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Hsp90 and Hsp40/Erdj3 are required for the expression and anti-apoptotic function of KSHV K1.

Wen KW, Damania B - Oncogene (2010)

Bottom Line: Kaposi sarcoma-associated herpesvirus (KSHV) is a member of the gammaherpesvirus family.We report that small-interfering RNAs directed against Hsp90 and Hsp40/Erdj3, as well as pharmacological inhibitors of Hsp90, dramatically reduced K1 expression, suggesting that K1 is a client protein of these chaperones.Finally, we report that the Hsp90 inhibitors, 17-AAG and 17-DMAG, can suppress the proliferation of KSHV-positive PEL cell lines and exhibited IC(50) values of 50 nM and below.

View Article: PubMed Central - PubMed

Affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA.

ABSTRACT
Kaposi sarcoma-associated herpesvirus (KSHV) is a member of the gammaherpesvirus family. It is the etiological agent of three different human cancers, Kaposi sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman disease. The far left end of the KSHV genome encodes a unique transmembrane glycoprotein called K1. K1 possesses the ability to transform rodent fibroblasts and block apoptosis. K1 has also been shown to activate the PI3K/Akt/mTOR pathway in different cells. Using tandem affinity purification, we identified heat shock protein 90beta (Hsp90beta) and endoplasmic reticulum-associated Hsp40 (Erdj3/DnaJB11), as cellular binding partners of K1. Interactions of K1 with Hsp90beta and Hsp40 were confirmed by co-immunoprecipitation in both directions. Furthermore, K1 also interacted with the Hsp90alpha isoform. We report that small-interfering RNAs directed against Hsp90 and Hsp40/Erdj3, as well as pharmacological inhibitors of Hsp90, dramatically reduced K1 expression, suggesting that K1 is a client protein of these chaperones. In addition, both Hsp90 and Hsp40/Erdj3 were essential for K1's anti-apoptotic function. Finally, we report that the Hsp90 inhibitors, 17-AAG and 17-DMAG, can suppress the proliferation of KSHV-positive PEL cell lines and exhibited IC(50) values of 50 nM and below.

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K1 interacts with endogenous Hsp90β and ER-associated Hsp40A. Protein lysates from stable 293 cells expressing empty control vector (left lanes) or FLAG-K1 (right lanes) were immunoprecipitated with anti-FLAG antibody. Immunoprecipitation reactions were subjected to SDS-PAGE followed by Western blotting with an anti-Hsp90β antibody. Input lysates showed the presence of Hsp90β in all cell lysates, and K1 protein in the 293-K1 cell lysate. B. Reverse co-immunoprecipitations were also performed. Lysates from 293-Vec or 293-K1 stable cells were immunoprecipitated with an anti-Hsp90β antibody. Immunoprecipitations were subjected to SDS-PAGE and WB analysis with an anti-FLAG antibody to detect K1 protein expression. Input lysates showed the presence of Hsp90β in all cell lysates, and K1 protein in the 293-K1 cell lysate. These data are representative of at least three independent experiments. C. & D. Identical co-immunoprecipitations were performed as indicated in panels A and B, respectively, except that anti-Hsp40 antibody was used instead of Hsp90β antibody.
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Figure 2: K1 interacts with endogenous Hsp90β and ER-associated Hsp40A. Protein lysates from stable 293 cells expressing empty control vector (left lanes) or FLAG-K1 (right lanes) were immunoprecipitated with anti-FLAG antibody. Immunoprecipitation reactions were subjected to SDS-PAGE followed by Western blotting with an anti-Hsp90β antibody. Input lysates showed the presence of Hsp90β in all cell lysates, and K1 protein in the 293-K1 cell lysate. B. Reverse co-immunoprecipitations were also performed. Lysates from 293-Vec or 293-K1 stable cells were immunoprecipitated with an anti-Hsp90β antibody. Immunoprecipitations were subjected to SDS-PAGE and WB analysis with an anti-FLAG antibody to detect K1 protein expression. Input lysates showed the presence of Hsp90β in all cell lysates, and K1 protein in the 293-K1 cell lysate. These data are representative of at least three independent experiments. C. & D. Identical co-immunoprecipitations were performed as indicated in panels A and B, respectively, except that anti-Hsp40 antibody was used instead of Hsp90β antibody.

Mentions: To confirm the TAP results, equivalent micrograms of pre-cleared 293-K1 and 293-Vec lysates were used to perform immunoprecipitation with anti-FLAG resin beads to immunoprecipitate FLAG-tagged K1 (Figs. 2A and 2C). The immunoprecipitates were subjected to Western blot analysis with anti-Hsp90β (Fig. 2A) or anti-Erdj3 antibody (Fig. 2C). Interactions of K1 with endogenous Hsp90β and Erdj3 were confirmed by reverse co-immunoprecipitation using anti-Hsp90β (Fig. 2B) or anti-Erdj3/Hsp40 (Fig. 2D), followed by immunoblotting with an anti-FLAG antibody to detect K1 expression. Immunoprecipitation assays performed in both directions strongly corroborated the TAP results, and demonstrated that K1 physically associates with Hsp90β and Erdj3/ER-associated Hsp40. To confirm specificity, we repeated the K1 immunoprecipitation assay with a normal mouse IgG antibody as a negative control. We found that immunoprecipitatons using an anti-Flag antibody to pull down K1 co-immunoprecipitated Hsp90β and Hsp40, while immunoprecipitation with the control normal mouse IgG antibody did not pull down K1, Hsp90β or Hsp40 (Supplemental Fig. 1).


Hsp90 and Hsp40/Erdj3 are required for the expression and anti-apoptotic function of KSHV K1.

Wen KW, Damania B - Oncogene (2010)

K1 interacts with endogenous Hsp90β and ER-associated Hsp40A. Protein lysates from stable 293 cells expressing empty control vector (left lanes) or FLAG-K1 (right lanes) were immunoprecipitated with anti-FLAG antibody. Immunoprecipitation reactions were subjected to SDS-PAGE followed by Western blotting with an anti-Hsp90β antibody. Input lysates showed the presence of Hsp90β in all cell lysates, and K1 protein in the 293-K1 cell lysate. B. Reverse co-immunoprecipitations were also performed. Lysates from 293-Vec or 293-K1 stable cells were immunoprecipitated with an anti-Hsp90β antibody. Immunoprecipitations were subjected to SDS-PAGE and WB analysis with an anti-FLAG antibody to detect K1 protein expression. Input lysates showed the presence of Hsp90β in all cell lysates, and K1 protein in the 293-K1 cell lysate. These data are representative of at least three independent experiments. C. & D. Identical co-immunoprecipitations were performed as indicated in panels A and B, respectively, except that anti-Hsp40 antibody was used instead of Hsp90β antibody.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2908282&req=5

Figure 2: K1 interacts with endogenous Hsp90β and ER-associated Hsp40A. Protein lysates from stable 293 cells expressing empty control vector (left lanes) or FLAG-K1 (right lanes) were immunoprecipitated with anti-FLAG antibody. Immunoprecipitation reactions were subjected to SDS-PAGE followed by Western blotting with an anti-Hsp90β antibody. Input lysates showed the presence of Hsp90β in all cell lysates, and K1 protein in the 293-K1 cell lysate. B. Reverse co-immunoprecipitations were also performed. Lysates from 293-Vec or 293-K1 stable cells were immunoprecipitated with an anti-Hsp90β antibody. Immunoprecipitations were subjected to SDS-PAGE and WB analysis with an anti-FLAG antibody to detect K1 protein expression. Input lysates showed the presence of Hsp90β in all cell lysates, and K1 protein in the 293-K1 cell lysate. These data are representative of at least three independent experiments. C. & D. Identical co-immunoprecipitations were performed as indicated in panels A and B, respectively, except that anti-Hsp40 antibody was used instead of Hsp90β antibody.
Mentions: To confirm the TAP results, equivalent micrograms of pre-cleared 293-K1 and 293-Vec lysates were used to perform immunoprecipitation with anti-FLAG resin beads to immunoprecipitate FLAG-tagged K1 (Figs. 2A and 2C). The immunoprecipitates were subjected to Western blot analysis with anti-Hsp90β (Fig. 2A) or anti-Erdj3 antibody (Fig. 2C). Interactions of K1 with endogenous Hsp90β and Erdj3 were confirmed by reverse co-immunoprecipitation using anti-Hsp90β (Fig. 2B) or anti-Erdj3/Hsp40 (Fig. 2D), followed by immunoblotting with an anti-FLAG antibody to detect K1 expression. Immunoprecipitation assays performed in both directions strongly corroborated the TAP results, and demonstrated that K1 physically associates with Hsp90β and Erdj3/ER-associated Hsp40. To confirm specificity, we repeated the K1 immunoprecipitation assay with a normal mouse IgG antibody as a negative control. We found that immunoprecipitatons using an anti-Flag antibody to pull down K1 co-immunoprecipitated Hsp90β and Hsp40, while immunoprecipitation with the control normal mouse IgG antibody did not pull down K1, Hsp90β or Hsp40 (Supplemental Fig. 1).

Bottom Line: Kaposi sarcoma-associated herpesvirus (KSHV) is a member of the gammaherpesvirus family.We report that small-interfering RNAs directed against Hsp90 and Hsp40/Erdj3, as well as pharmacological inhibitors of Hsp90, dramatically reduced K1 expression, suggesting that K1 is a client protein of these chaperones.Finally, we report that the Hsp90 inhibitors, 17-AAG and 17-DMAG, can suppress the proliferation of KSHV-positive PEL cell lines and exhibited IC(50) values of 50 nM and below.

View Article: PubMed Central - PubMed

Affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA.

ABSTRACT
Kaposi sarcoma-associated herpesvirus (KSHV) is a member of the gammaherpesvirus family. It is the etiological agent of three different human cancers, Kaposi sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman disease. The far left end of the KSHV genome encodes a unique transmembrane glycoprotein called K1. K1 possesses the ability to transform rodent fibroblasts and block apoptosis. K1 has also been shown to activate the PI3K/Akt/mTOR pathway in different cells. Using tandem affinity purification, we identified heat shock protein 90beta (Hsp90beta) and endoplasmic reticulum-associated Hsp40 (Erdj3/DnaJB11), as cellular binding partners of K1. Interactions of K1 with Hsp90beta and Hsp40 were confirmed by co-immunoprecipitation in both directions. Furthermore, K1 also interacted with the Hsp90alpha isoform. We report that small-interfering RNAs directed against Hsp90 and Hsp40/Erdj3, as well as pharmacological inhibitors of Hsp90, dramatically reduced K1 expression, suggesting that K1 is a client protein of these chaperones. In addition, both Hsp90 and Hsp40/Erdj3 were essential for K1's anti-apoptotic function. Finally, we report that the Hsp90 inhibitors, 17-AAG and 17-DMAG, can suppress the proliferation of KSHV-positive PEL cell lines and exhibited IC(50) values of 50 nM and below.

Show MeSH
Related in: MedlinePlus