Title: Phosphoproteomic Analysis as an Approach for Understanding Molecular Mechanisms of cAMP-dependent Actions.

In recent years, highly sensitive mass spectrometry-based phosphoproteomic analysis is beginning to be applied to identification of protein kinase substrates altered downstream of increased cyclic 39,59adenosine monophosphate (cAMP). Such studies identify a very large number of phosphorylation sites regulated in response to increased cAMP. Therefore, we now are tasked with the challenge of determining how many of these altered phosphorylation sites are relevant to regulation of function in the cell. This mini-review describes the use of phosphoproteomic analysis to monitor the effects of cyclic nucleotide phosphodiesterase (PDE) inhibitors on cAMP-dependent phosphorylation events. More specifically, it describes two examples of this approach carried out in the authors9 laboratories using the selective PDE inhibitor approach. After a short discussion of several likely conclusions suggested by these analyses of cAMP function in steroid hormone producing cells and also in T-cells, it expands into a discussion about some newer and more speculative interpretations of the data. These include the idea that multiple phosphorylation sites and not a single rate limiting step likely regulate these and by analogy many other cAMP-dependent pathways. In addition, the idea that meaningful regulation requires a high stoichiometry of phosphorylation in order to be important is discussed and suggested to be untrue in many instances. These new interpretations have important implications for drug design, especially for targeting pathway agonists. Significance Statement Phosphoproteomic analyses identify thousands of altered phosphorylation sites upon drug treatment providing many possible regulatory targets, but also highlight questions about which phosphosites are functionally important. These data imply that multi-step processes are regulated by phosphorylation at not one, but rather many sites. Most previous studies assumed single or very few rate-limiting steps were changed by phosphorylation. This concept should be changed. Previous interpretations also assumed sub-stoichiometric phosphorylation was not of regulatory importance. This assumption also should be changed.