Regulation of membrane traffic by ubiquitination

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The relevance of the ubiquitin(Ub)-proteasome system is widely accepted in a variety of cell system. We previously reported a MS-based proteomic technology involving multi-step immunoaffinity purification to characterize signaling complexes in a large scale. In this study we described a novel procedure that combined the technology with newly developed Ub derivatives that inserted a His tag within its C-terminal region. Using this procedure, we successfully identified in vivo ubiquitination sites of over 200 proteins with high confidence. In another experiment, we investigated the crosstalk between phosphorylation and ubiquitination. We found that the ubiquitin E3 ligase TRIM32 binds 14-3-3 proteins in a PKA-catalyzed phosphorylation-dependent manner. Subsequent studies demonstrated that this interaction negatively regulates the catalytic activity and intracellular transport of TRIM32.The findings described herein constitute the first evidence for the regulatory mechanism of the TRIM member.