The Akt1 isoform is an essential mediator of ischaemic preconditioning

Phosphatidyl-inositol-3-kinase (PI3K)-Akt pathway is essential for conferring cardioprotection in response to ischaemic preconditioning (IPC) stimulus. However, the role of the individual Akt isoforms expressed in the heart in mediating the protective response to IPC is unknown. In this study, we investigated the specific contribution of Akt1 and Akt2 in cardioprotection against ischaemia-reperfusion (I-R) injury. Mice deficient in Akt1 or Akt2 were subjected to in vivo regional myocardial ischaemia for 30 min. followed by reperfusion for 2 hrs with or without a prior IPC stimulus. Our results show that mice deficient in Akt1 were resistant to protection with either one or three cycles of IPC stimulus (42.7 ± 6.5% control versus 38.5 ± 1.9% 1 χ IPC, N = 6, NS; 41.4 ± 6.3% control versus 32.4 ± 3.2% 3 χ IPC, N = 10, NS). Western blot analysis, performed on heart samples taken from Akt1(-/-) mice subjected to IPC, revealed an impaired phosphorylation of GSK-3β, a downstream effector of Akt, as well as Erk1/2, the parallel component of the reperfusion injury salvage kinase pathway. Akt2(-/-) mice, which exhibit a diabetic phenotype, however, were amenable to protection with three but not one cycle of IPC (46.4 ± 5.6% control versus 35.9 ± 5.0% in 1 χ IPC, N = 6, NS; 47.0 ± 6.0% control versus 30.8 ± 3.3% in 3 χ IPC, N = 6; *P = 0.039). Akt1 but not Akt2 is essential for mediating a protective response to an IPC stimulus. Impaired activation of GSK-3β and Erk1/2 might be responsible for the lack of protective response to IPC in Akt1(-/-) mice. The rise in threshold for protection in Akt2(-/-) mice might be due to their diabetic phenotype.     

Rapamycin causes activation of protein phosphatase-2A1 and nuclear translocation of PCNA in CD4+ T cells

Rapamycin is a powerful immunosuppressant that causes cell cycle arrest in T cells and several other cell types. Despite its important clinical role, the mechanism of action of rapamycin is not fully understood. Here, we show that rapamycin causes the activation of protein phosphatase-2A1 which forms a complex with proliferation cell nuclear antigen (PCNA) in a CD4+ T cell line. Rapamycin also induces PCNA translocation from the cytoplasm to the nucleus, an effect which is antagonized by okadaic acid, an inhibitor of type 2A protein phosphatases. These findings provide evidence for the existence of a signal transduction pathway that links a rapamycin-activated type 2A protein phosphatase to the control of DNA synthesis, DNA repair, cell cycle, and cell death via PCNA.     

cAMP mediated proteolysis of the catalytic subunit of cAMP-dependent protein kinase

The cAMP-dependent protein kinase from LLC-PK1 cells can be activated in vivo by calcitonin and vasopressin, or forskolin. Continuous treatment of cells with these agents results in a decrease of total cAMP-PK activity. The loss of kinase activity was enhanced when either of these three agents was incubated in the presence of isobutylmethylxanthine. Results obtained using affinity purified antibodies to the catalytic subunit show that the loss of kinase was due to specific proteolysis of this subunit.     

Protein Kinase B Localization and Activation Differentially Affect S6 Kinase 1 Activity and Eukaryotic Translation Initiation Factor 4E-Binding Protein 1 Phosphorylation

Recent studies indicate that phosphatidylinositide-3OH kinase (PI3K)-induced S6 kinase (S6K1) activation is mediated by protein kinase B (PKB). Support for this hypothesis has largely relied on results obtained with highly active, constitutively membrane-localized alleles of wild-type PKB, whose activity is independent of PI3K. Here we set out to examine the importance of PKB signaling in S6K1 activation. In parallel, glycogen synthase kinase 3beta (GSK-3beta) inactivation and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation were monitored as markers of the rapamycin-insensitive and -sensitive branches of the PI3K signaling pathway, respectively. The results demonstrate that two activated PKBalpha mutants, whose basal activity is equivalent to that of insulin-induced wild-type PKB, inhibit GSK-3beta to the same extent as a highly active, constitutively membrane-targeted wild-type PKB allele. However, of these two mutants, only the constitutively membrane-targeted allele of PKB induces S6K1 activation. Furthermore, an interfering mutant of PKB, which blocks insulin-induced PKB activation and GSK-3beta inactivation, has no effect on S6K1 activation. Surprisingly, all the activated PKB mutants, regardless of constitutive membrane localization, induce 4E-BP1 phosphorylation and the interfering PKB mutant blocks insulin-induced 4E-BP1 phosphorylation. The results demonstrate that PKB mediates S6K1 activation only as a function of constitutive membrane localization, whereas the activation of PKB appears both necessary and sufficient to induce 4E-BP1 phosphorylation independently of its intracellular location.     

Novel domain packing in the crystal structure of a thiosulphate-oxidizing enzyme

A key component of the oxidative biogeochemical sulphur cycle involves the utilization by bacteria of reduced inorganic sulphur compounds as electron donors to photosynthetic or respiratory electron transport chains. The SoxAX protein of the photosynthetic bacterium Rhodovulum sulfidophilum is a heterodimeric c-type cytochrome that is involved in the oxidation of thiosulphate and sulphide. The recently solved crystal structure of the SoxAX complex represents the first structurally characterized example of a productive electron transfer complex between haemoproteins where both partners adopt the c-type cytochrome fold. The packing of c-type cytochrome domains both within SoxA and at the interface between the subunits of the complex has been compared with other examples and found to be unique.     

Two splice variants of protein kinase B gamma have different regulatory capacity depending on the presence or absence of the regulatory phosphorylation site serine 472 in the carboxyl-terminal hydrophobic domain

We have reported previously the cloning and characterization of human and mouse protein kinase B gamma (PKB gamma), the third member of the PKB family of second messenger-regulated serine/threonine kinases (Brodbeck, D., Cron, P., and Hemmings, B. A. (1999) J. Biol. Chem. 274, 9133--9136). Here we report the isolation of human and mouse PKB gamma 1, a splice variant lacking the second regulatory phosphorylation site Ser-472 in the hydrophobic C-terminal domain. Expression of PKB gamma 1 is low compared with PKB gamma, and it is regulated in different human tissues. We show that PKB gamma and PKB gamma 1 differ in their response to stimulation by insulin, pervanadate, peroxide, or okadaic acid. Activation of PKB gamma 1 requires phosphorylation at a single regulatory site Thr-305. Interestingly, this site is phosphorylated to a higher extent in PKB gamma compared with PKB gamma 1 upon maximal stimulation by pervanadate, and this is reflected in the respective specific kinase activities. Furthermore, upon insulin stimulation of transfected cells, PKB gamma 1 translocates to the plasma membrane to a lesser extent than PKB gamma. Taken together, these results suggest that phosphorylation of the hydrophobic motif at the extreme C terminus of PKB gamma may facilitate translocation of the kinase to the membrane and/or its phosphorylation on the activation loop site by phosphoinositide-dependent protein kinase-1.     

The anxiety-like phenotype of 5-HT receptor null mice is associated with genetic background-specific perturbations in the prefrontal cortex GABA-glutamate system

A deficit in the serotonin 5-HT(1A) receptor has been found in panic and post-traumatic stress disorders, and genetic inactivation of the receptor results in an anxiety-like phenotype in mice on both the C57Bl6 and Swiss-Webster genetic backgrounds. Anxiety is associated with increased neuronal activity in the prefrontal cortex and here we describe changes in glutamate and GABA uptake of C57Bl6 receptor null mice. Although these alterations were not present in Swiss-Webster null mice, we have previously reported reductions in GABA(A) receptor expression in these but not in C57Bl6 null mice. This demonstrates that inactivation of the 5-HT(1A) receptor elicits different and genetic background-dependent perturbations in the prefrontal cortex GABA/glutamate system. These perturbations can result in a change in the balance between excitation and inhibition, and indeed both C57Bl6 and Swiss-Webster null mice show signs of increased neuronal excitability. Because neuronal activity in the prefrontal cortex controls the extent of response to anxiogenic stimuli, the genetic background-specific perturbations in glutamate and GABA neurotransmission in C57Bl6 and Swiss-Webster 5-HT(1A) receptor null mice may contribute to their shared anxiety phenotype. Our study shows that multiple strains of genetically altered mice could help us to understand the common and individual features of anxiety.     

Structural parsimony in endonuclease active sites: should the number of homing endonuclease families be redefined?

Homing endonucleases are classified into four families based on active site sequence motifs. Through structural comparisons we have found structural similarities between the endonuclease domain of colicin E9, an H-N-H motif-containing enzyme, and both the non-specific nuclease from Serratia and I-PpoI, a His-Cys box-containing homing endonuclease. Our comparison identifies conservation at the heart of all three enzyme active sites and so argues for a re-classification of H-N-H and His-Cys box homing endonucleases as a single family. We suggest the 'betabetaalpha-Me family' of homing enzymes to reflect the three elements of secondary structure and the metal ion that define the motif.     

A family of leukemia inhibitory factor-binding peptides that can act as antagonists when conjugated to poly(ethylene glycol)

A panel of six na?ve 14-residue random peptide libraries displayed polyvalently on M13 phage was pooled and sorted against human leukemia inhibitory factor (LIF). After four rounds of selection, a single large family of peptides with the consensus sequence XCXXXXG(A/S)(D/E)(W/F)WXCF was found to bind specifically to LIF. Peptides within this family did not bind related members of the interleukin-6 family of cytokines, nor to murine LIF that has 80% sequence identity with human LIF. A representative peptide from this family was synthesized and found to bind to LIF with an affinity of approximately 300 nM. The phage-displayed form of this peptide was able to compete with the LIF receptor alpha chain (LIFR) for binding to LIF; however, the free synthetic peptide was unable to inhibit LIF-LIFR binding or inhibit LIF bioactivity in vitro. Using a panel of human/murine chimeric LIF molecules, the peptide-binding site on LIF was mapped to a groove located between the B and the C helices of the LIF structure, which is distinct from the surfaces involved in binding to receptor. To mimic the effect of the phage particle and convert the free peptide into an antagonist of LIFR binding, a 40 kDa poly(ethylene glycol) (PEG) moiety was conjugated to the synthetic LIF-binding peptide. This PEG-peptide conjugate was found to be both an antagonist of LIF-LIFR binding and of LIF signaling in engineered Ba/F3 cells expressing LIFR and the gp130 coreceptor.