In the molecular scheme of living organisms, adenosine 3′,5′-monophosphate (cyclic AMP or cAMP) has been a universal second messenger. In eukaryotic cells, the primary receptors for cAMP are the regulatory subunits of cAMP-dependent protein kinase. The crystal structure of a 1-91 deletion mutant of the type Iα regulatory subunit was refined to 2.8 Å resolution. Each of the two tandem cAMP binding domains provides an extensive network of hydrogen bonds that buries the cyclic phosphate and the ribose between two β strands that are linked by a short α helix. Each adenine base stacks against an aromatic ring that lies outside the β barrel. This structure provides a molecular basis for understanding how cAMP binds cooperatively to its receptor protein, thus mediating activation of the kinase.

There is a growing appreciation that the cyclic adenosine monophosphate (cAMP)–protein kinase A (PKA) signaling pathway is organized to form transduction units that function to deliver specific messages. Such organization results in the local activation of PKA subsets through the generation of confined intracellular gradients of cAMP, but the mechanisms responsible for limiting the diffusion of cAMP largely remain to be clarified. In this study, by performing real-time imaging of cAMP, we show that prostaglandin 1 stimulation generates multiple contiguous, intracellular domains with different cAMP concentration in human embryonic kidney 293 cells. By using pharmacological and genetic manipulation of phosphodiesterases (PDEs), we demonstrate that compartmentalized PDE4B and PDE4D are responsible for selectively modulating the concentration of cAMP in individual subcellular compartments. We propose a model whereby compartmentalized PDEs, rather than representing an enzymatic barrier to cAMP diffusion, act as a sink to drain the second messenger from discrete locations, resulting in multiple and simultaneous domains with different cAMP concentrations irrespective of their distance from the site of cAMP synthesis.

Import of matrix proteins into peroxisomes requires two targeting signal-specific import receptors, Pex5p and Pex7p, and their binding partners at the peroxisomal membrane, Pex13p and Pex14p. Several constructs of human PEX5 have been overexpressed and purified by affinity chromatography in order to determine functionally important interactions and provide initial structural information. Sizing chromatography and electron microscopy suggest that the two isoforms of the human PTS1 receptor, PEX5L and PEX5S, form homotetramers. Surface plasmon resonance analysis indicates that PEX5 binds to the N-terminal fragment of PEX14-(1–78) with a very high affinity in the low nanomolar range. Stable complexes between recombinant PEX14-(1–78) and both the full-length and truncated versions of PEX5 were formed in vitro. Analysis of these complexes revealed that PEX5 possesses multiple binding sites for PEX14, which appear to be distributed throughout its N-terminal half. Coincidentally, this part of the molecule is also responsible for oligomerization, whereas the C-terminal half with its seven tetratricopeptide repeats has been reported to bind PTS1-proteins. A pentapeptide motif that is reiterated seven times in PEX5 is proposed as a determinant for the interaction with PEX14.

Significance: Leucine-rich repeat kinase 2 (LRRK2) is a multidomain protein implicated in Parkinson disease, and cAMP-dependent protein kinase (PKA) has been suggested to act as an upstream kinase phosphorylating LRRK2. Using a phosphoproteomics approach, we identified several novel PKA phosphorylation sites on LRRK2. We could demonstrate that one PKA phosphosite comprises the second most common mutation in LRRK2 (R1441C/G/H) attributed to Parkinson disease. Our findings reveal that this site is mandatory for subsequent 14-3-3 binding and affects LRRK2 kinase activity. These data provide a mechanistic insight into the regulation of LRRK2 kinase activity and its perturbation by disease-associated mutations.