Abstract
We have previously shown that the protein kinase inhibitor beta (PKIbeta) form of the cAMP-dependent protein kinase inhibitor exists in multiple isoforms, some of which are specific inhibitors of the cAMP-dependent protein kinase, whereas others also inhibit the cGMP-dependent enzyme [Kumar, Van Patten and Walsh (1997), J. Biol. Chem. 272, 20011-20020]. We have now demonstrated that the switch from a cAMP-dependent protein kinase (PKA)-specific inhibitor to one with dual specificity arises as a consequence of alternate gene splicing. We have confirmed using bacterially produced pure protein that a single inhibitor species has dual specificity for both PKA and cGMP-dependent protein kinase (PKG), inhibiting each with very high and closely similar inhibitory potencies. The gene splicing converted a protein with 70 amino acids into one of 109 amino acids, and did not change the inhibitory potency to PKA, but changed it from a protein that had no detectable PKG inhibitory activity to one that now inhibited PKG in the nanomolar range.
Full Text
The Full Text of this article is available as a PDF (174.5 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Glass D. B., Cheng H. C., Kemp B. E., Walsh D. A. Differential and common recognition of the catalytic sites of the cGMP-dependent and cAMP-dependent protein kinases by inhibitory peptides derived from the heat-stable inhibitor protein. J Biol Chem. 1986 Sep 15;261(26):12166–12171. [PubMed] [Google Scholar]
- Glass D. B., Cheng H. C., Mende-Mueller L., Reed J., Walsh D. A. Primary structural determinants essential for potent inhibition of cAMP-dependent protein kinase by inhibitory peptides corresponding to the active portion of the heat-stable inhibitor protein. J Biol Chem. 1989 May 25;264(15):8802–8810. [PubMed] [Google Scholar]
- Glass D. B., Feller M. J., Levin L. R., Walsh D. A. Structural basis for the low affinities of yeast cAMP-dependent and mammalian cGMP-dependent protein kinases for protein kinase inhibitor peptides. Biochemistry. 1992 Feb 18;31(6):1728–1734. doi: 10.1021/bi00121a021. [DOI] [PubMed] [Google Scholar]
- Glass D. B., Krebs E. G. Protein phosphorylation catalyzed by cyclic AMP-dependent and cyclic GMP-dependent protein kinases. Annu Rev Pharmacol Toxicol. 1980;20:363–388. doi: 10.1146/annurev.pa.20.040180.002051. [DOI] [PubMed] [Google Scholar]
- Hanks S. K., Quinn A. M. Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members. Methods Enzymol. 1991;200:38–62. doi: 10.1016/0076-6879(91)00126-h. [DOI] [PubMed] [Google Scholar]
- Kemp B. E., Pearson R. B. Intrasteric regulation of protein kinases and phosphatases. Biochim Biophys Acta. 1991 Aug 13;1094(1):67–76. doi: 10.1016/0167-4889(91)90027-u. [DOI] [PubMed] [Google Scholar]
- Knighton D. R., Zheng J. H., Ten Eyck L. F., Xuong N. H., Taylor S. S., Sowadski J. M. Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):414–420. doi: 10.1126/science.1862343. [DOI] [PubMed] [Google Scholar]
- Kumar P., Van Patten S. M., Walsh D. A. Multiplicity of the beta form of the cAMP-dependent protein kinase inhibitor protein generated by post-translational modification and alternate translational initiation. J Biol Chem. 1997 Aug 8;272(32):20011–20020. doi: 10.1074/jbc.272.32.20011. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Reed J., De Ropp J. S., Trewhella J., Glass D. B., Liddle W. K., Bradbury E. M., Kinzel V., Walsh D. A. Conformational analysis of PKI(5-22)amide, the active inhibitory fragment of the inhibitor protein of the cyclic AMP-dependent protein kinase. Biochem J. 1989 Dec 1;264(2):371–380. doi: 10.1042/bj2640371. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Scarpetta M. A., Uhler M. D. Evidence for two additional isoforms of the endogenous protein kinase inhibitor of cAMP-dependent protein kinase in mouse. J Biol Chem. 1993 May 25;268(15):10927–10931. [PubMed] [Google Scholar]
- Slice L. W., Taylor S. S. Expression of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli. J Biol Chem. 1989 Dec 15;264(35):20940–20946. [PubMed] [Google Scholar]
- Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
- Szmigielski A., Guidotti A., Costa E. Endogenous protein kinase inhibitors. Purification, characterization, and distribution in different tissues. J Biol Chem. 1977 Jun 10;252(11):3848–3853. [PubMed] [Google Scholar]
- Van Patten S. M., Howard P., Walsh D. A., Maurer R. A. The alpha- and beta-isoforms of the inhibitor protein of the 3',5'-cyclic adenosine monophosphate-dependent protein kinase: characteristics and tissue- and developmental-specific expression. Mol Endocrinol. 1992 Dec;6(12):2114–2122. doi: 10.1210/mend.6.12.1491692. [DOI] [PubMed] [Google Scholar]
- Van Patten S. M., Ng D. C., Th'ng J. P., Angelos K. L., Smith A. J., Walsh D. A. Molecular cloning of a rat testis form of the inhibitor protein of cAMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5383–5387. doi: 10.1073/pnas.88.12.5383. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Whitehouse S., Walsh D. A. Inhibitor protein of the cAMP-dependent protein kinase: characteristics and purification. Methods Enzymol. 1983;99:80–93. doi: 10.1016/0076-6879(83)99044-4. [DOI] [PubMed] [Google Scholar]