It is widely accepted that the functional pleiotropy of cyclic AMP (cAMP) depends on its ability to generate intracellular messenger “signatures” that the cell “decodes” to specific functions. Indeed, it has been postulated that cAMP generating stimuli (e.g. hormones) result in the activation of spatially distinct effector moieties (microdomains) that are coupled to specific cellular responses. Despite decades of intense research, the molecular mechanisms by which cAMP achieves the spatio-temporal organization of its cascade remain poorly understood and hotly debated. The leading idea associates the selective activation of effector proteins to the local availability of the messenger. Specifically, the subcellular heterogeneity in cAMP levels has been attributed to the localization or activity of either adenylate cyclases (ACs) and phosphodiesterases (PDEs) in different cellular sub compartments. However, arguing against cAMP microdomains being solely dependent on the messenger’s distribution are computational models which consistently show that the distribution of cAMP cannot be sufficiently restricted by PDEs implicating the need for additional factors in the generation of functionally distinct cAMP microdomains. In order to investigate this issue my group generated a series of FRET-based sensors that allow the measurement in real time in living cells of both, the levels of cAMP and the actions of its main effector Protein Kinase A (PKA) (PKA-dependent phosphorylation). As proof of principle we targeted our sensors to the outer mitochondrial membrane (OMM), a well-recognised functional PKA microdomain, and compared the levels of cAMP and PKA-dependent phosphorylation in response to cAMP generating agonists between OMM and cytosol. We found that, at equivalent levels of cAMP, PKA located at the OMM responded differently than the cytosol and that was due to differential action of the terminators of the cAMP/PKA axis, the phosphatases. Our work unveiled phosphatases as major, hitherto unrecognized contributors to the compartmentalization of PKA activity. This work demonstrated that differences in local phosphatase activity fully explain functional data assessed using readouts of cAMP signalling in the cytosolic and OMM compartments

KONSTANTINOS LEFKIMMIATIS

  • 2017-present VIMM Junior Principal Investigator
  • 2017-present Researcher (Tenured) Neuroscience Institute CNR
  • 2013-2016 Intermediate Fellow (University of Oxford)
  • 2010-2013 Instructor (Harvard Medical School)
  • 2006-2010 Postdoc (Harvard Medical School)
  • 2003-2006 Ph.D. in Genetics and Molecular Evolution (University of Bari)
  • 1993-1999 Master’s degree in Biology (University of Bari)