Journal of Biological Chemistry
Volume 270, Issue 45, 10 November 1995, Pages 27380-27388
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Enzymology
The Type II Isoform of cGMP-dependent Protein Kinase Is Dimeric and Possesses Regulatory and Catalytic Properties Distinct from the Type I Isoforms (*)

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The type I cGMP-dependent protein kinases (cGK Iα and Iβ) form homodimers (subunit Mr ∼ 76,000), presumably through conserved, amino-terminal leucine zipper motifs. Type II cGMP-dependent protein kinase (cGK II) has been reported to be monomeric (Mr ∼ 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 Mr values for holoenzymes of cGK Iα (168,000) and cGK II (152,500), which suggest that both are dimers. Native cGK Iα possessed significantly lower Ka values for cGMP (8-fold) and β-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 Iα 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.

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*

This work was supported by National Institutes of Health Grants GM 38788 and MH 42652 (to M. D. U.) and DK 40029 (to J. D. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.