Effect of retinyl acetate on cAMP‐dependent protein kinase in transformed mouse 10t1/2 cells

Abstract

The effect of retinyl acetate (RAC) on the activity of cAMP-dependent protein kinase (PKA) was studied in mouse 10T1/2 cells. The studies revealed that normal 10T1/2 cells had about 13-fold more PKA activity than did methylcholanthrene-transformed cells (MCA cells). The addition of RAC to MCA cells increased the activity of PKA about 3-fold as measured by the in vitro phosphorylation of a specific site in H1 histone (site A) or Kemptide. The increased PKA activity coincided with a reduction in the rate of cell replication of MCA cells, about 24 hr after exposure to the retinoid. Addition of forskolin to RAC-treated MCA cells resulted in a further reduction in the rate of cell replication, and this suggested that the enhanced PKA activity was also capable of action in vivo. To test this notion, MCA cells were grown with and without RAC, and the phosphorylation of the H1 histone at site A, a site known to be phosphorylated by PKA in cells treated with hormones or other agonists which activate PKA, was studied in vivo. RAC, by itself, was capable of causing an increase in the phosphorylation of the H1 histone at site A, demonstrating that the retinoid-mediated increase in PKA activity was sufficient to cause the enhanced phosphorylation of a known substrate.

Retinola induced changes in cAMP‐dependent protein kinase activity detected by a new minigel assay

“…As shown in table 1, the addition of RAC lowered the growth rate and saturation density of the cells over the 48 h period. The total PKA activity, measured in solution as described previously [6] was about 75% higher in the RACtreated cultures (data not shown, but compare to about a 3-fold increase in our previous work [a].) As shown in fig.2A, there is no difference in the PKA isoforms after 18 h. In fig.2 we have included two lanes wherein the protein kinase extracts have been preincubated with CAMP for 30 min prior to electrophoresis.…”

“…Based on our previous observations, it takes at least 24 h for the retinoid-dependent increase in PKA activity to become detectable in MCA cells [6]. As shown in table 1, the addition of RAC lowered the growth rate and saturation density of the cells over the 48 h period.…”

“…These commercial PKA preparations can readily be seen to be mixtures of the two MCA cells were cultured at a 9-fold dilution, and allowed to establish in BME for 24 h. At this time (0 h) the medium in the experimental flasks was changed to BME containing 0.5 pg/ml RAC. The DNA content, and specific activity, was determined at 18, 36 and 48 h after time 0 [6] isoform classes. The bovine heart preparation is predominantly type I.…”

“…Recent experiments from this laboratory have shown that retinyl acetate can increase the PKA activity of transformed mouse lOT1/2 cells (MCA cells) about 3-fold after about 36-48 h in culture [6]. PKA, however, is comprised of two major isoforms, type I and type II, which, in turn, have further subforms which may differ in the structure either of their regulatory 171 or catalytic [S] subunits.…”

“…MCA cells were grown as monolayers in 75 cm' flasks using BME (Basal Medium, Eagle) supplemented with 5% heat-inactivated fetal bovine serum (Gibco) as previously described [6]. The standard protocol consisted of subculturing confluent monolayers at a 1: 9 dilution.…”