Control of specificity in cAMP signaling is achieved by A-kinase anchoring proteins (AKAPs), which assemble cAMP effectors such as protein kinase A (PKA) into multiprotein signaling complexes in the cell. AKAPs tether the PKA holoenzymes at subcellular locations to favor the phosphorylation of selected substrates. PKA anchoring is mediated by an amphipathic helix of 14 -18 residues on each AKAP that binds to the R subunit dimer of the PKA holoenzymes. Using a combination of bioinformatics and peptide array screening, we have developed a high affinity-binding peptide called RIAD (RI anchoring disruptor) with >1000-fold selectivity for type I PKA over type II PKA. Cell-soluble RIAD selectively uncouples cAMP-mediated inhibition of T cell function and inhibits progesterone synthesis at the mitochondria in steroid-producing cells. This study suggests that these processes are controlled by the type I PKA holoenzyme and that RIAD can be used as a tool to define anchored type I PKA signaling events.The cAMP signaling pathway synchronizes a variety of physiological responses including cell proliferation and differentiation, microtubule dynamics, reproductive function, modulation of immune responses, and steroidogenesis (1-3). Many of these responses require activation of the cAMP-dependent protein kinase (PKA).3 The dormant PKA holoenzyme is a heterotetramer composed of two catalytic (C) subunits held in an inactive conformation by a regulatory (R) subunit dimer. Upon activation with cAMP, the C subunits are released from their interaction with the R subunit dimer and are free to phosphorylate a plethora of target substrates. Individual cells express a range of PKA isozymes differing in R (RI␣, RI␤, RII␣, RII␤) and C (C␣, C␤, C␥) subunit composition. The type I and type II PKA isozymes, which are classified on the basis of their R subunit dimer, possess slightly different physical and biological properties including a differential sensitivity to cAMP.Spatiotemporal regulation of PKA phosphorylation events is facilitated by A-kinase anchoring proteins (AKAPs). This family of structurally diverse but functionally related proteins act as molecular scaffolds to cluster PKA with specific substrates and signal termination enzymes such as phosphatases (4, 5) and cAMP-specific phosphodiesterases (6 -9). The number of AKAPs has been estimated to more than 75, of which ϳ50 have been identified to date (3, 10). Although the majority of AKAPs described to date bind type II PKA via the RII regulatory subunit, several dual function AKAPs are capable of interacting with both the type I and the type II PKA holoenzyme (11-15). There are also examples of anchoring proteins such as AKAPce (16), PAP7 (17), and merlin (18) that selectively interact with the RI subunit of PKA.The molecular basis for PKA anchoring is the interaction of an amphipathic helical motif of 14 -18 residues on the AKAP with a hydrophobic groove in the N-terminal dimerization domain of the R subunit. High affinity peptides mimicking the amphipathic helix bind to the R...

We have solved, by X-ray crystallography to a resolution of 1.8 A, the structure of a protein capable of mimicking approximately 20 base pairs of B-form DNA. This ocr protein, encoded by gene 0.3 of bacteriophage T7, mimics the size and shape of a bent DNA molecule and the arrangement of negative charges along the phosphate backbone of B-form DNA. We also demonstrate that ocr is an efficient inhibitor in vivo of all known families of the complex type I DNA restriction enzymes. Using atomic force microscopy, we have also observed that type I enzymes induce a bend in DNA of similar magnitude to the bend in the ocr molecule. This first structure of an antirestriction protein demonstrates the construction of structural mimetics of long segments of B-form DNA.

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The mechanism by which classical cadherins mediate cell adhesion and, in particular, the roles played by calcium and Trp2, the second amino acid in the N-terminal domain, have long been controversial. We have used antibodies to investigate the respective contributions of Trp2 and calcium to the stability of the N-terminal domain of N-cadherin. Using a peptide antibody to the βB strand in domain 1, which detects a disordered structure, we show that both Trp2 and calcium play crucial parts in regulating stability of the domain. The epitope for another antibody, mAb GC4, has been mapped to the base of domain 1. Binding of GC4 to this epitope was shown to depend on intramolecular `docking' of Trp2 into the domain 1 structure. Using this property, we provide evidence that calcium regulates a dynamic equilibrium between docked and undocked Trp2. Finally, a novel technique has been developed to test whether Trp2 cross-intercalation between cadherin molecules from adjacent cells (strand exchange) is central to cadherin-mediated cell adhesion. Guided by crystal structures showing strand exchange, we have introduced single cysteine point mutations into N-cadherin domain 1 in such a way that a disulphide bond will form between opposing N-cadherin molecules during cell adhesion if strand exchange occurs. The bond requires complementary cysteines to be precisely juxtaposed according to the strand exchange model. Our results demonstrate that the disulphide bond forms as predicted. This provides compelling evidence that strand exchange is indeed a primary event in cell adhesion by classical cadherins.

Atomic force microscopy (AFM) has been used to examine perturbations in the tertiary structure of DNA induced by the binding of ditercalinium, a DNA bis-intercalator with strong anti-tumour properties. We report AFM images of plasmid DNA of both circular and linearised forms showing a difference in the formation of supercoils and plectonemic coils caused at least in part by alterations in the superhelical stress upon bis-intercalation. A further investigation of the effects of drug binding performed with 292 bp mixed-sequence DNA fragments, and using increment in contour length as a reliable measure of intercalation, revealed saturation occurring at a point where sufficient drug was present to interact with every other available binding site. Moment analysis based on the distribution of angles between segments along single DNA molecules showed that at this level of bis-intercalation, the apparent persistence length of the molecules was 91.7 ± 5.7 nm, approximately twice as long as that of naked DNA. We conclude that images of single molecules generated using AFM provide a valuable supplement to solution-based techniques for evaluation of physical properties of biological macromolecules.

Engagement of the T-cell receptor (TCR) in human primary T cells activates a cyclic AMP (cAMP)-T-cell receptor (TCR) stimulation alone is insufficient for activation of T cells, and sustainable T-cell immune responses require a second signal in addition to the TCR-mediated signal. The second signal is typically elicited by ligands B7-1 or B7-2 on antigen-presenting cells engaging the coreceptor CD28 to prevent anergy and apoptosis and enhancing interleukin-2 (IL-2) production and clonal expansion (4). Although CD28 plays a central role in T-cell activation in vivo (5), relatively little is known about the molecular basis for the increased efficacy of T-cell activation upon TCR and CD28 costimulation. Involvement of Lck, Itk, phosphatidylinositol 3-kinase (PI3K), SLP-76, Vav-1, and phospholipase C-␥ (PLC-␥) has, however, been reported (43). CD28-mediated signals are transmitted via a short intracellular stretch in the receptor containing a conserved YMNM motif (32). Phosphorylation of Tyr173 in this motif by Lck and Fyn following CD28 ligation is key to efficient signal transduction (41), generating a binding site for the SH2 domain of the p85 regulatory subunit of PI3K (37, 40). CD28 may also contribute to TCR-dependent PI3K activity without recruiting PI3K directly (18). Whether engagement of CD28 alone can also induce PI3K activity has been a matter of controversy. However, recent reports confirming phosphorylation of the protein kinase B (PKB) substrate glycogen synthase kinase 3 (GSK3) upon CD28 ligation has demonstrated that this is indeed the case (6, 15). In addition, CD28 can recruit growth factor receptor-bound protein 2 (Grb2), and such association of Grb2 occurs via the phosphorylated YMNM motif as well as via the C-terminal PXXP motif (22,35). The PXXP motif also binds and regulates Src family kinases (SFKs) (21, 47), and knock-in mice mutated in this motif were recently reported to have impaired IL-2 secretion (16).Ligation of the TCR induces cyclic AMP (cAMP) production (27). However, the significance of this observation is still not fully understood, as it is well established that cAMP potently inhibits T-cell function and proliferation (2,45,46,50). The spatiotemporal dynamics of the activation-induced cAMP gradient also are not completely appreciated. We have previously shown that cAMP is rapidly produced in lipid rafts following engagement of the TCR in primary T cells (3). This activates a pool of PKA type I targeted to rafts by association with the anchoring protein Ezrin, forming part of a supramolecular complex where Ezrin, EBP50, and PAG provide a scaffold that is able to coordinate PKA phosphorylation and activation of Csk, thereby inhibiting T-cell activation (44, 50). In addition, we have demonstrated that CD3/CD28 costimulation leads to recruitment of type 4 phosphodiesterase (PDE4) isoforms to rafts, resulting in degradation of the TCR-induced cAMP pool (3). Thus, we envisage that TCR-induced cAMP production constitutes a negative feedback loop capable of abrogating T-cell activation ...

Bacterial type I restriction/modification systems are capable of performing multiple actions in response to the methylation pattern on their DNA recognition sequences. The enzymes making up these systems serve to protect the bacterial cells against viral infection by binding to their recognition sequences on the invading DNA and degrading it after extensive ATP-driven translocation. DNA cleavage has been thought to occur as the result of a collision between two translocating enzyme complexes. Using atomic force microscopy (AFM), we show here that EcoKI dimerizes rapidly when bound to a plasmid containing two recognition sites for the enzyme. Dimerization proceeds in the absence of ATP and is also seen with an EcoKI mutant (K477R) that is unable to translocate DNA. Only monomers are seen when the enzyme complex binds to a plasmid containing a single recognition site. Based on our results, we propose that the binding of EcoKI to specific DNA target sequences is accompanied by a conformational change that leads rapidly to dimerization. This event is followed by ATP-dependent translocation and cleavage of the DNA.

Full T-cell activation critically depends on the engagement of the TCR (T-cell receptor) in conjunction with a second signal by co-stimulatory receptors that boost the immune response. In the present study we have compared signalling patterns induced by the two co-receptors CD2 and CD28 in human peripheral blood T-cells. These co-receptors were previously suggested to be redundant in function. By a combination of multi-parameter phosphoflow cytometry, phosphokinase arrays and Western blot analyses, we demonstrate that CD2 co-stimulation induces phosphorylation of the TCR-proximal signalling complex, whereas CD28 activates distal signalling molecules, including the transcription factors NF-κB (nuclear factor κB), ATF (activating transcription factor)-2, STAT3/5 (signal transducer and activator of transcription 3/5), p53 and c-Jun. These signalling patterns were conserved in both naïve and effector/memory T-cell subsets. We show that free intracellular Ca(2+) and signalling through the PI3K (phosphoinositide 3-kinase)/Akt pathway are required for proper CD28-induced NF-κB activation. The signalling patterns induced by CD2 and CD28 co-stimulation lead to distinct functional immune responses in T-cell proliferation and cytokine production. In conclusion, CD2 and CD28 co-stimulation induces distinct signalling responses and functional outcomes in T-cells.