Protein kinases

Oxidant stress and injury is inherent in many human diseases such as ischemic vascular and respiratory diseases, heart failure, myocardial infarction, stroke, perinatal and placental insufficiencies, diabetes, cancer, and numerous psychiatric and neurodegenerative disorders. Finding novel therapeutics to combat the deleterious effects of oxidative stress is critical to create better therapeutic strategies for many conditions that have few treatment options. This study used the anoxia-tolerant fruit fly, Drosophila melanogaster, to investigate endogenous cellular protection mechanisms and potential interactions to determine their ability to regulate synaptic functional tolerance and cell survival during acute oxidative stress. The Drosophila larval neuromuscular junction (NMJ) was used to analyze synaptic transmission and specific motor axon contributions. Drosophila Schneider 2 (S2) cells were used to assess viability. Acute oxidative stress was induced using p harmacological paradigms that generate physiologically relevant oxidant species: mitochondrial superoxide production induced by sodium azide (NaN3) and hydroxyl radical formation via hydrogen peroxide (H2O2). A combination of genetic and pharmacological approaches were used to explore the hypothesis that endogenous protection mechanisms control cellular responses to stress by manipulating ion channel conductance and neurotransmission. Furthermore, this study analyzed a group of marine natural products, pseudopterosins, to identify compounds capable of modulating synaptic transmission during acute oxidative stress and potential novel neuromodulatory agents.

Anoxia is characterized by an absence of oxygen supply to a tissue (Dawson- Scully et al., 2010). Unlike humans, Drosophila melanogaster is an organism that can survive low oxygen levels for hours without showing any pathology (Lutz et al., 2003) Under anoxia, the fruit fly loses locomotive activity, resulting in an anoxic coma (Haddad et al., 1997). In this study we investigate the influence of five variables for anoxic tolerance in adult Drosophila: 1) anoxic environment (gas vs. drowning), 2) anoxia duration, 3) temperature (cold [3ÀC] or room temperature [21ÀC]), 4) age (young 2-9 days and old 35-39 days), and 5) PKG variation. Tolerance to anoxia is measured by the time of recovery and survival of the fruit fly from the anoxic coma. The results from this study show that short stress, low temperature, young age, and low PKG activity increased anoxic tolerance. Our findings will lay the foundation to investigate different variables, genes or pharmacological compounds that can modulate neuronal anoxic tolerance.

p-21-activated kinase 6 (PAK6) is a serine-threonine protein kinase originally identified as an Androgen Receptor (AR) interacting protein. In current study, we determined the subcellular localization of PAK6 through mutational analysis. We have found that the N-terminal CRIB domain is partly responsible for plasma membrane targeting, the region between amino acid residues #292 to #368 is functionally relevant to plasma membrane localization and that amino acid residues #119 through #190 are responsible for nuclear targeting of PAK6, in addition to a stretch of positively charged N-terminal residues (#2-#11) since mutants lacking this sequence mis-localizes to cytoplasm. In junction forming epithelial cells, PAK6 is demonstrated to co-localize with B-catenin at adherens junctions, suggesting that PAK6 is an activation-dependent event and that PAK6 translocates from plasma membrane to the cytoplasm in response activation via the PKA signal pathway.

Two protein tyrosine phosphatases, dual specificity phosphatase PVP and low molecular weight phosphatase WZB were purified and characterized. PVP was expressed as inclusion bodies and a suitable purification and refolding method was devised. Enzyme kinetics revealed that p-nitrophenylphosphate and (Sb(B-naphthyl phosphate were substrates with KM of 4.0mM and 8.1mM respectively. PVP showed no reactivity towards phosphoserine. Kinetic characterization of WZB showed that only pnitrophenylphosphate was a substrate with no affinity for Ç-naphthyl phosphate and phosphoserine. Optimal conditions for activity with PNPP were found at a pH of 5 with a KM of 1.1mM, kcat of 35.4s-1 and kcat/KM of 32.2s-1mM-1. Inhibition studies showed that phosphate, fluoride, and molybdate were competitive inhibitors with Ki of 3.2mM, 71.7mM, and 50.4(So(BM respectively and hydrogen peroxide abolished activity. Active site mutants of WZB Cys9Ser and Asp115Asn showed no activity.