Since the discovery of cyclic AMP (cAMP) as an essential second messenger by Sutherland and colleagues, the actions of its receptor protein, the cAMP-dependent protein kinase (PKA) have been extensively studied. An important early finding was that cyclic AMP inhibited the growth of many cell types, including those transformed by oncogenes such as Ras. Despite this well known observation, this phenomena remains poorly understood to this day. To further complicate analysis, cAMP can be mitogenic in some cells, the mechanism of this proliferative action is unknown. Recently, it was found that cAMP could prevent the activation of a major Ras-dependent, growth factor-stimulated protein kinase cascade known as the MAP kinase pathway. This finding detailed the first physiological example of inhibition of Ras signaling and suggested at least one explanation for the effects of cAMP on cell proliferation. However, the means by which this is achieved and the implications for this regulation on cell growth remains to be elucidated. The objective of this proposal is to determine the mechanism by which PKA inhibits the MAPK pathway and to understand its relevance to the regulation of cell proliferation. We will focus on the regulation of specific components within the MAP kinase cascade and in particular, the effects of PKA on Raf kinase regulation. Whether Raf is the direct target of PKA, or if other aspects of the regulation of activity are involved is at present, a matter of controversy. We will investigate PKA substrates that may regulate Raf- dependent, growth factor-stimulated signals and whether the specificity and localization of PKA influences Raf regulation. Finally, we will attempt to establish a link between the antagonism of MAPK signaling and inhibition of cell growth by cAMP.
The specific aims of this proposal are to: (1) determine the mechanism by which PKA regulates Raf activity, (2) identify PKA targets involved in growth factor signaling, (3) investigate the importance of PKA isozymes and localized PKA activity in the control of MAPK signaling, and (4) determine the consequence of inhibiting MAPK signaling by PKA on cell proliferation. The objective of this research is to gain a better understanding of how cell proliferation is stimulated by oncogenes or by mitogens, with a particular emphasis on the signals that inhibit this process. The recent finding that cAMP inhibits the activation of the MAP cascade has opened up many new possibilities and this research will undoubtedly contribute to our knowledge of how diverse types of signals (e.g. hormone, heterotrimeric G protein, growth factor etc.) can modify normal and pathologic growth states.
