Cullin 4A and 4B ubiquitin ligases interact with γ-tubulin and induce its polyubiquitination

Abstract

Regulated polyubiquitination is a key step for controlling protein degradation and maintaining proper balance between the proliferation of normal and uncontrolled cells. Addition of ubiquitin to the proteins by E3 ubiquitin ligases targets them for degradation by the 26S proteosome machinery. Discrepancies in ubiquitination and/or proteosome degradation might lead to multiple genetic disorders in humans. It is reported that CUL1 and BRCA1 ubiquitin ligases localize on centrosome region and regulate the centrosome duplication cycle for genomic stability. In the current study, we predicted the possible interaction of E3 ubiquitin ligase CUL4A complex with γ-tubulin, a centrosome-specific protein, using bioinformatic protein-protein docking analysis. We also confirmed their interaction by performing co-immunoprecipitation studies using endogenous CUL4A/B and stable cell lines that overexpress Flag-CUL4A or Flag-CUL4B. We additionally noted that the γ-tubulin was polyubiquitinated by CUL4A or 4B immune complex indicating that CUL4A or CUL4B may regulate the stability of γ-tubulin. Furthermore, the inhibition of proteosomal degradation pathway using MG132 or LLNV drugs resulted in accumulation and co-localization of CUL4A with γ-tubulin in the centrosome region. Overall, our observation has identified γ-tubulin as a novel target for E3 ubiquitin ligase CUL4 complex, and might lead to the establishment of a unique mechanism for controlling centrosome stability.

Cullin E3 Ligase Activity Is Required for Myoblast Differentiation

“…Our data indicate that a defective ubiquitylation activity through CRL inhibition might either interfere with the abundance of proteins that are important for neuromuscular junction development, or with cytoskeletal remodeling that has been shown to be important for the agrin-induced AchR clustering process [74–79]. Indeed, several members of the cullin protein family associate with, and are capable of remodeling actin and tubulin filaments in non-muscle cells [80–84]. The biological function of these CRL at the cytoskeleton may also extend to myoblasts, as we find cullin-3 associated with the tubulin cytoskeleton in skeletal muscle myoblasts (Supplemental Figure S3).…”

N-terminal truncations of human bHLH transcription factor Twist1 leads to the formation of aggresomes

“…S2). In addition, we also noted the reported localization of HDAC6 (aggresome marker) [6], γ-tubulin (centrosome marker), CUL4A (Centrosome associated E3 ligase) [29] in the juxta nuclear position when cells were treated with MG132 (Supplementary Fig. S2).…”

“…Primers employed for cloning is tabulated in Supplementary (S1). CUL4A-GFP construct was described in our earlier report [29].…”

The preclinical discovery and development of bortezomib for the treatment of mantle cell lymphoma

“…Ub is a highly conserved and small (76 amino acids, 8.5 kDa) regulatory protein moiety which, through a series of molecular processes, is linked to lysine residues of targeted proteins. The fate of the target proteins is dependent on the dynamic pattern of ubiquitin branching [13–15]. …”

“…Ub conjugation occurs via an enzymatic cascade, involving three distinct enzymes: (i) Ub-activating (E1), (ii) Ub-conjugating (E2), and (iii) Ub-ligating enzymes (E3 Ligases) (Figure 2B). Protein ubiquitination is initiated by the ATP-dependent formation of a thioester linkage between the C-terminus of the Ub moiety and a cysteine residue of the E1-activating enzyme [13–14]. The Ub moiety is then transferred to an E2-Ubiquitin-conjugating enzyme through the formation of an E1-Ub-E2 complex via the formation of a thio -ester intermediate complex [15].…”