Regulation of protein kinase a activation and prostaglandin E2‐stimulated migration of lewis lung carcinoma clones

Lewis lung carcinoma (LLC) clones were used in in vitro models for dissemination to identify mechanisms regulating the stimulation of metastatic LLC-LN7 migration by prostaglandin E2 (PGE2) or forskolin plus 3-isobutyl-I-methylxanthine (IBMX), and the lack of responsiveness to generated cAMP in non-metastatic LLC-C8 cells. The regulatory subunits of protein kinase A (PKA) from LLC-LN7 cells bound more 8-N3-32P-cAMP, even though production of regulatory subunits was equal to that in LLC-C8 cells. Protein kinase C (PKC) differentially regulated PKA activation in the LLC variants. PKC activation inhibited PGE2-stimulated migration by LLC-LN7 cells. Inhibition of PKC with staurosporine stimulated LLC-LN7 cell migration to a level comparable with that induced by PGE2. However, PGE2 did not further stimulate the migration of staurosporine-treated cells. The PGE2 or staurosporine stimulation of LLC-LN7 cell migration was dependent on PKA activation. The effects that modulation of PKA and PKC had on LLC-LN7 cell migration paralleled the effects on endogenous protein phosphorylation. LLC-LN7 cell autophosphorylation was stimulated to a similar degree by PGE2, forskolin plus IMBX, staurosporine, or the combination of staurosporine and forskolin plus IBMX. In contrast, neither migration nor autophosphorylation was stimulated in non-metastatic LLC-C8 cells by cAMP elevation or by PKC inhibition. Autophosphorylation, although not migration, of LLC-C8 cells was stimulated by forskolin plus IBMX when PKC activity was inhibited. These results suggest that the increased PKA response of metastatic LLC-LN7 cells is contributed by an increased binding of cAMP by the PKA regulatory subunits and a reduced level of regulation by PKC.