Abstract B68: Ubiquitin E3 ligase, tripartite motif protein 68 (TRIM68) inhibits TCP-1β function and can be a target of imatinib-resistance
Authors: Kuniyoshi Ryoko , Terui Yasuhito , Mishima Yuji , Matsusaka Satoshi , Hatake Kiyohiko 
Published: June 2012

Abstract
Background: Resistance to imatinib mesylate is a major problem in chronic myeloid leukaemia (CML) treatment. Most of the studies about the resistance have focused on point mutations in BCR/ABL. However, other mechanisms of resistance that do not imply mutations in BCR/ABL have been also described. The identification of new proteins induced by imatinib may lead to find the novel potent molecular targets in imatinib-resistant CML.
Methods: K562 cells were treated with or without 1 µM imatinib for 24 hours, and then differential display between them was performed. TRIM68 expression was examined by RT-PCR, and in vivo ubiquitination or sumoylation assay was performed by transfection experiment and Western blot analysis. The substrates for TRIM68 were analyzed by mass spectorometry.
Results: From the results of RNA differential display, we found that the expression of TRIM68 mRNA was increased when the K562 cells were treated with 1 µM imatinib for 24 hours. TRIM68 protein possesses a RING finger domain at its N-terminal site. Since many RING-finger proteins have been identified as E3 ligases for ubiquitination or sumoylation, we examined whether TRIM68 functions as an E3 ligase for ubiquitination or sumoylation. To examine the function of TRIM68 as an E3 ligase, wild type TRIM68 and a RING domain deletion mutant of TRIM68 (TRIM68/ΔR) genes were constructed into a mammalian expression vector and they were transfected into MCF7 cells. TRIM68 had auto-ubiquitination activity but not autosumoylation activity in vivo assays. This suggests that TRIM68 is could be an ubiquitin E3 ligase but not sumo E3 ligase. Moreover, wild type TRIM68 promoted the whole ubiqutination in the cells, whereas TRIM68/ΔR prevented the ubiquitination inside of the cells. To identify the TRIM68-interacting proteins, we transfected FLAG-tagged wild type TRIM68 gene or B30.2/SPRY domain of TRIM68 gene into MCF7 cells, and immunoprecipitation with FLAG-M2 agarose was performed and mass spectrometric analysis was performed. As the results, we identified the members of molecular chaperone T-complex polypeptide 1 (TCP-1) complex, TCP-1β and heat shock protein 70 interacting with TRIM68 at the B30.2/SPRY domain. Then, we examined whether TCP-1β is one of the substrates for TRIM68- related ubiqutination. TCP-1β was ubiquitinated by wild type TRIM68, but not by TRIM68/ΔR. Furthermore, the ubiquitination of TCP-1β was accumulated by the treatment with a proteasome inhibitor MG132. These suggested that TCP-1β is one of the substrates for TRIM68.
Conclusion: We found that TRIM68 is induced by the treatment with imatinib and functions as an ubiquitin E3 ligase. Furthermore, we identified that TCP-1β is a substrate of TRIM68. TRIM68 may inhibit the function of TCP-1β as a chaperone by ubiquitination and proteasome-mediated degradation. TRIM68 could be a new target in the imatinib-resistant CML.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B68.