Biochemical characterization of extracellular cAMP-dependent protein kinase as a tumor marker.

In a recent report (Cho et al., Proc. Natl. Acad. Sci. USA 97, 835-840, 2000), we showed that cancer cells of various cell types secrete cAMP-dependent protein kinase (PKA) into the conditioned medium and that in the serum of cancer patients this extracellular PKA (ECPKA) is upregulated 10-fold as compared with normal serum. Here, we characterized the enzymatic properties of ECPKA that is present in the conditioned medium of PC3M prostate cancer cells and in the serum of cancer patients, and we compared ECPKA with PKA found in the cell extracts of PC3M cells. ECPKA present in the conditioned medium and human serum was not activated by cAMP addition, but intracellular PKA activity was totally dependent on the addition of cAMP. This indicates that the ECPKA is present in active, free C subunit form, whereas intracellular PKA is present in inactive holoenzyme form. ECPKA activity increased in a substrate concentration- and time-dependent manner, as did intracellular PKA. Both ECPKA and intracellular PKA activities were specifically inhibited by the PKA inhibitor protein, PKI. However, ECPKA activity was more temperature-sensitive than intracellular PKA; after two cycles of freezing/thawing, only 20% of initial ECPKA activity was detected compared with over 40% of intracellular PKA activity. Western blot analysis revealed the presence of a 40 kDa C(alpha) subunit of PKA in both conditioned medium and in the serum of cancer patients. These results suggest that ECPKA, out of the context of cAMP regulation, may function as a growth factor promoting cell growth and transformation; thus, it may serve as a tumor biomarker.

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