Nuclear localization signal sequence is required for VACM-1/CUL5-dependent regulation of cellular growth

Abstract

VACM-1/CUL5 is a member of the cullin family of proteins involved in the E3 ligase-dependent degradation of diverse proteins that regulate cellular proliferation. The ability of VACM-1/CUL5 to inhibit cellular growth is affected by its posttranslational modifications and its localization to the nucleus. Since the mechanism of VACM-1/CUL5 translocation to the nucleus is not clear, the goal of this project was to determine the role that the putative nuclear localization signal (NLS) we identified in the VACM-1/CUL5 (PKLKRQ) plays in the cellular localization of VACM-1/CUL5 and its effect on cellular growth. We used site-directed mutagenesis to change Lys642 and Lys644 to Gly and the mutated cDNA constructs were transfected into COS-1 cells. Mutation of the NLS in VACM-1/CUL5 significantly reduced its localization to the nucleus and compromised its effect on cellular growth. We have shown previously that the antiproliferative effect of VACM-1/CUL5 could be reversed by mutation of PKA-specific phosphorylation sequence (VACM-1/CUL5), which was associated with its increased nuclear localization and modification by NEDD8. Thus, we examined whether these properties can be controlled by the NLS. The mutation of NLS in VACM-1/CUL5 cDNA compromised its proliferative effect and reduced its localization to the nucleus. The immunocytochemistry results showed that, in cells transfected with the mutant cDNAs, the nuclear NEDD8 signal was decreased. Western blot analysis of total cell lysates, however, showed that VACM-1/CUL5 neddylation was not affected. Together, these results suggest that the presence of the NLS, both in VACM-1/CUL5 and inVACM-1/CUL5 sequences, is critical for their control of cell proliferation.

SENP8 limits aberrant neddylation of NEDD8 pathway components to promote cullin-RING ubiquitin ligase function

“…Accompanying these deficiencies, SENP8 knockout cells also showed aberrant G1/S cell cycle progression and accelerated cell growth (Figure 6). Interestingly, these cell cycle alterations partially phenocopy previously reported defects associated with the loss of either Cul1 (Dealy et al, 1999; Wang et al, 1999; Chen and Li, 2010) or Cul5 function (Burnatowska-Hledin et al, 2001; Buchwalter et al, 2008; Bradley et al, 2010; Ma et al, 2013a; Willis et al, 2017). Through an unbiased proteomic screen in which we enriched for K-ε-GG remnant-containing peptides in MG132-treated cells, we were able to identify several candidate substrates with deregulated ubiquitylation status and stability in the SENP8 knockout cells that could potentially contribute to G1/S progression defects.…”

Types of nuclear localization signals and mechanisms of protein import into the nucleus

“…identified a putative NLS ( 640 P K L KR Q 646 ) in vasopressin-activated calcium-mobilizing protein/cullin5 (VACM-1/CUL5), which is necessary for its nuclear localization and inhibitory effect on cellular growth. This sequence starts with a proline (P) and is followed by an amino acid sequence containing three basic residues out of four (P K L KR ) [ 19 ]. An analogous NLS, found in the 146th-149th amino acids of chemokine receptor CXCR4, is composed of Arg-Pro-Arg-Lys ( R P RK ) [ 20 ].…”

Comparative Analysis of cul5 and rbx2 Expression in the Developing and Adult Murine Brain and Their Essentiality During Mouse Embryogenesis

“…Our data also indicate that despite Cul5 and Rbx2 being expressed in the same cells, their subcellular localization is, in general, different, with Cul5 mostly detected in the cytoplasm and Rbx2 mostly detected in the nucleus. Accordingly, Cul5 has been postulated to regulate cellular growth when localized in the nucleus, and preventing nuclear localization disrupts the ability of Cul5 to regulate cellular proliferation, which suggests that Cul5 localization is regulated and important for its function (Willis et al, 2017). Further work will elucidate whether Cul5 and Rbx2 compartmentalization responds to a regulatory control of CRL5 activity or indicates a preference in CRL5 substrate localization.…”