Fig. 3: UBE2T induces ubiquitination and proteasomal degradation of RACK1 at K172, K225, and K257 independent of any E3 ligase. | Oncogene

Fig. 3: UBE2T induces ubiquitination and proteasomal degradation of RACK1 at K172, K225, and K257 independent of any E3 ligase.

From: A novel UBE2T inhibitor suppresses Wnt/β-catenin signaling hyperactivation and gastric cancer progression by blocking RACK1 ubiquitination

Fig. 3: UBE2T induces ubiquitination and proteasomal degradation of RACK1 at K172, K225, and K257 independent of any E3 ligase.

A HEK-293T cells were transiently transfected with plasmids encoding HA-tagged RACK1, along with the indicated amounts of a plasmid encoding Flag-tagged UBE2T 24 h after transfection, cell lysates were analyzed by western blot with indicated antibodies. Top: the RACK1 protein level in wild or UBE2T−/− AGS cells was analyzed by western blot with indicated antibodies (bottom). B Correlational analysis between UBE2T and RACK1 immunohistochemistry (IHC) score in GC tissues. Spearman correlation coefficient = −0.5112; P < 0.0001. n = 155. C HEK-293T cells were transiently transfected with plasmids encoding Flag-tagged RACK1 and UBE2T, along with plasmids encoding HA-tagged wild ubiquitin or indicated mutant ubiquitin. Sixteen hours after transfection, cells were treated with MG132 for 8 h (10 μM). Cell lysates were analyzed by immunoprecipitation with anti-Flag and western immunoblotting with indicated antibodies. D HEK-293T cells were transiently transfected with plasmids encoding HA-tagged RACK1, along with a plasmid encoding Flag-tagged wild-type UBE2T or UBE2TC86A mutant. C86A: the 86th cysteine was substituted to alanine. Twenty-four hours after transfection, cell lysates were analyzed by western blot with indicated antibodies. E HEK-293T cells were transiently transfected with plasmids expressing Flag-tagged RACK1 and HA-tagged ubiquitin, along with plasmids encoding wild UBE2T or UBE2TC86A mutant. Sixteen hours after transfection, cells were treated with MG132 for 8 h (10 μM). Cell lysates were analyzed by immunoprecipitation with anti-Flag and western immunoblotting with indicated antibodies. F Recombinant RACK1 proteins were subjected to in vitro ubiquitination assay in the absence or presence of in vitro-translated wild-type UBE2T, and western immunoblotting with indicated antibodies. G The schematic diagram of RACK1 domain. H HEK-293T cells were transiently transfected with plasmids expressing Flag-tagged the deletion of the indicated domain of RACK1 and HA-tagged ubiquitin, along with plasmid expressing UBE2T. I HEK-293T cells were transiently transfected with plasmids expressing Flag-tagged of indicated RACK1 mutant plasmids and HA-tagged ubiquitin plasmids, along with plasmid expressing UBE2T. J Wild-type and lysine residual mutated Flag-tagged RACK1 plasmids were individually transfected into HEK-293T cells, with or without HA-tagged UBE2T. Cell lysates were analyzed by western blot with indicated antibodies. K A plasmid expressing Flag-tagged wild type, indicated mutant RACK1 or GFP were transfected into HEK-293T cells with a plasmid expressing HA-tagged UBE2T. Sixteen hours after transfection, cells were treated with MG132 for 8 h (10 μM). Cell lysates were analyzed by immunoprecipitation with anti-Flag and western immunoblotting with indicated antibodies.

Back to article page