Nuclear localization signal sequence is required for VACM-1/CUL5-dependent regulation of cellular growth.
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 inS730AVACM-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 inS730AVACM-1/CUL5 sequences, is critical for their control of cell proliferation.
Departments of Chemistry and Biology, Paul A. Schaap Science Center, Hope College, Holland, MI, 49422-9000, USA.
Angelica N
AN
Willis
Departments of Chemistry and Biology, Paul A. Schaap Science Center, Hope College, Holland, MI, 49422-9000, USA.
Shirley E Bradley
SE
Dean
Departments of Chemistry and Biology, Paul A. Schaap Science Center, Hope College, Holland, MI, 49422-9000, USA.
Joe A
JA
Habbouche
Departments of Chemistry and Biology, Paul A. Schaap Science Center, Hope College, Holland, MI, 49422-9000, USA.
Brian T
BT
Kempers
Departments of Chemistry and Biology, Paul A. Schaap Science Center, Hope College, Holland, MI, 49422-9000, USA.
Megan L
ML
Ludwig
Departments of Chemistry and Biology, Paul A. Schaap Science Center, Hope College, Holland, MI, 49422-9000, USA.
Aaron D
AD
Sayfie
Departments of Chemistry and Biology, Paul A. Schaap Science Center, Hope College, Holland, MI, 49422-9000, USA.
Steven P
SP
Lewis
Departments of Chemistry and Biology, Paul A. Schaap Science Center, Hope College, Holland, MI, 49422-9000, USA.
Stephanie
S
Harrier
Departments of Chemistry and Biology, Paul A. Schaap Science Center, Hope College, Holland, MI, 49422-9000, USA.
Zachary J
ZJ
DeBruine
Departments of Chemistry and Biology, Paul A. Schaap Science Center, Hope College, Holland, MI, 49422-9000, USA.
Richard
R
Garrett
Departments of Chemistry and Biology, Paul A. Schaap Science Center, Hope College, Holland, MI, 49422-9000, USA. hledin@hope.edu.
Maria A
MA
Burnatowska-Hledin
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D049109:Cell Proliferation; D002522:Chlorocebus aethiops; D044842:Cullin Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport; D020539:Sequence Analysis, Protein; D013329:Structure-Activity Relationship; D014162:Transfection
D044842:Cullin Proteins; D019913:Nuclear Localization Signals
Cellular proliferation; NEDD8; Nuclear localization signal; VACM-1/CUL5