Previous results from our laboratory have shown that phosphorylation of type VI adenylyl cyclase (ACVI) by protein kinase C (PKC) caused suppression of adenylyl cyclase activity. In the present study, we investigated the role of the N terminus cytosolic domain of ACVI in this PKC-mediated inhibition of ACVI. Removal of amino acids 1 to 86 of ACVI or mutation of Ser(10) (a potential PKC phosphorylation site) into alanine significantly relieved the PKC-mediated inhibition and markedly reduced the PKC-evoked protein phosphorylation. PKC also effectively phosphorylated a recombinant N terminus cytosolic domain (amino acids 1-160) protein of ACVI and a synthetic peptide representing Ser(10). In addition, the amino acids 1 to 86 truncated mutant exhibited kinetic properties similar to those of the wild type. Taken together, these data demonstrate that the highly variable N terminus cytoplasmic domain of ACVI is a regulatory domain with a critical role in PKC-mediated suppression, which is a hallmark of this adenylyl cyclase isozyme. In addition, Ser(10) was found to serve as an acceptor for the PKC-mediated phosphorylating transfer of ACVI.

The NADPH oxidases (Noxs) are a family of superoxide-generating enzymes implicated in a variety of biological processes. Full activity of Nox1, -2, and -3 requires the action of a Rac GTPase. A direct regulatory interaction of Rac with Nox2 has been proposed as part of a two-step mechanism for regulating electron transfer during superoxide formation. Using truncation analysis of Rac binding to the cytoplasmic tail of Nox2, along with peptides derived from this region in cell-free assays, we identify a Rac interaction site within amino acids 419 -430 of Nox2. This region is required for binding Rac2 but not p47 phox or p67 phox cytosolic regulatory factors. A cell-permeant version of the peptide encompassing amino acids 419 -430 specifically inhibits NADPH oxidase activation in intact human neutrophils. Mutational analysis of the putative Rac-binding site revealed specific residues, particularly Lys-421, Tyr-425, and Lys-426, individually required for Rac-dependent NADPH oxidase activity that are conserved in the Rac-regulated Nox1, Nox2, and Nox3 enzymes but not in Nox4 or Nox5. Mutation of the conserved residues in the Rac-binding site of Nox1 also result in the loss of Rac-dependent activity. Our data identify a functional Rac interaction site conserved in Rac-dependent Noxs and support a direct regulatory interaction of Rac GTPases to promote activation of these NADPH oxidases.

In the present study, we used the N terminus (amino acids 1ϳ160) of type VI adenylyl cyclase (ACVI) as bait to screen a mouse brain cDNA library and identified Snapin as a novel ACVI-interacting molecule. Snapin is a binding protein of SNAP25, a component of the SNARE complex. Co-immunoprecipitation analyses confirmed the interaction between Snapin and full-length ACVI. Mutational analysis revealed that the interaction domains of ACVI and Snapin were located within amino acids 1ϳ86 of ACVI and 33-51 of Snapin, respectively. Co-localization of ACVI and Snapin was observed in primary hippocampal neurons. Moreover, expression of Snapin specifically eliminated protein kinase C (PKC)-mediated suppression of ACVI, but not that of cAMP-dependent protein kinase (PKA) or calcium. Mutation of the potential PKC and PKA phosphorylation sites of Snapin did not affect the ability of Snapin to reverse the PKC inhibitory effect on ACVI. Phosphorylation of Snapin by PKC or PKA therefore might not be crucial for Snapin action on ACVI. In contrast, Snapin ⌬33-51 , which harbors an internal deletion of amino acids 33-51 did not affect PKC-mediated inhibition of ACVI, supporting that amino acids 33-51 of Snapin comprises the ACVI-interacting region. Consistently, Snapin exerted no effect on PKC-mediated inhibition of an ACVI mutant (ACVI-⌬A87), which lacked the Snapin-interacting region (amino acids 1-86). Snapin thus reverses its action via direct interaction with the N terminus of ACVI. Collectively, we demonstrate herein that in addition to its association with the SNARE complex, Snapin also functions as a regulator of an important cAMP synthesis enzyme in the brain. Adenylyl cyclases (ACs)1 are a family of enzymes that produce cyclic AMP (cAMP) from ATP upon extracellular stimulation. To date, at least 9 membrane-bound ACs have been isolated and characterized (1). These enzymes are capable of integrating positive and negative signals that act directly through stimulation of G protein-coupled receptors (GPCRs) or indirectly via intracellular signaling molecules in isozyme-specific patterns. In addition, the regulatory properties and expression patterns of different AC isoforms greatly diverge and may account for the distinctive cell-and tissue-specific responsiveness of ACs. Recently, several different proteins, including RGS2 and the protein associated with Myc (PAM), have been shown to interact and modulate activity of different AC isozymes (2, 3), adding additional dimensions to the isozymespecific regulation of the AC superfamily.Except for the newly identified soluble AC, all membranebound AC members share a primary structure consisting of 12 transmembrane regions and 3 large cytoplasmic domains (N, C1a/b, and C2). The C1a and C2 domains, which form the catalytic core complex, are highly conserved and are homologous to each other. The N-terminal domains of ACs, in contrast, are variable among ACs, and have been demonstrated to play mostly regulatory roles (4, 5). Among the AC isozymes, ACVI is of particular interest, because...

We previously showed that phosphorylation of Ser 10 of the N terminus domain of the type VI adenylyl cyclase (ACVI) partly mediated protein kinase C (PKC)-induced inhibition of ACVI. We now report that phosphorylation of the other two cytosolic domains (C1 and C2), which form the catalytic core complex of ACVI, also contributes to PKC-mediated inhibition. to PKC-mediated regulation. Based on these results, we propose that the three cytosolic domains of ACVI might form a regulatory complex. Phosphorylation of this regulatory complex at different sites might induce a finetuning of the catalytic core complex and subsequently lead to alternation in the catalytic activity of ACVI.Genes of nine mammalian membrane-bound adenylyl cyclases (ACs) 1 have been isolated and characterized (1-3). All of these ACs contain two hydrophobic spans, composed of six transmembrane helices, and three large cytoplasmic domains (N, C1a/b, and C2, see Fig. 1A). The crystal structure of the catalytic domains of AC has been resolved and analyzed in detail (4,5). It was clearly demonstrated that two cytoplasmic domains (C1a and C2) of ACs form the catalytic core. In addition, G␣ s and/or forskolin increase the affinity between C1a and C2 and activate cyclase by changing the relative orientation of the C1 and C2 domains to an active conformation (4, 5). The functional roles of the C1b domain of ACs are relatively more variable. For ACII and ACVII, the C1b domain suppresses the catalytic activity by holding ACs in their basal non-stimulated state (6). Moreover, the C1b domain modulates calmodulin-elicited activation of ACI-and PKA-evoked inhibition of ACVI (7,8). The N terminus domains of ACs are highly variable among ACs and have been demonstrated to play a regulatory role (9, 10). The twelve transmembrane segments are responsible for linking together the two catalytic domains (C1a and C2) to achieve a proper functional conformation and are important for membrane targeting of ACs (4, 11).We have previously reported that prolonged activation of the A 2A adenosine receptor (A 2A -R) in PC12 cells significantly inhibits the activity of type VI adenylyl cyclase (ACVI), which in turn causes a lower response to subsequent stimulation by A 2A -R (12, 13). In addition, stimulation by A 2A -R activates the calcium-independent protein kinase C (PKC) that phosphorylates and inhibits ACVI. Suppression of ACVI by protein phosphorylation thus produces a lower response of ACVI to subsequent stimulation by A 2A -R in PC12 cells (14). Further biochemical analyses reveal that the apparent maximal forskolin-and G␣ s -stimulated activities of PKC-treated ACVI are significantly lower than those of the non-treated enzyme. However, treatment with PKC does not alter the K m of the substrate, nor does it markedly affect the EC 50 values of forskolin or G␣ s (10). Interestingly, the glycosylation state of ACVI affects the ability of ACVI to be inhibited by PKC (15), further suggesting that post-translational modification of ACVI is important for its activity. Analysis...

Mammalian MDC1 interacts with CHK2 in the regulation of DNA damage-induced S-phase checkpoint and apoptosis, which is directed by the association of MDC1-FHA and CHK2-pThr68. However, different ligand specificities of MDC1-FHA have been reported, and no structure is available. Here we report the crystal structures of MDC1-FHA and its complex with a CHK2 peptide containing pThr68. Unlike other FHA domains, MDC1-FHA exists as an intrinsic dimer in solution and in crystals. Structural and binding analyses support pThr+3 ligand specificity and provide structural insight into MDC1-CHK2 interaction.

The mammalian adenylyl cyclase (AC) 1 superfamily consists of nine membrane-bound isoforms. All of these possess three large cytosolic domains (designated N, C1a, and C2 domains; Fig. 1A), which are separated by two sets of six-transmembrane domains (1-3). The C1a and C2 domains among the nine AC members are highly homologous (with 50 -90% similarity in amino acids). In addition, the C1a and C2 domains of each AC share ϳ50% similarity and form the catalytic core complex, which can be stimulated by forskolin or G␣ s proteins (4 -6). Crystallographic analysis of the catalytic complex consisting of the C1a domain, the C2 domain, and the GTP␥S-bound G␣ s protein revealed that forskolin and/or G␣ s stimulate ACs by enhancing the interaction between the C1a and C2 domains and by stabilizing the C1a-C2 catalytic core complex (7).Although most ACs can be activated by G␣ s and forskolin, regulation of each AC isozyme differs. Studies of the morevariable N and C1b domains reveal that these two domains may play important regulatory roles. For example, the C1b domain has been implicated in the regulation of AC isozymes mediated by Ca 2ϩ /calmodulin, calcineurin, or protein kinase A (8 -11). In addition, the C1b domain of ACV and ACVII has been shown to modulate G␣ s -evoked activation by interacting with catalytic core complexes (12, 13). Others and we have shown that another variable region, the N-terminal domain, also significantly contributes to the regulation of AC activity (14, 15). Specifically, the N-terminal domain of ACVI (amino acids 1-160) plays an important role in the protein kinase C (PKC)-mediated inhibition and phosphorylation of ACVI (14). Removal of the first 86 amino acids (aa) of ACVI reduced the inhibitory effect of PKC on ACVI activity without affecting the basic enzymatic properties, including the affinities toward its substrate and two stimuli (forskolin and G␣ s protein (14)). The N-terminal domain of ACVI therefore functions as a regulatory domain. Further biochemical analyses revealed that at least four PKC phosphorylation sites (Ser-10, Ser-568, Ser-674, and Thr-931), located in the three large cytosolic domains of ACVI, significantly contribute to PKC-mediated inhibition of ACVI (16). Intramolecular interactions among the N, C1a/b, and C2 domains of ACVI therefore appear to be important for regulation of ACVI activity.G␣ i -mediated inhibition is a major regulatory feature of the AC superfamily. Among AC members, only ACI, ACV, and ACVI can effectively be inhibited by G␣ i proteins (17, 18). Based on results obtained from an in vitro binding assay, the C1a domain was shown to bind myristoylated G␣ i proteins and form stable complexes (19,20). Mutagenesis of full-length ACV in the ␣2 and ␣3 helices of the C1a domain revealed several residues important for the inhibition by G␣ i proteins. It was postulated that G␣ i may exert its inhibitory effect through binding to the C1a domain at the site just opposite the G␣ sbinding site on the C2 domain and, subsequently, causes reduced interaction between ...

No abstract

No abstract