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.

We have selected designed ankyrin repeat proteins (DARPins) from a synthetic library by using ribosome display that selectively bind to the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase 2) in either its nonphosphorylated (inactive) or doubly phosphorylated (active) form. They do not bind to other kinases tested. Crystal structures of complexes with two DARPins, each specific for one of the kinase forms, were obtained. The two DARPins bind to essentially the same region of the kinase, but recognize the conformational change within the activation loop and an adjacent area, which is the key structural difference that occurs upon activation. Whereas the rigid phosphorylated activation loop remains in the same form when bound by the DARPin, the more mobile unphosphorylated loop is pushed to a new position. The DARPins can be used to selectively precipitate the cognate form of the kinases from cell lysates. They can also specifically recognize the modification status of the kinase inside the cell. By fusing the kinase with Renilla luciferase and the DARPin to GFP, an energy transfer from luciferase to GFP can be observed in COS-7 cells upon intracellular complex formation. Phosphorylated ERK2 is seen to increase by incubation of the COS-7 cells with FBS and to decrease upon adding the ERK pathway inhibitor PD98509. Furthermore, the anti-ERK2 DARPin is seen to inhibit ERK phosphorylation as it blocks the target inside the cell. This strategy of creating activation-state-specific sensors and kinase-specific inhibitors may add to the repertoire to investigate intracellular signaling in real time.intrabodies | X-ray crystallography

During early stages of cotranslational protein translocation across the endoplasmic reticulum (ER) membrane the ribosome is targeted to the heterotrimeric Sec61p complex, the major component of the proteinconducting channel. We demonstrate that this interaction is mediated by the 28S rRNA of the eukaryotic large ribosomal subunit. Bacterial ribosomes also bind via their 23S rRNA to the bacterial homolog of the Sec61p complex, the SecYEG complex. Eukaryotic ribosomes bind to the SecYEG complex, and prokaryotic ribosomes to the Sec61p complex. These data indicate that rRNA-mediated interaction of ribosomes with the translocation channel occurred early in evolution and has been conserved. Keywords: protein translocation/ribosome/rRNA/SecY/ Sec61 IntroductionProtein translocation across the ER membrane can occur either co-or post-translationally. In the cotranslational pathway, a polypeptide chain is synthesized on a membrane-bound ribosome; the elongating chain is transferred from a tunnel in the ribosome through a channel in the membrane into the lumen of the ER (Crowley et al., 1994;Mothes et al., 1994;Beckmann et al., 1997). In the posttranslational pathway, the chain is ®rst completed in the cytosol before being transported across the membrane. In both cases the same membrane channel is used, formed by three or four copies of the evolutionarily conserved heterotrimeric Sec61p complex. In the cotranslational pathway, the Sec61p channel associates with the ribosome (Go Èrlich and Rapoport, 1993;Kalies et al., 1994;Hanein et al., 1996;Beckmann et al., 1997), and in the posttranslational pathway, at least in the yeast Saccharomyces cerevisiae, it associates with the tetrameric Sec62/63p membrane protein complex to form a heptameric Sec complex (Deshaies et al., 1991;Panzner et al., 1995;Hanein et al., 1996).How the ribosome binds to the Sec61p complex during cotranslational translocation is unknown, and it is unclear whether the interaction involves ribosomal RNA (rRNA), protein or both. Non-translating mammalian ribosomes can bind with high af®nity to the Sec61p complex in mammalian ER membranes (Rolleston, 1972;Borgese et al., 1974;Kalies et al., 1994). This interaction is salt sensitive, and the binding site is likely to be located in the large ribosomal subunit (Rolleston, 1972;Borgese et al., 1974;Unwin, 1977). The association of non-translating ribosomes with the Sec61p complex mimics early stages of cotranslational protein translocation (Jungnickel and Rapoport, 1995;Beckmann et al., 1997;Neuhof et al., 1998). When the signal sequence of a growing nascent chain has just emerged from the ribosome, the ribosome± nascent chain complex is targeted by the signal recognition particle (SRP) to the membrane (Walter and Blobel, 1981) and initially binds to the Sec61p complex in a saltsensitive manner (Jungnickel and Rapoport, 1995). After further chain elongation, the signal sequence is recognized by the channel and inserts into it, and the ribosome± membrane interaction becomes salt resistant (Jungnickel and Rapopor...

PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIalpha subunits with high affinity is AKAP7delta [AKAP18delta; K(d) (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7delta mutant binds RIIalpha subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654-26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7delta bind RIIalpha subunits with higher affinity (K(d)=0.4+/-0.3 nM) than either full-length or N-terminally truncated AKAP7delta, or peptides derived from other RII binding domains. The AKAP7delta-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP-RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RII-binding domain of AKAP7delta, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond- and salt-bridge-forming amino acid residues increase the affinity of the interaction.

Knowledge about the molecular structure of protein kinase A (PKA) isoforms is substantial. In contrast, the dynamics of PKA isoform activity in living primary cells has not been investigated in detail. Using a high content screening microscopy approach, we identified the RIIb subunit of PKA-II to be predominantly expressed in a subgroup of sensory neurons. The RIIb-positive subgroup included most neurons expressing nociceptive markers (TRPV1, NaV1.8, CGRP, IB4) and responded to pain-eliciting capsaicin with calcium influx. Isoform-specific PKA reporters showed in sensory-neuronderived F11 cells that the inflammatory mediator PGE 2 specifically activated PKA-II but not PKA-I. Accordingly, pain-sensitizing inflammatory mediators and activators of PKA increased the phosphorylation of RII subunits (pRII) in subgroups of primary sensory neurons. Detailed analyses revealed basal pRII to be regulated by the phosphatase PP2A. Increase of pRII was followed by phosphorylation of CREB in a PKA-dependent manner. Thus, we propose RII phosphorylation to represent an isoform-specific readout for endogenous PKA-II activity in vivo, suggest RIIb as a novel nociceptive subgroup marker, and extend the current model of PKA-II activation by introducing a PP2A-dependent basal state.