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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 Hsp40 through its NterminusA. Co-immunoprecipitation of a panel of K1 constructs with endogenous Hsp90β. 293 cells were transfected with the empty vector or a panel of FLAG-K1 domain deletion mutants (ΔC: C-terminal deletion; ΔTM: transmembrane deletion; ΔN: N-terminal deletion), or the full-length FLAG-K1. Lysates were immunoprecipitated with anti-FLAG resin. Immunoprecipitation reactions were subjected to SDS-PAGE and Western blotting with an anti-Hsp90β antibody. Input lysates showed the presence of Hsp90β in all cell lysates and the presence of K1 mutants in the K1 mutant transfected cells. B. Reverse co-immunoprecipitations were also performed. Lysates from cells transfected with the vector control or the K1 panel of mutants were immunoprecipitated with an anti-Hsp90β antibody. Immunoprecipitation reactions were subjected to Western blot analysis with an anti-FLAG antibody to detect K1 protein expression. Input lysates showed the presence of Hsp90β in all cell lysates and the presence of K1 mutants in the K1 mutant transfected cells. 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. In panels A–D, arrows indicate the N-terminal deletion mutant of K1.
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Figure 4: K1 interacts with endogenous Hsp90β and ER-associated Hsp40 through its NterminusA. Co-immunoprecipitation of a panel of K1 constructs with endogenous Hsp90β. 293 cells were transfected with the empty vector or a panel of FLAG-K1 domain deletion mutants (ΔC: C-terminal deletion; ΔTM: transmembrane deletion; ΔN: N-terminal deletion), or the full-length FLAG-K1. Lysates were immunoprecipitated with anti-FLAG resin. Immunoprecipitation reactions were subjected to SDS-PAGE and Western blotting with an anti-Hsp90β antibody. Input lysates showed the presence of Hsp90β in all cell lysates and the presence of K1 mutants in the K1 mutant transfected cells. B. Reverse co-immunoprecipitations were also performed. Lysates from cells transfected with the vector control or the K1 panel of mutants were immunoprecipitated with an anti-Hsp90β antibody. Immunoprecipitation reactions were subjected to Western blot analysis with an anti-FLAG antibody to detect K1 protein expression. Input lysates showed the presence of Hsp90β in all cell lysates and the presence of K1 mutants in the K1 mutant transfected cells. 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. In panels A–D, arrows indicate the N-terminal deletion mutant of K1.

Mentions: To gain insight into the function and topology of the interaction, we used a panel of FLAG-tagged K1 domain deletion constructs ((ΔC: C-terminal deletion; ΔTM: transmembrane deletion; ΔN: N-terminal deletion)) to determine the K1 domain that interacted with Hsp90β and the ER-associated Hsp40. Expression plasmids for the K1 deletion mutants, the FLAG-tagged wild-type K1, and the control pcDNA3 vector, were transiently transfected into 293 cells. Protein lysates were harvested 48 h post-transfection. Co-immunoprecipitations for K1 with Hsp90β and Hsp40 were performed as described above for Fig. 2. FLAG-tagged K1 was immunoprecipitated with anti-FLAG beads, followed by immunoblotting with anti-Hsp90β (Fig. 4A) or anti-Hsp40/Erdj3 antibody (Fig. 4C). Hsp90β and Hsp40 were detected in all of the samples expressing K1 except for the sample expressing the K1ΔN mutant. Additionally, the reverse immunoprecipitation of Hsp90β (Fig. 4B) or Hsp40 (Fig. 4D) followed by Western blot analysis with anti-FLAG antibody also suggested that the K1ΔN mutant protein does not interact with Hsp90β or Hsp40. Collectively, our data suggests that the N-terminal domain of K1 interacts with the Hsp90β and Hsp40/Erdj3 molecular chaperones.


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 Hsp40 through its NterminusA. Co-immunoprecipitation of a panel of K1 constructs with endogenous Hsp90β. 293 cells were transfected with the empty vector or a panel of FLAG-K1 domain deletion mutants (ΔC: C-terminal deletion; ΔTM: transmembrane deletion; ΔN: N-terminal deletion), or the full-length FLAG-K1. Lysates were immunoprecipitated with anti-FLAG resin. Immunoprecipitation reactions were subjected to SDS-PAGE and Western blotting with an anti-Hsp90β antibody. Input lysates showed the presence of Hsp90β in all cell lysates and the presence of K1 mutants in the K1 mutant transfected cells. B. Reverse co-immunoprecipitations were also performed. Lysates from cells transfected with the vector control or the K1 panel of mutants were immunoprecipitated with an anti-Hsp90β antibody. Immunoprecipitation reactions were subjected to Western blot analysis with an anti-FLAG antibody to detect K1 protein expression. Input lysates showed the presence of Hsp90β in all cell lysates and the presence of K1 mutants in the K1 mutant transfected cells. 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. In panels A–D, arrows indicate the N-terminal deletion mutant of K1.
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Figure 4: K1 interacts with endogenous Hsp90β and ER-associated Hsp40 through its NterminusA. Co-immunoprecipitation of a panel of K1 constructs with endogenous Hsp90β. 293 cells were transfected with the empty vector or a panel of FLAG-K1 domain deletion mutants (ΔC: C-terminal deletion; ΔTM: transmembrane deletion; ΔN: N-terminal deletion), or the full-length FLAG-K1. Lysates were immunoprecipitated with anti-FLAG resin. Immunoprecipitation reactions were subjected to SDS-PAGE and Western blotting with an anti-Hsp90β antibody. Input lysates showed the presence of Hsp90β in all cell lysates and the presence of K1 mutants in the K1 mutant transfected cells. B. Reverse co-immunoprecipitations were also performed. Lysates from cells transfected with the vector control or the K1 panel of mutants were immunoprecipitated with an anti-Hsp90β antibody. Immunoprecipitation reactions were subjected to Western blot analysis with an anti-FLAG antibody to detect K1 protein expression. Input lysates showed the presence of Hsp90β in all cell lysates and the presence of K1 mutants in the K1 mutant transfected cells. 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. In panels A–D, arrows indicate the N-terminal deletion mutant of K1.
Mentions: To gain insight into the function and topology of the interaction, we used a panel of FLAG-tagged K1 domain deletion constructs ((ΔC: C-terminal deletion; ΔTM: transmembrane deletion; ΔN: N-terminal deletion)) to determine the K1 domain that interacted with Hsp90β and the ER-associated Hsp40. Expression plasmids for the K1 deletion mutants, the FLAG-tagged wild-type K1, and the control pcDNA3 vector, were transiently transfected into 293 cells. Protein lysates were harvested 48 h post-transfection. Co-immunoprecipitations for K1 with Hsp90β and Hsp40 were performed as described above for Fig. 2. FLAG-tagged K1 was immunoprecipitated with anti-FLAG beads, followed by immunoblotting with anti-Hsp90β (Fig. 4A) or anti-Hsp40/Erdj3 antibody (Fig. 4C). Hsp90β and Hsp40 were detected in all of the samples expressing K1 except for the sample expressing the K1ΔN mutant. Additionally, the reverse immunoprecipitation of Hsp90β (Fig. 4B) or Hsp40 (Fig. 4D) followed by Western blot analysis with anti-FLAG antibody also suggested that the K1ΔN mutant protein does not interact with Hsp90β or Hsp40. Collectively, our data suggests that the N-terminal domain of K1 interacts with the Hsp90β and Hsp40/Erdj3 molecular chaperones.

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