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The subunit composition and intracellular location of the two forms of cAMP-dependent protein kinase of Paramecium cilia were determined using antibodies against the 40-kDa catalytic (C) and 44-kDa regulatory (R44) subunits of the 70-kDa cAMP-dependent protein kinase purified from deciliated cell bodies. Both C and R44 were present in soluble and particulate fractions of cilia and deciliated cells. Crude cilia and a soluble ciliary extract contained a 48-kDa protein (R48) weakly recognized by one of several monoclonal antibodies against R44, but not recognized by an anti-R44 polyclonal serum. Gel-filtration chromatography of a soluble ciliary extract resolved a 220-kDa form containing C and R48 and a 70-kDa form containing C and R44. In the large enzyme, R48 was the only protein to be autophosphorylated under conditions that allow autophosphorylation of R44. The subunits of the large enzyme subsequently were purified to homogeneity by cAMP-agarose chromatography. Both C and R48 were retained by the column and eluted with I M NaCl; no other proteins were purified in this step. These results confirm that the ciliary cAMP-dependent protein kinases have indistinguishable C subunits, but different R subunits. The small ciliary enzyme, like the cell-body enzyme, contains R44, whereas R48 is the R subunit of the large enzyme.

The 44-kDa regulatory subunit (R44) of one form of cAMP-dependent protein kinase of Paramecium was purified, and two partial internal amino acid sequences from it were used to clone the corresponding cDNA. This R44 cDNA clone was 1022-bp long, including 978 bp of coding sequence and 7 bp and 37 bp of 5' and 3' untranslated sequences, respectively. A 1.1-kb mRNA was labeled on a Northern blot. The deduced R44 amino acid sequence had 31%-38% positional identity to the sequences of other cloned cAMP-dependent protein kinase regulatory subunits. R44 sequence showed equal sequence similarity to mammalian types I and II regulatory subunits. The N-terminal sequence encoding the regulatory subunit dimerization domain found in most regulatory subunits is not present in the R44 clone, confirming the lack of regulatory subunit dimer formation previously reported for the Paramecium cAMP-dependent protein kinase. The putative autophosphorylation site of R44 contains the amino acid sequence TRTS, distinct from the consensus sequence RRXS, where X is any residue, found in other autophosphorylated cAMP-dependent protein kinase regulatory subunits and many cAMP-dependent protein kinase substrates.