Types I alpha and I beta isozymes of cGMP-dependent protein kinase: alternative mRNA splicing may produce different inhibitory domains.
We recently described a novel isozyme of cGMP-dependent protein kinase (type I beta). It has a structure and peptide substrate specificity which is similar to that of type I alpha, but it has a different cGMP binding behavior, and autophosphorylation occurs almost entirely in serine instead of in both serine and threonine residues (Wolfe, L., Corbin, J.D., and Francis, S.H. (1989) J. Biol. Chem. 264, 7734-7741). An amino-terminal sequence of 31 amino acids derived from three proteolytic fragments of type I beta had 45% homology with a sequence beginning at type I alpha-47. However, sequences of three CNBr peptides of type I beta were identical to sequences of type I alpha beginning at type I alpha-202, -213, and -576 of 11, 27, and 30 residues. These sequences include portions of the catalytic domain and at least one cGMP-binding domain (site 1). Thus, types I alpha and I beta may be produced by alternative splicing of two unique mRNA segments to generate different amino acid sequences in the protein in a region that is amino-terminal to type I alpha-202. This segment in type I beta corresponds to the region in type I alpha that includes the major autophosphorylation site (Thr-58) which is within the domain that is proposed to inhibit catalytic activity. This region presumably interacts with the cGMP-binding site(s) to account for the differences in cGMP-binding behavior between types I alpha and I beta. Even though the sequence of type I beta in the variable region lacks the residue corresponding to Thr-58, it includes a consensus phosphorylation site (KRQAISA) beginning at type I alpha-59, which is absent in type I alpha. The results imply flexibility in the design of the autophosphorylation site and, hence, of the inhibitory domain.
Francis
S H
SH
Department of Molecular Physiology and Biophysics, Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, TN 37232-0295.
Woodford
T A
TA
Wolfe
L
L
Corbin
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000201:enzymology; D002417:Cattle; D007527:Isoenzymes / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D012326:RNA Splicing
D007527:Isoenzymes; D011494:Protein Kinases