Beacon interacts with cdc2/cdc28-like kinases

Previously we found elevated beacon gene expression in the hypothalamus of obese Psammomys obesus. Beacon administration into the lateral ventricle of P. obesus stimulated food intake and body weight gain. In the current study we used yeast two-hybrid technology to screen for proteins in the human brain that interact with beacon. CLK4, an isoform of cdc2/cdc28-like kinase family of proteins, was identified as a strong interacting partner for beacon. Using active recombinant proteins and a surface plasmon resonance based detection technique, we demonstrated that the three members of this subfamily of kinases (CLK1, 2, and 4) all interact with beacon. Based on the known sequence and functional properties of beacon and CLKs, we speculate that beacon could either modulate the function of key regulatory molecules such as PTP1B or control the expression patterns of specific genes involved in the central regulation of energy metabolism.     

Generation of an active monomer of rabbit muscle creatine kinase by site-directed mutagenesis: the effect of quaternary structure on catalysis and stability

Cytosolic creatine kinase exists in native form as a dimer; however, the reasons for this quaternary structure are unclear, given that there is no evidence of active site communication and more primitive guanidino kinases are monomers. Three fully conserved residues found in one-half of the dimer interface of the rabbit muscle creatine kinase (rmCK) were selectively changed to alanine by site-directed mutagenesis. Four mutants were prepared, overexpressed, and purified: R147A, R151A, D209A, and R147A/R151A. Both the R147A and R147A/R151A were confirmed by size-exclusion chromatography and analytical ultracentrifugation to be monomers, whereas R151A was dimeric and D209A appeared to be an equilibrium mixture of dimers and monomers. Kinetic analysis showed that the monomeric mutants, R147A and R147A/R151A, showed substantial enzymatic activity. Substrate binding affinity by R147A/R151A was reduced approximately 10-fold, although k(cat) was 60% of the wild-type enzyme. Unlike the R147A/R151A, the kinetic data for the R147A mutant could not be fit to a random-order rapid-equilibrium mechanism characteristic of the wild-type, but could only be fit to an ordered mechanism with creatine binding first. Substrate binding affinities were also significantly lower for the R147A mutant, but k(cat) was 11% that of the native enzyme. Fluorescence measurements using 1-anilinonaphthalene-8-sufonate showed that increased amounts of hydrophobic surface area are exposed in all of the mutants, with the monomeric mutants having the greatest amounts of unfolding. Thermal inactivation profiles demonstrated that protein stability is significantly decreased in the monomeric mutants compared to wild-type. Denaturation experiments measuring lambda(max) of the intrinsic fluorescence as a function of guanidine hydrochloride concentration helped confirm the quaternary structures and indicated that the general unfolding pathway of all the mutants are similar to that of the wild-type. Collectively, the data show that dimerization is not a prerequisite for activity, but there is loss of structure and stability upon formation of a CK monomer.     

Characterization of a lactose permease mutant that binds IIAGlc in the absence of ligand

Enzyme IIA(Glc) of the Escherichia coli phosphoenolpyruvate:glucose phosphotransferase system plays a direct role in regulating inducible transport systems. Dephosphorylated IIA(Glc) binds directly to lactose permease in a reaction that requires binding of a galactosidic substrate. A double-Cys mutation (Ile129 --> Cys/Lys131 --> Cys) was introduced into helix IV of the permease near the IIA(Glc) binding site in cytoplasmic loop IV/V and in the vicinity of the galactoside binding site at the interface of helices IV, V, and VIII. The mutant no longer requires galactoside for IIA(Glc) binding as demonstrated by both a [(125)I]IIA(Glc) binding assay and a newly developed fluorescence anisotropy assay. Further characterization of the mutant shows that it binds substrate with high affinity, but is almost completely defective in all modes of translocation across the cytoplasmic membrane. The data are consistent with the interpretation that the double mutant is locked in an inward-facing conformation.     

Role for the BRCA1 C-terminal repeats (BRCT) protein 53BP1 in maintaining genomic stability

p53-binding protein-1 (53BP1) is phosphorylated in response to DNA damage and rapidly relocalizes to presumptive sites of DNA damage along with Mre11 and the phosphorylated histone 2A variant, gamma-H2AX. 53BP1 associates with the BRCA1 tumor suppressor, and knock-down experiments with small interfering RNA have revealed a role for the protein in the checkpoint response to DNA damage. By generating mice defective in m53BP1 (m53BP1(tr/tr)), we have created an animal model to further explore its biochemical and genetic roles in vivo. We find that m53BP1(tr/tr) animals are growth-retarded and show various immune deficiencies including a specific reduction in thymus size and T cell count. Consistent with a role in responding to DNA damage, we find that m53BP1(tr/tr) mice are sensitive to ionizing radiation (gamma-IR), and cells from these animals exhibit chromosomal abnormalities consistent with defects in DNA repair. Thus, 53BP1 is a critical element in the DNA damage response and plays an integral role in maintaining genomic stability.     

Effect of electroacupuncture on pressor reflex during gastric distension

The effect of electroacupuncture (EA) on the reflex cardiovascular response induced by mechanical distension of the stomach was studied in ventilated male Sprague-Dawley rats anesthetized by ketamine and alpha-chloralose. Repeated balloon inflation of the stomach to produce 20 mmHg tension on the gastric wall induced a consistent rise in mean arterial pressure, while heart rate (372 +/- 22 beats/min) was unchanged. This response was reversed by transection of the splanchnic nerves. Bilateral application of EA (1-2 mA, 2 Hz) at Neiguan-Jianshi acupoints (pericardial meridian, Pe 5-6) over the median nerve for 30 min significantly decreased the pressor response from 33 +/- 6 to 18 +/- 4 mmHg (n = 7, P < 0.05). This effect began after 10 min of EA and continued for 40 min after termination of EA. EA at Zusanli-Shangquxu acupoints (stomach meridian, St 36-37) over the deep peroneal nerve similarly inhibited the pressor response. The effect lasted for 10 min after EA was stopped (n = 6, P < 0.05), while EA at Guangming-Xuanzhong acupoints (gallbladder meridian, GB 37-39) over the superficial peroneal nerve did not inhibit the pressor response. Naloxone injected intravenously (n = 6) immediately after termination of EA or administered by microinjection into the rostral ventrolateral medulla (rVLM) 25 min after initiation of EA (n = 6) reversed the inhibition by EA, suggesting an opiate mechanism, including the rVLM, was involved.     

Identification of chemosensory sensilla activating antennular grooming behavior in the Caribbean spiny lobster,Panulirus argus

Crustaceans such as crabs and lobsters clean or 'groom' their olfactory organ, the antennule, by wiping it through a pair of mouthpart appendages, the third maxillipeds. In the lobster, only a few chemicals found in prey extracts, especially glutamate, elicit grooming. Chemosensory input driving grooming is likely to be mediated via sensilla located on antennules and third maxillipeds. Chemosensory antennular sensilla are innervated by neurons with central projections either to the glomerular olfactory lobe (aesthetasc sensilla) or to non-glomerular antennular neuropils (nonaesthetasc sensilla). By selectively ablating the chemosensory sensilla on the antennules and the third maxillipeds we have determined that the aesthetascs are necessary and sufficient to drive grooming behavior. Chemosensory activation of antennular grooming behavior likely follows a 'labeled-line' model in that aesthetasc neurons tuned to glutamate provide adequate input via the olfactory lobe to motor centers in the brain controlling antennular movements.     

Modulation of HeLa cells spreading by the non-receptor tyrosine kinase ACK-2

The CDC42 regulated non-receptor tyrosine kinase ACK-2 has been associated with integrin signaling. In this report, the effect of ACK-2 on the modulation of cell spreading and motility was examined. HeLa cells expressing epitope-tagged wild type ACK-2 showed a slower rate of spreading on fibronectin when compared with untransfected cells. An ACK-2 protein lacking its SH3 domain was still capable of modulating HeLa cell spreading suggesting that its tyrosine kinase activity is sufficient to induce the observed phenotype. The ACK-2 effect on the rate of cell spreading did not involve inhibition of integrin-mediated activation of PI-3K signaling, since it did not alter membrane translocation of a GFP-PH-AKT domain (AKT pleckstrin homology domain) used as a reporter for PI-3K products induced by cell adhesion. The ACK-2 effect appears to be upstream from the adapter protein CrkII, since co-expression of CrkII and ACK-2 results in a neutralization of ACK-2 mediated effects on HeLa cell spreading. Similarly, co-expression of p130Cas, which interacts with the adapter protein CrkII, with ACK-2, also results in a partial reversion of the ACK-2 effects on cell spreading. CrkII mediated reversal of the ACK-2 induced phenotype requires the activity of the small GTPase, Rap1. Co-expression of ACK-2 and CrkII with a dominant negative form of Rap1 reverses the neutralization by CrkII suggesting that CrkII mediated activation of Rap1 is required. However, an active form of Rap1 is not sufficient to reverse the ACK-2 phenotype by itself. A role for Rac1 in ACK-2 effects was also established. An activated Rac1 protein neutralized the ACK-2 mediated inhibition of cell spreading. A direct measurement of cell motility by either a modified Boyden chamber or wounding assay demonstrates that ACK-2 overexpression increases the motility of the cells. These results suggest that ACK-2 modulates HeLa cells spreading upstream of pathways regulated by CrkII and that ACK-2 may regulate cell motility by controlling the activation of small GTPases such as Rap1 and Rac1.     

Further evidence that periodate cleavage of heparin occurs primarily through the antithrombin binding site

Porcine mucosal heparin was fragmented into low-molecular-weight (LMW) heparin by treatment of periodate-oxidized heparin with sodium hydroxide, followed by reduction with sodium borohydride and acid hydrolysis. Gradient polyacrylamide gel electrophoresis analysis showed a mixture of heparin fragments with an average size of eight disaccharide units. 1D 1H NMR showed two-thirds of the N-acetyl groups were lost on periodate cleavage, suggesting cleavage had occurred at the glucopyranosyluronic acid (GlcpA) and idopyranosyluronic acid (IdopA) residues located within and adjacent to the antithrombin III (ATIII) binding site. The N-acetyl glucopyranose (GlcpNAc) residue was lost on workup. The GlcpA residue, within the ATIII binding site, is on the non-reducing side of the N-sulfo, 3, 6-O-sulfo glycopyranosylamine (GlcpNS3S6S) residue. Thus, periodate cleaved heparin should be enriched in GlcpNS3S6S residues. Two-dimensional correlation spectroscopy (2D COSY) confirmed that LMW heparin prepared through periodate cleavage contained GlcpNS3S6S at its non-reducing end. As expected, this LMW heparin also showed reduced ATIII mediated anti-factor IIa and anti-factor Xa activities.