THE TYPE-II ISOFORM OF CGMP-DEPENDENT PROTEIN-KINASE IS DIMERIC AND POSSESSES REGULATORY AND CATALYTIC PROPERTIES DISTINCT FROM THE TYPE-I ISOFORMS

The type I cGMP-dependent protein kinases (cGK I alpha and I beta) for m homodimers (subunit M(r) similar to 76,000), presumably through cons erved, amino-terminal leucine zipper motifs, Type II cGMP-dependent pr otein kinase (cGK II) has been reported to be monomeric (M(r) similar to 86,000), but recent cloning and sequencing of mouse brain cGK II cD NA revealed a leucine zipper motif near its amino terminus, In the pre sent study, recombinant mouse brain cGK II was expressed, purified, an d characterized, Sucrose gradient centrifugation and gel filtration ch romatography were used to determine M(r) values for holoenzymes of cGK I alpha (168,000) and cGK II (152,500), which suggest that both are d imers. Native cGK I alpha possessed significantly lower K-a values for cGMP (8-fold) and beta-phenyl-1,N-2-etheno-cGMP (300-fold) than did r ecombinant cGK II, Conversely, the Sp- and Rp-isomers of 8-(4-chloro-p henylthio)-guanosine-3',5'-cyclic monophosphorothioate demonstrated se lectivity toward cGK II in assays of kinase activation or inhibition, respectively, A peptide substrate derived from histone f(2B) had a 20- fold:greater V-max/K-m ratio for cGK I alpha than for cGK II, whereas a peptide based upon a cAMP response element binding protein phosphory lation site exhibited a greater V-max/K-m ratio for cGK II, Finally, g el filtration of extracts of mouse intestine partially resolved two cG K activities, one of which had properties similar to those demonstrate d by recombinant cGK II, The combined results show that both cGK I and cGK II form homodimers but possess distinct cyclic nucleotide and sub strate specificities.