8-Cl-cAMP, a site-selective cAMP analog, induces growth inhibition in a variety of cell types of human
cancer cell lines. This inhibitory effect of
8-Cl-cAMP was related to its ability to differentially regulate type I versus type II
cAMP-dependent protein kinase. In the present study we demonstrated a unique mechanism of action of
8-Cl-cAMP in the regulation of these
kinase isozymes in HL-60 human promyelocytic
leukemia cells. High-performance liquid chromatography (HPLC) resolved various
isoforms of
protein kinase present in HL-60 cells. In control cells, type I
protein kinase (PKI) comprised more than 90% and type II
protein kinase (PKII) less than 10% of the total cAMP-stimulated
kinase activity. Treatment with
8-Cl-cAMP (5 microM, 72 h) decreased PKI to a level below 30% of that in untreated control cells and markedly increased PKII composed of three peaks. Photoaffinity labeling/SDS-
polyacrylamide gel electrophoresis of column fractions identified the molecular species of regulatory (R) subunits present in
protein kinases. Control cells contained high levels of the 48-kDa
protein (RI) that composed PKI and low levels of the 50-kDa RII associated with PKII.
8-Cl-cAMP treatment brought about a decrease in the 48-kDa RI along with an increased formation of the truncated 34-kDa RI associated with PKI and an increase in the 50-54-kDa species of RII associated with PKII. A similar
protein kinase profile as that shown by
8-Cl-cAMP treatment was observed in cells infected with the human RII beta retroviral vector: the 48-kDa RI of PKI decreased and the 52- and 54-kDa RII associated with PKII increased as compared with uninfected control cells. However, unlike
8-Cl-cAMP treatment, RII beta retroviral vector
infection brought about no increase in the 34-kDa-truncated RI but exhibited an increase in the free 48-kDa RI subunit. As the 48-kDa RI and the 50-kDa RII were present in control cells, the enhanced expression of the 52- and 54-kDa RII
proteins was due to overexpression of the RII beta gene. We identified the 48-kDa RI as RI alpha, the 50-kDa RII as RII alpha, the 52-kDa RII as RII beta, and the 54-kDa RII as the phosphorylated form of either the RII alpha or RII beta subunit. In vivo labeling experiments using [3H]8-Cl-cAMP demonstrated that
8-Cl-cAMP enters cells and binds to both PKI and PKII.(ABSTRACT TRUNCATED AT 400 WORDS)