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 (640PKLKRQ646) 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 (S730AVACM-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 S730AVACM-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 in S730AVACM-1/CUL5 sequences, is critical for their control of cell proliferation.
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Abbreviations
- NEDD8:
-
Neural precursor cell expressed, developmentally down-regulated protein 8
- NLS:
-
Nuclear localization signal
- PKA:
-
Protein kinase A
- RAMEC:
-
Rat adrenal medullary endothelial cells
- VACM-1/CUL5:
-
Vasopressin-activated calcium-mobilizing (VACM-1) protein/cullin5
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Acknowledgments
This work was supported by NIH (RO1DK47199) and by NCI (R15CA104014). In addition, we acknowledge a Beckman Foundation Award to S. Dean and grants from the National Science Foundation Research Experience for Undergraduates Programs (DBI-0754293, CHE-851194) that supported A. Willis. We also acknowledge the editorial assistance by Elizabeth Brigger.
Authors’ contributions
ANW and JH designed and constructed all mutant cDNAs and SH prepared and screened all plasmids. SEBD and MLL performed growth assays while BTK, ADS and SPL performed transfections, Western blot analysis and immuno staining. RG performed western blot analysis and MABH performed cytotoxicity assay. The first three authors were involved in writing and editing the manuscript. All authors read and approved the final manuscript.
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Angelica N. Willis, Shirley E. Bradley Dean and Joe A. Habbouche contributed equally to this work.
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Willis, A.N., Dean, S.E.B., Habbouche, J.A. et al. Nuclear localization signal sequence is required for VACM-1/CUL5-dependent regulation of cellular growth. Cell Tissue Res 368, 105–114 (2017). https://doi.org/10.1007/s00441-016-2522-7
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DOI: https://doi.org/10.1007/s00441-016-2522-7