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Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4(CRBN.).

Gandhi AK, Kang J, Havens CG, Conklin T, Ning Y, Wu L, Ito T, Ando H, Waldman MF, Thakurta A, Klippel A, Handa H, Daniel TO, Schafer PH, Chopra R - Br. J. Haematol. (2013)

Bottom Line: We confirm that Aiolos and Ikaros are transcriptional repressors of interleukin-2 expression.The findings link lenalidomide- or pomalidomide-induced degradation of these transcriptional suppressors to well documented T cell activation.In conclusion, we present a molecular model in which drug binding to cereblon results in the interaction of Ikaros and Aiolos to CRL4(CRBN) , leading to their ubiquitination, subsequent proteasomal degradation and T cell activation.

View Article: PubMed Central - PubMed

Affiliation: Celgene Corporation, Summit, NJ, USA.

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Cereblon binds Aiolos and Ikaros and requires the thalidomide binding domain on Cereblon. (A) HEK-293T cells were transfected with Flag-WT Cereblon or Flag-YW/AA Cereblon with or without HA-Aiolos for 48 h with pomalidomide added to cells for the last hour. Lysates were made and immunoprecipitation with Flag antibody followed by SDS-PAGE separation and immunoblotting with anti-Aiolos or anti–Cereblon was performed. (B) HEK-293T cells were transfected with Flag-WT Cereblon or Flag-YW/AA Cereblon with or without HA-Ikaros for 48 h with pomalidomide added to cells for the last hour. Lysates were made and immunoprecipitation with Flag antibody followed by SDS-PAGE separation and immunoblotting with anti–Cereblon or anti-Ikaros was performed. IP, immunoprecipitation; CRBN, Cereblon; WT, wildtype; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis.
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fig03: Cereblon binds Aiolos and Ikaros and requires the thalidomide binding domain on Cereblon. (A) HEK-293T cells were transfected with Flag-WT Cereblon or Flag-YW/AA Cereblon with or without HA-Aiolos for 48 h with pomalidomide added to cells for the last hour. Lysates were made and immunoprecipitation with Flag antibody followed by SDS-PAGE separation and immunoblotting with anti-Aiolos or anti–Cereblon was performed. (B) HEK-293T cells were transfected with Flag-WT Cereblon or Flag-YW/AA Cereblon with or without HA-Ikaros for 48 h with pomalidomide added to cells for the last hour. Lysates were made and immunoprecipitation with Flag antibody followed by SDS-PAGE separation and immunoblotting with anti–Cereblon or anti-Ikaros was performed. IP, immunoprecipitation; CRBN, Cereblon; WT, wildtype; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis.

Mentions: To determine if Aiolos and Cereblon physically interact, co-immunoprecipitation experiments were performed in HEK-293T cells overexpressing Flag-CRBN and HA-Aiolos (Fig 3A). After immunoprecipitation with anti-Flag antibody, Aiolos protein was co-precipitated, but only in the presence of pomalidomide. Furthermore, overexpression of a mutant CRBN (YW/AA), which can no longer bind to the pomalidomide structural analogue thalidomide (Ito et al, 2010) abrogated the CRL4CRBN co-precipitation with Aiolos. Fig 3B shows that binding of Ikaros to Cereblon similarly occurred in a pomalidomide-dependent manner, with enhanced recovery of Ikaros. Taken together, drug binding promotes Cereblon engagement in interactions with the transcription factors Aiolos and Ikaros, proteins that are not readily detected as substrates in the absence of drug. These interactions lead to their degradation in T cells.


Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4(CRBN.).

Gandhi AK, Kang J, Havens CG, Conklin T, Ning Y, Wu L, Ito T, Ando H, Waldman MF, Thakurta A, Klippel A, Handa H, Daniel TO, Schafer PH, Chopra R - Br. J. Haematol. (2013)

Cereblon binds Aiolos and Ikaros and requires the thalidomide binding domain on Cereblon. (A) HEK-293T cells were transfected with Flag-WT Cereblon or Flag-YW/AA Cereblon with or without HA-Aiolos for 48 h with pomalidomide added to cells for the last hour. Lysates were made and immunoprecipitation with Flag antibody followed by SDS-PAGE separation and immunoblotting with anti-Aiolos or anti–Cereblon was performed. (B) HEK-293T cells were transfected with Flag-WT Cereblon or Flag-YW/AA Cereblon with or without HA-Ikaros for 48 h with pomalidomide added to cells for the last hour. Lysates were made and immunoprecipitation with Flag antibody followed by SDS-PAGE separation and immunoblotting with anti–Cereblon or anti-Ikaros was performed. IP, immunoprecipitation; CRBN, Cereblon; WT, wildtype; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232904&req=5

fig03: Cereblon binds Aiolos and Ikaros and requires the thalidomide binding domain on Cereblon. (A) HEK-293T cells were transfected with Flag-WT Cereblon or Flag-YW/AA Cereblon with or without HA-Aiolos for 48 h with pomalidomide added to cells for the last hour. Lysates were made and immunoprecipitation with Flag antibody followed by SDS-PAGE separation and immunoblotting with anti-Aiolos or anti–Cereblon was performed. (B) HEK-293T cells were transfected with Flag-WT Cereblon or Flag-YW/AA Cereblon with or without HA-Ikaros for 48 h with pomalidomide added to cells for the last hour. Lysates were made and immunoprecipitation with Flag antibody followed by SDS-PAGE separation and immunoblotting with anti–Cereblon or anti-Ikaros was performed. IP, immunoprecipitation; CRBN, Cereblon; WT, wildtype; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis.
Mentions: To determine if Aiolos and Cereblon physically interact, co-immunoprecipitation experiments were performed in HEK-293T cells overexpressing Flag-CRBN and HA-Aiolos (Fig 3A). After immunoprecipitation with anti-Flag antibody, Aiolos protein was co-precipitated, but only in the presence of pomalidomide. Furthermore, overexpression of a mutant CRBN (YW/AA), which can no longer bind to the pomalidomide structural analogue thalidomide (Ito et al, 2010) abrogated the CRL4CRBN co-precipitation with Aiolos. Fig 3B shows that binding of Ikaros to Cereblon similarly occurred in a pomalidomide-dependent manner, with enhanced recovery of Ikaros. Taken together, drug binding promotes Cereblon engagement in interactions with the transcription factors Aiolos and Ikaros, proteins that are not readily detected as substrates in the absence of drug. These interactions lead to their degradation in T cells.

Bottom Line: We confirm that Aiolos and Ikaros are transcriptional repressors of interleukin-2 expression.The findings link lenalidomide- or pomalidomide-induced degradation of these transcriptional suppressors to well documented T cell activation.In conclusion, we present a molecular model in which drug binding to cereblon results in the interaction of Ikaros and Aiolos to CRL4(CRBN) , leading to their ubiquitination, subsequent proteasomal degradation and T cell activation.

View Article: PubMed Central - PubMed

Affiliation: Celgene Corporation, Summit, NJ, USA.

Show MeSH
Related in: MedlinePlus