKBP51 and found that the interaction between PHLPP and Akt increased in cells with FKBP51 overexpression (). Furthermore, there was less interaction between Akt and PHLPP1 in FKBP51−/− cells, than in FKBP51+/+ cells (). To test whether FKBP51 directly 7promotes the Akt-PHLPP interaction, we expressed and affinity purified FKBP51, PHLPP , and Akt. As shown in , FKBP51 can increase the interaction between Akt and PHLPP in vitro.
In the absence of FKBP51, Akt becomes hyperphosphorylated at Ser473 due to inefficient binding of PHLPP to Akt, which may contribute to the chemoresistance observed in cells depleted of FKBP51. Indeed, in cells expressing AktS473D, which mimics Ser473 phosphorylation, cells became resistant to gemcitabine (Figure S1B
). Similarly, depletion of PHLPP rendered cells resistant to gemcitabine (Figure S1C
). These results support the hypothesis that FKBP51 regulates chemoresistance through the Akt pathway.
To further confirm that FKBP51 regulates Akt Ser473 phosphorylation through PHLPP, we overexpressed FKBP51 while downregulating PHLPP. As we demonstrated earlier, overexpression of FKBP51 alone decreased Akt Ser473 phosphorylation and downstream GSK-3β phosphorylation. However, these effects were reversed by reducing PHLPP ( and Figure S1D
). Overexpression of FKBP51 did not have further effect on Akt Ser473 phosphorylation in cells depleted of PHLPP. Consistent with these observations, although FKBP51 overexpression alone sensitized cells to gemcitabine treatment, downregulation of PHLPP reversed this sensitizing effect (). Furthermore, PHLPP overexpression blocked the effects of FKBP51 knockdown on Akt Ser473 and GSK-3β phosphorylation (). These results establish that FKBP51 regulates Akt Ser473 phosphorylation through PHLPP.
It is possible that FKBP51 not only increases the Akt-PHLPP interaction but also enhances the dephosphorylation of Akt by stimulating PHLPP activity. FKBP51 has peptidylprolyl isomerase activity thus could affect PHLPP activity. However, overexpression of the FKBP51 mutant FD67/68DV, which lacks peptidylprolyl isomerase activity (Barent et al., 1998
), had similar effect on Akt Ser473 phosphorylation as WT FKBP51 (Figure S3A
), suggesting that FKBP51 regulates Akt phosphorylation in a peptidylprolyl isomerase-independent manner. Although it is possible that the binding of FKBP51 to PHLPP directly stimulates PHLPP activity, we found that overexpression or depletion of FKBP51 did not affect PHLPP phosphatase activity in vitro
). PHLPP has been shown to regulate the levels of PKC βII by dephosphorylating the hydrophobic motif of PKC βII, resulting in rapid degradation of PKC βII (Gao et al., 2008
). However, FKBP51 did not interact with and did not significantly affect the phosphorylation or levels of PKC βII, (Figure S3D–E
). These results further confirm that FKBP51 regulates Akt Ser473 phosphorylation mostly through its scaffolding function.
Akt has three isoforms (Akt1, Akt2 and Akt3) (Manning and Cantley, 2007
). The antibodies that we used in previous experiments recognize all three isoforms. In addition, PHLPP has 2 isoforms (PHLPP1 and PHLPP2) (Brognard et al., 2007
; Gao et al., 2005
). Previous studies have established that both PHLPP1 and PHLPP2 dephosphorylate the same hydrophobic phosphorylation motif on Akts (Ser473 on Akt1), but they inhibit Akt signaling differently by interacting with distinct Akt isoforms (Brognard et al., 2007
). PHLPP1 specially regulates Akt2 and Akt3, and PHLPP2 regulates Akt1 and Akt3. We next examined whether the isoform-specific effects of PHLPP on Akt are regulated by FKBP51. Consistent with previous studies (Brognard et al., 2007
), Akt1 and Akt3 were coimmunoprecipitated with PHLPP2; while Akt2 and Akt3 were coimmunoprecipitated with PHLPP1 (). Overexpression of FKBP51 increased the interaction between both PHLPP isoforms with their corresponding Akt isoforms (); while downregulation of FKBP51 decreased these interactions (). These results suggest that FKBP51 facilitates isoform-specific interaction between Akt and PHLPP.
FKBP51 scaffolding function regulates Akt phosphorylation and cell survival
To investigate how FKBP51 enhances the Akt-PHLPP interaction, we generated a series of deletion mutants of FKBP51. As shown in , we found that deletion of either the FKBP1 (Residues 1–138) or FKBP2 (138–251) domain abolished the interaction between FKBP51 and PHLPP, suggesting that both domains are required for this interaction. In addition, we found that the C-terminal TPR domain of FKBP51 was essential for the binding of FKBP51 to Akt. These results suggest that FKBP51 binds PHLPP and Akt using distinct domains, consistent with our hypothesis that FKBP51 acts as a scaffolding protein to promote the Akt-PHLPP interaction. If so, we expected that FKBP51 deletion mutants that could not bind either Akt or PHLPP would not enhance the Akt-PHLPP interaction. Indeed, deletion of the FKBP1 domain or the TPR domain abolished the ability of FKBP51 to enhance the Akt-PHLPP interaction ().
To further confirm that the scaffolding function of FKBP51 is important for the regulation of Akt phosphorylation and cell survival, we reconstituted FKBP51−/− MEFs with wild type FKBP51 (FK), FKBP51 deleted of the FKBP1 domain (ΔN2) or FKBP51 deleted of the TPR domain (ΔC2). As in , pSer473, pGSK-3β and pFOXO1 levels were higher in FKBP51−/− cells than in FKBP51+/+ cells (). Reconstitution of the WT FKBP51 in FKBP51−/− cells returned the phosphorylation levels of AktSer473, GSK-3β, and FOXO1 to those observed in FKBP51+/+ cells, whereas reconstitution with either mutant had no effect. This observation correlated with a decreased Akt-PHLPP interaction in the absence of WT FKBP51 (). Importantly, reconstitution of WT FKBP51 in FKBP51−/− MEFS restored cell sensitivity to gemcitabine to a level similar to that of FKBP51+/+ MEFS (), whereas neither mutants had this rescue effect. These results confirmed that the scaffolding function of FKBP51 is important for the regulation of Akt phosphorylation and cell survival.
Since decrease or loss of FKBP51 expression results in Akt hyperactivation, which has been observed in many cancers, it is possible that FKBP51 expression is downregulated in cancer cells. Consistent with this notion, we found that FKBP51 expression is lost or significantly decreased in a high percentage of the pancreatic cancer cell lines and breast cancer cell lines that we examined (). Reconstitution of FKBP51 in Miapaca2 or BxPC3 cells decreased Akt phosphorylation at Ser473 (), and sensitized these cells to Ara-C (), supporting the hypothesis that loss of FKBP51 expression renders these cells resistant to chemotherapy. Furthermore, overexpression of FKBP51 in a pancreatic cancer cell line (Panc0403) having high endogenous levels of FKBP51 did not affect Akt phosphorylation (). These results suggest that cancer cell lines having high FKBP51 expression might depend less on the PI3K-Akt pathway to survive. To test this possibility, we treated Miapaca2 (low levels of FKBP51 expression) and Panc0403 (high levels of FKBP51 expression) with the PI3K inhibitor, wortmannin, together with gemcitabine. As shown in Figure S4A
, Miapaca2 cells are more sensitive to PI3K inhibition, consistent with the notion that cells expressing low levels of FKBP51 might be more dependent on the PI3K-Akt pathway. However, these two cell lines have different genetic backgrounds, so factors other than FKBP51 expression may contribute to their different responses to the PI3K inhibitor.
Loss of FKBP51 expression in cancer cells and tissues
Our results also imply that FKBP51 might function as a tumor suppressor. As an initial step to test this hypothesis, we performed microarray analysis using RNA isolated from 36 pancreatic tumor and 19 normal tissue samples. These were fresh frozen samples obtained during surgical procedures. The microarray data revealed that expression levels of FKBP51
were significantly lower in pancreatic tumor tissue than in normal pancreatic tissue (). The comparison of expression profile between normal and tumor tissues identified genes expressed significantly differently (P<10−6
) between the two (Figure S4B
). Network analysis using Ingenuity Pathway analysis software of the most differentially expressed genes showed that a network surrounding Akt was the top network (Figure S4C
). We also performed real time quantitative RT-PCR (QRTPCR) to validate the microarray results and found these results to be similar with the correlation coefficient between the RT-PCR and microarray data approximately 0.8 (P<0.0001) (). Furthermore, lower or loss of FKBP51 protein levels was found in selected pancreatic cancer samples, many of which had increased Akt/GSK-3β phosphorylation ( and Figure S4D–E
). Decreased expression levels of FKBP51 was also found in ovarian, head and neck, seminoma, leukemia and prostate cancer tissues based on expression data obtained through the Oncomine (www. Oncomine.org
). Overall, our results suggest that FKBP51 negatively regulates Akt activation through its scaffolding function and that hyperactivation of Akt caused by the loss of FKBP51 might contribute to tumorigenesis and cancer cell resistance to chemotherapy.