Regulation of Cell Growth by VACM‐1/cul5 is Dependent on its Phosphorlyation by PKA and PKC

Abstract

Expression of VACM‐1/cul‐5 gene in endothelial and in cancer cell lines in vitro inhibits cellular proliferation and decreases phosphorylation of mitogen‐activated protein kinase (MAPK). Structure‐function analysis of VACM‐1 protein sequence identified consensus sites specific for phosphorylation by protein kinases PKA and PKC. Mutations at the PKA specific site in VACM‐1 (S730AVACM‐1) sequence resulted in increased cellular growth of rat endothelial cells, RAMEC. The aim of our study was to examine if PKC activity also regulates VACM‐1 dependent cell growth. To induce PKC activity, cells were treated with Phorbol 12‐myristate 13‐acetate (PMA) for 16 hours and growth was monitored. PKC specific phosphorylation of VACM‐1 was examined using anti‐PKC substrate specific antibody. Our results show that cells transfected with S730AVACM‐1 cDNA and treated with PMA for 16 hours grew significantly faster than control cells treated with PMA. Further, Western blot analysis of cell lysates from both groups probed with anti‐PKC substrate specific antibody revealed a higher level of VACM‐1 phosphorylation in S730AVACM‐1 cDNA transfected cells when compared to controls. These results suggest that the antiproliferative effect of VACM‐1/cul5 is dependent on its posttranslational phosphorylation by PKA and PKC.