THE WAYS IN WHICH HORMONES CHANGE CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE-DEPENDENT PROTEIN-KINASE SUBUNITS, AND HOW SUCH CHANGES AFFECT CELL BEHAVIOR

HORMONES and cytokines regulate many cellular functions by activating the ubiquitous cAMP-dependent protein kinase (A kinase) system. Newly synthesized cAMP molecules bind to regulatory (R) subunits in A kinase holoenzymes, causing them to release their catalytic (C) subunits. These free C subunits then phosphorylate proteins until the cAMP level falls, whereupon the R subunits regain their affinity for free C subunits, and thus form inactive holoenzymes again. However if cAMP levels remain persistently elevated, many cells change their A kinase system. Some cells alter the rate of degradation of subunits, and some cells change the level or stability of the messages encoding subunits. Cellular behavior often changes if cAMP levels remain elevated: many cells differentiate, some cells proliferate, and some cells die, depending on the stage of the cell cycle. The two forms of A kinase holoenzyme (type I and type II) contain identical C subunits, but contain either an RI dimer or an RII dimer. In some tissues, type II holoenzyme is compartmentalized to subcellular organelles via specific anchoring proteins, whereas type I holoenzyme is generally cytosolic. Free RI subunits tum over more rapidly than free RII subunits in most cells, but all free subunits are degraded more rapidly than when they are associated together in holoenzymes. Free C subunits can phosphorylate a broad spectrum of proteins in both the cytoplasm and nucleus, depending on the type of cell, its state of differentiation, and the hormonal milieux. If free C subunit is microinjected into the cytoplasm of some intact cells, it migrates to the nucleus, whereas if free R subunit is microinjected, it remains in the cytoplasm. If both subunits are coinjected, R subunit blocks the nuclear migration of the C subunit. A major nuclear target for free C subunits is the CREB family of nuclear proteins, which bind to cAMP response elements (CREs) in the promoter regions of cAMP-responsive genes. Phosphorylation of CREB proteins alters their ability to form dimers and to interact with CREs. Many CREB proteins can be phosphorylated by other kinases as well, indicating this is one means by which cells coordinate cAMP- and non-cAMP-mediated gene responses. However, interactions between CREB and a number of other nuclear proteins with which they can dimerize, especially proteins whose levels are rapidly altered in response to hormones, provide an even higher degree of complexity of gene regulation than is possible from various kinases phosphorylating the different sites in CREB proteins. Genes encoding different components of the A kinase system have been transfected into a variety of cells. One common observation obtained by transfecting cells with C subunit gene is that it causes more type I holoenzyme to form, yet the level of the messenger RNA (mRNA) encoding RII does not change substantially. When the transfected cells make a higher-than-normal level of C subunits, free RI subunits that normally would be degraded instead bind to the extra C subunits that are being synthesized and form the additional type I holoenzyme. However, if so much C subunit is expressed that it outstrips the cell's output of free RI subunits, then the unbound C subunit causes cAMP-responsive genes to respond as if cAMP levels had risen. When DNA encoding RII subunit genes is overexpressed, it causes more type II holoenzyme to form, whereas it causes RI subunit levels to fall, indicating that free RI subunit is being degraded more rapidly, probably because the C subunit that normally would be available to bind to (and protect) this RI is used up in forming the type II holoenzyme. In contrast, overexpressing the RI subunit gene had little effect on the level of any subunit, again indicating that RII has a greater affinity for C subunits than RI does in such cells. Thus, when additional RI is expressed, it is simply degraded since it cannot compete for C subunits bound to RII. Transfection studies with mutant genes have provided a more detailed understanding of the specific sites involved in subunit interactions and also of the mechanisms that cells use to regulate the levels of free A kinase subunits. Some tissue-specific A kinase responses have been uncovered which illustrate the different ways cells utilize components of the A kinase system, and suggest some important areas for further investigation.