Compartmentalization of the cyclic nucleotide -dependent protein kinases and their substrates.

Abstract

Many hormones elevate cellular concentrations of the cyclic nucleotides, cAMP and cGMP. The cAMP- and cGMP-dependent protein kinases (cAKs and cGKs) comprise a family of kinases that are activated by cyclic nucleotides and regulate many functions, including transcription and proliferation. This dissertation examines the role of cellular compartmentalization in determining the function of the cAKs and cGKs. Upon cAMP elevation, cAMP binds to the regulatory (R) subunits of the inactive, cytoplasmic cAK holoenzyme causing the release of active catalytic (C) subunits. The C subunit, because of its small size, can diffuse into the nucleus and phosphorylate specific transcription factors. Protein kinase inhibitor (PKI) proteins provide another mechanism for regulating cAK activity. Attempts to understand the cellular roles of PKIs have been hampered by the heterogeneity of PKI activity. To resolve this problem, we sought to identify novel PKI isoforms. The identification of PKIgamma, a widely expressed PKI isoform suggests that PKI activity may be critical to cell function. In addition to inhibiting C subunit, PKIgamma also serves to localize C subunit in the cell by exporting C subunit from the nucleus. Unlike the cAKs, the regulatory and catalytic domains of the cGKs are contained within the same polypeptide chain. Analyses of cGKs subcellular localizations indicate that cGKs are restricted to the cytoplasm regardless of their activation state. Although both cAKs and cGKs phosphorylate the cytoplasmic substrate VASP to a similar extent, cyclic nucleotide regulation of transcription was higher in cAK-transfected cells. The inability of cGKs to translocate to the nucleus renders them relatively weak activators of transcription. In different cell types, cAMP treatment either blocks or promotes cell cycle progression. LKB1, a tumor suppressor, possesses potential cAK phosphorylation sites. Analyses of LKB1 showed that it is phosphorylated by cAK and prenylated <italic> in vivo</italic>. LKB1's prenylation motif is capable of targeting proteins to membranes. Taken together, the results of this thesis work suggest that the regulatory mechanisms controlling cyclic nucleotide dependent protein kinase function are more diverse than known previously and that additional mechanisms exist for cyclic nucleotide control of cell cycle regulation.