Guanylate cyclase in Escherichia coli. I. Purification of the enzyme and evidence for an inhibitor]

Guanylate cyclase activity is present in crude E. coli extract. Guanylate cyclase has been purified 3500 fold from this extract, through ammonium sulfate fractionation, DEAE-cellulose chromatography. Sephadex G-75 gel-filtration and polyacrylamide gel preparative microelectrophoresis. During the purification a guanylate cyclase inhibitor has been separated.     

Yotiao protein, a ligand for the NMDA receptor, binds and targets cAMP-dependent protein kinase II(1)

A yeast two-hybrid screen revealed that regulatory subunits (RII) of PKAII bind the Yotiao protein. Yotiao interacts with the NR1 subunit of the NMDA receptor. A purified C-terminal fragment of Yotiao binds PKAII, via an RII binding site constituted by amino acid residues 1452-1469, with a dissociation constant (K(d)) between 50 and 90 nM in vitro. A stable complex composed of Yotiao, RII and NR1 was immunoprecipitated from whole rat brain extracts. Immunostaining analysis disclosed that Yotiao, RIIbeta and NR1 colocalize in striatal and cerebellar neurons. Co-assembly of Yotiao/PKAII complexes with NR1 subunits may promote cAMP-dependent modulation of NMDA receptor activity at synapses, thereby influencing brain development and synaptic plasticity.     

The length of polyglutamine tract, its level of expression, the rate of degradation, and the transglutaminase activity influence the formation of intracellular aggregates.

A common feature of CAG-expansion neurodegenerative diseases is the presence of intranuclear aggregates in neuronal cells. We have used a synthetic fusion protein containing at the NH2 terminus the influenza hemoagglutinin epitope (HA), a polyglutamine stretch (polyQ) of various size (17, 36, 43 CAG) and a COOH tail encoding the green fluorescent protein (GFP). The fusion proteins were expressed in COS-7 and neuroblastoma SK-N-BE cells. We found that the formation of aggregates largely depends on the length of polyglutamine tracts and on the levels of expression of the fusion protein. Moreover, transglutaminase overexpression caused an increase of insoluble aggregates only in cells expressing the mutant expanded protein. Conversely, treatment of cells with cystamine, a transglutaminase inhibitor, reduced the percentage of aggregates. We found also that the inhibition of the proteasome ubiquitin-dependent degradation increased the formation of intranuclear aggregates. These data suggest that length of polyglutamine tract, its expression, unbalance between cellular transglutaminase activity, and the ubiquitin-degradation pathway are key factors in the formation of intranuclear aggregates.     

Essential role of A-kinase anchor protein 121 for cAMP signaling to mitochondria

A-Kinase anchor proteins (AKAPs) immobilize and concentrate protein kinase A (PKA) isoforms at specific subcellular compartments. Intracellular targeting of PKA holoenzyme elicits rapid and efficient phosphorylation of target proteins, thereby increasing sensitivity of downstream effectors to cAMP action. AKAP121 targets PKA to the cytoplasmic surface of mitochondria. Here we show that conditional expression of AKAP121 in PC12 cells selectively enhances cAMP.PKA signaling to mitochondria. AKAP121 induction stimulates PKA-dependent phosphorylation of the proapoptotic protein BAD at Ser(155), inhibits release of cytochrome c from mitochondria, and protects cells from apoptosis. An AKAP121 derivative mutant that localizes on mitochondria but does not bind PKA down-regulates PKA signaling to the mitochondria and promotes apoptosis. These findings indicate that PKA anchored by AKAP121 transduces cAMP signals to the mitochondria, and it may play an important role in mitochondrial physiology.