INVOLVEMENT OF THE CA2-DEPENDENT PHOSPHORYLATION OF A 20-KDA PROTEIN IN THE PROLIFERATIVE EFFECT OF HIGH-DENSITY-LIPOPROTEINS (SUBCLASS-3) ON THE ADENOCARCINOMA CELL-LINE A549()

Previous studies from our laboratory demonstrated that high-density li poproteins (subclass 3; HDL3) bind to sites specific for apolipoprotei n AI on the human adenocarcinoma cell line A549 and that HDL3 binding promotes a mitogenic effect [Favre, Tazi, Le Gaillard, Bennis, Hachem and Soula (1993) J. Lipid Res. 34, 1093-1106]. In the present study, w e have examined the cell proteins that showed modified phosphorylation after binding of HDL3 to specific sites, and the roles of Ca2+ and pr otein kinase C. Native HDL3 (but not tetranitromethane-modified HDL3) and Ca2+ ionophore A23187 strongly enhanced the phosphorylation of a 2 0 kDa protein (x 3.6) and, to a lower extent, the phosphorylation of 2 4 and 28 kDa proteins (x 2.2 and 2.6 respectively). The two effecters were equally able to stimulate cell growth. Down-regulation of protein kinase C by a 24 h incubation of cells with phorbol myristate acetate prevented the effects of HDL3 on the phosphorylation of 24 and 28 kDa proteins. However, the extent of phosphorylation of the 20 kDa protei n was not affected. In contrast, activation of protein kinase C by a s hort incubation with phorbol myristate acetate resulted in inhibition of proliferation and an increase in 24 and 28 kDa (but not 20 kDa) pro tein phosphorylation. These results suggest that HDL3 putative recepto rs exert their proliferative effect on A549 cells through activation o f a Ca2+-dependent protein kinase. This kinase activity is not modulat ed by phorbol ester and thus may be a calmodulin kinase or an isoenzym e of protein kinase C that is independent of phorbol ester. It allows a subsequent 20 kDa protein to be phosphorylated.