Characterization of cyclic nucleotide-dependent protein kinase isoforms and their inhibitors.

Abstract

The cyclic nucleotide-dependent signal transduction pathways regulate a large and diverse array of cellular processes, due mainly to the broad substrate specificities of the cAMP- and cGMP-dependent protein kinases (cAK and cGK). However, the pleiotropic nature of cAK and cGK necessitates the existence of mechanisms to specify individual cellular responses to hormone and neurotransmitter binding in different cell types. One such mechanism may involve the differential expression of biochemically distinct isoforms of cAK, cGK and the protein kinase inhibitor (PKI). Studies described in Chapter II demonstrate that the two major isoforms of the catalytic (C) subunit of cAK, C$\alpha$ and C$\beta1,$ participate in discrete interactions with substrates and inhibitors. In addition, type II cAK holoenzymes containing C$\beta1$ were activated at significantly lower cAMP concentrations in vitro and in vivo than were those containing C$\alpha.$ These results provide evidence that type II holoenzymes formed with C$\beta1$ have distinct regulatory and catalytic properties, which may allow cAK isoforms to regulate separate functions in certain cells. Further control of C subunit activity is provided by the endogenous cAK inhibitor isoforms, PKI$\alpha$ and PKI$\beta1.$ Data from Chapter III reveal that PKI$\beta1$ has a 32-fold higher $K\sb{i}$ value than does PKI$\alpha.$ Subsequent analyses of chimeric and site-directed PKI mutants implicate the presence of a tyrosine at position 7 in PKI$\alpha$ as a major contributor to its enhanced inhibitory potency. These experiments suggest that PKI$\alpha$ and PKI$\beta1$ are capable of differentially regulating cAK activity in vivo. Lastly, Chapter IV provides a detailed biophysical and biochemical characterization of a novel cGK isoform (cGK II) found in intestine and brain. The data show that cGK II is dimeric and possesses cyclic nucleotide and substrate specificities distinct from the type I cGK isoforms. These differences were then used to devise procedures to differentiate cGK II and cGK I activities from partially purified tissue extracts. Together, these studies raise the possibility that cellular responses to cAMP- and cGMP-dependent signaling can be specified in part by the differential expression of cAK, PKI and cGK isoforms.