Phosphorylation of VACM-1/Cul5 by protein kinase A regulates its neddylation and antiproliferative effect.
Expression of the VACM-1/cul5 gene in endothelial and in cancer cell lines in vitro inhibits cellular proliferation and decreases phosphorylation of MAPK. Structure-function analysis of the VACM-1 protein sequence identified consensus sites specific for phosphorylation by protein kinases A and C (PKA and PKC) and a Nedd8 protein modification site. Mutations at the PKA-specific site in VACM-1/Cul5 ((S730A)VACM-1) sequence resulted in increased cellular growth and the appearance of a Nedd8-modified VACM-1/Cul5. The aim of this study was to examine if PKA-dependent phosphorylation of VACM-1/Cul5 controls its neddylation status, phosphorylation by PKC, and ultimately growth. Our results indicate that in vitro transfection of rat adrenal medullary endothelial cells with anti-VACM-1-specific small interfering RNA oligonucleotides decreases endogenous VACM-1 protein concentration and increases cell growth. Western blot analysis of cell lysates immunoprecipitated with an antibody directed against a PKA-specific phosphorylation site and probed with anti-VACM-1-specific antibody showed that PKA-dependent phosphorylation of VACM-1 protein was decreased in cells transfected with (S730A)VACM-1 cDNA when compared with the cytomegalovirus-transfected cells. This change was associated with increased modification of VACM-1 protein by Nedd8. Induction of PKA activity with forskolin reduced modification of VACM-1 protein by Nedd8. Finally, rat adrenal medullary endothelial cells transfected with (S730A)VACM-1/cul5 cDNA and treated with phorbol 12-myristate 13-acetate (10 and 100 nm) to induce PKC activity grew significantly faster than the control cells. These results suggest that the antiproliferative effect of VACM-1/Cul5 is dependent on its posttranslational modifications and will help in the design of new anticancer therapeutics that target the Nedd8 pathway.
Bradley
Shirley E
SE
Department of Biology, Hope College, Holland, Michigan 49422-9000, USA.
Johnson
Alyssa E
AE
Le
Isabelle P
IP
Oosterhouse
Elizabeth
E
Hledin
Michael P
MP
Marquez
Gabriel A
GA
Burnatowska-Hledin
Maria
M
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D047468:Immunoprecipitation; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000235:genetics / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics / Q000502:physiology; D051381:Rats; D017483:Receptors, Vasopressin / Q000235:genetics / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014452:Ubiquitins / Q000378:metabolism
C094547:Cul5 protein, rat; D044842:Cullin Proteins; D034741:RNA, Small Interfering; D017483:Receptors, Vasopressin; D014452:Ubiquitins; D017868:Cyclic AMP-Dependent Protein Kinases; D013755:Tetradecanoylphorbol Acetate