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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 Hsp90β and Erdj3/Hsp40 in both 293 and BJAB cellsA. Protein lysates from stable 293 cells expressing empty pcDNA3.1 control vector or FLAG-K1 were immunoprecipitated with anti-FLAG or control anti-normal mouse Ig antibody. Immunoprecipitation reactions were subjected to SDS-PAGE followed by Western blotting with an anti-Hsp70 antibody. Input lanes show the presence of Hsp70 in all protein lysates and K1 protein in the 293-K1 lysate only. B. Protein lysates from stable 293-Vec or 293-K1 cells after treatment with vehicle or apyrase were immunoprecipitated with anti-FLAG antibody. Immunoprecipitation reactions were subjected to SDS-PAGE followed by Western blotting with an anti-Hsp90β antibody. C. Identical co-immunoprecipitations were performed as described in Fig. 2A, except that protein lysates from BJAB B cells transfected with pcDNA3.1 vector or K1 expression plasmid were immunoprecipitated with anti-FLAG antibody. Immunoprecipitates 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. Immunoprecipitation was also performed with species-matched normal IgG antibody as a negative control.
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Figure 3: K1 interacts with Hsp90β and Erdj3/Hsp40 in both 293 and BJAB cellsA. Protein lysates from stable 293 cells expressing empty pcDNA3.1 control vector or FLAG-K1 were immunoprecipitated with anti-FLAG or control anti-normal mouse Ig antibody. Immunoprecipitation reactions were subjected to SDS-PAGE followed by Western blotting with an anti-Hsp70 antibody. Input lanes show the presence of Hsp70 in all protein lysates and K1 protein in the 293-K1 lysate only. B. Protein lysates from stable 293-Vec or 293-K1 cells after treatment with vehicle or apyrase were immunoprecipitated with anti-FLAG antibody. Immunoprecipitation reactions were subjected to SDS-PAGE followed by Western blotting with an anti-Hsp90β antibody. C. Identical co-immunoprecipitations were performed as described in Fig. 2A, except that protein lysates from BJAB B cells transfected with pcDNA3.1 vector or K1 expression plasmid were immunoprecipitated with anti-FLAG antibody. Immunoprecipitates 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. Immunoprecipitation was also performed with species-matched normal IgG antibody as a negative control.

Mentions: We next investigated whether K1 interacts with heat shock proteins other than Hsp40 and Hsp90. We immunoprecipitated K1 from 293-K1 stable cells with an anti-FLAG antibody. The immunoprecipitates were subjected to Western blot analysis with an anti-Hsp70 antibody (Fig. 3A). We found that K1 failed to interact with Hsp70 suggesting that K1 specifically interacts with Hsp90 and Erdj3/Hsp40.


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 Hsp90β and Erdj3/Hsp40 in both 293 and BJAB cellsA. Protein lysates from stable 293 cells expressing empty pcDNA3.1 control vector or FLAG-K1 were immunoprecipitated with anti-FLAG or control anti-normal mouse Ig antibody. Immunoprecipitation reactions were subjected to SDS-PAGE followed by Western blotting with an anti-Hsp70 antibody. Input lanes show the presence of Hsp70 in all protein lysates and K1 protein in the 293-K1 lysate only. B. Protein lysates from stable 293-Vec or 293-K1 cells after treatment with vehicle or apyrase were immunoprecipitated with anti-FLAG antibody. Immunoprecipitation reactions were subjected to SDS-PAGE followed by Western blotting with an anti-Hsp90β antibody. C. Identical co-immunoprecipitations were performed as described in Fig. 2A, except that protein lysates from BJAB B cells transfected with pcDNA3.1 vector or K1 expression plasmid were immunoprecipitated with anti-FLAG antibody. Immunoprecipitates 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. Immunoprecipitation was also performed with species-matched normal IgG antibody as a negative control.
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Related In: Results  -  Collection

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Figure 3: K1 interacts with Hsp90β and Erdj3/Hsp40 in both 293 and BJAB cellsA. Protein lysates from stable 293 cells expressing empty pcDNA3.1 control vector or FLAG-K1 were immunoprecipitated with anti-FLAG or control anti-normal mouse Ig antibody. Immunoprecipitation reactions were subjected to SDS-PAGE followed by Western blotting with an anti-Hsp70 antibody. Input lanes show the presence of Hsp70 in all protein lysates and K1 protein in the 293-K1 lysate only. B. Protein lysates from stable 293-Vec or 293-K1 cells after treatment with vehicle or apyrase were immunoprecipitated with anti-FLAG antibody. Immunoprecipitation reactions were subjected to SDS-PAGE followed by Western blotting with an anti-Hsp90β antibody. C. Identical co-immunoprecipitations were performed as described in Fig. 2A, except that protein lysates from BJAB B cells transfected with pcDNA3.1 vector or K1 expression plasmid were immunoprecipitated with anti-FLAG antibody. Immunoprecipitates 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. Immunoprecipitation was also performed with species-matched normal IgG antibody as a negative control.
Mentions: We next investigated whether K1 interacts with heat shock proteins other than Hsp40 and Hsp90. We immunoprecipitated K1 from 293-K1 stable cells with an anti-FLAG antibody. The immunoprecipitates were subjected to Western blot analysis with an anti-Hsp70 antibody (Fig. 3A). We found that K1 failed to interact with Hsp70 suggesting that K1 specifically interacts with Hsp90 and Erdj3/Hsp40.

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