Masking of a Nuclear Signal Motif by Monoubiquitination Leads to Mislocalization and Degradation of the Regulatory Enzyme Cytidylyltransferase

摘要: Protein monoubiquitination has recently emerged as an important posttranslational modification regulating transcription, endocytic vesicle trafficking, histone modification, and DNA repair (12, 26). Conjugation of one (monoubiquitination) or multiple (multiubiquitination) ubiquitin molecules to lysines within target proteins serves signal for internalization targeting various ion channels, membrane cargo receptors, junctional the pathway (8). Monoubiquitination receptors triggers recruitment ubiquitin-binding that bridge receptor ligands cell sorting elements are further degraded recycled plasma (18). The addition a monoubiquitin tag p53, example, is sufficient its nuclear export; p53 may cooperatively interact with sumoylation involve export (NES) cytoplasmic (5). also regulates import some proteins. tumor suppresser phosphatase tensin homolog on chromosome ten (PTEN) requires despite lacking canonical localization (NLS) (27). Hence, in contrast polyubiquitination short-lived cytosolic usually destined rapid degradation proteasome, appears be means exquisitely regulate availability membrane-associated protein complexes. CTP:phosphocholine cytidylyltransferase (CCT) membrane-activated enzyme catalyzes penultimate rate-limiting step biosynthesis major eukaryotic phospholipid, phosphatidylcholine (PtdCho) (17). CCTα, predominant species lung epithelia, comprised 367 amino acids (aa) four functional domains: basic residue NH2-terminal NLS, catalytic core (C), membrane-binding domain (M), carboxyl-terminal phosphorylation (P) CCTα amphitrophic thus can switch between inactive soluble form active, membrane-bound nucleus. In fact, ability reversibly translocate endoplasmic membranes after stimulation by lipid activators well established remains central activation (13, 17). A second, more recent mode regulatory control involves turnover rate molecules. ubiquitinated response cytokines, such necrosis factor alpha (TNF-α), death effector neutral proteinases (19, 21). However, CCTα's extended half-life (∼8 h) vivo exceeds other comparable enzymes because it stabilized, part, calmodulin (6). long projects there cooperative layer complex regulation additional coadaptor modificational events primary structure impact enzyme's stability. This model would strike delicate balance remain activated state yet preserve phospholipid homeostasis eliminating population when PtdCho presented excess. In present study, we demonstrate monoubiquitinated at single molecular site (K57), thereby marking exclusion lysosome. K57R mutant exhibited greater stability, was proteolytically resistant actions TNF-α, retained nucleus, underscoring relevance this residue. Because K57 close proximity NLS tested hypothesis masking impede association importin-α mislocalize lysosome degradation. execution studies using CCTα-ubiquitin hybrid results unveil conceptually novel whereby cells constitutive behavior levels hindrance motif via monoubiquitination.