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) form homodimers (subunit M(r) approximately 76,000), presumably through conserved, amino-terminal leucine zipper motifs. Type II cGMP-dependent protein kinase (cGK II) has been reported to be monomeric (M(r) approximately 86,000), but recent cloning and sequencing of mouse brain cGK II cDNA revealed a leucine zipper motif near its amino terminus. In the present study, recombinant mouse brain cGK II was expressed, purified, and characterized. Sucrose gradient centrifugation and gel filtration chromatography 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 dimers. Native cGK I alpha possessed significantly lower K alpha values for cGMP (8-fold) and beta-phenyl-1,N2-etheno-cGMP (300-fold) than did recombinant cGK II. Conversely, the Sp- and Rp-isomers of 8-(4-chloro-phenylthio)-guanosine-3',5'-cyclic monophosphorothioate demonstrated selectivity toward cGK II in assays of kinase activation or inhibition, respectively. A peptide substrate derived from histone f2B had a 20-fold greater Vmax/Km ratio for cGK I alpha than for cGK II, whereas a peptide based upon a cAMP response element binding protein phosphorylation site exhibited a greater Vmax/Km ratio for cGK II. Finally, gel filtration of extracts of mouse intestine partially resolved two cGK activities, one of which had properties similar to those demonstrated by recombinant cGK II. The combined results show that both cGK I and cGK II form homodimers but possess distinct cyclic nucleotide and substrate specificities.
Gamm
D M
DM
Neuroscience Program, University of Michigan, Ann Arbor 48109, USA.
Francis
S H
SH
Angelotti
T P
TP
Corbin
J D
JD
Uhler
M D
MD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D003001:Cloning, Molecular; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009712:Nucleotides, Cyclic / Q000494:pharmacology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D009712:Nucleotides, Cyclic; D011994:Recombinant Proteins; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP