PIP 3 BP is a phosphatidylinositol 3,4,5-trisphosphatebinding protein (PIP 3 BP) abundant in brain, containing a zinc finger motif and two pleckstrin homology (PH) domains. Staining of rat brain cells with anti-PIP 3 BP antibody and determination of localization of PIP 3 BP fused to the green fluorescent protein (GFP-PIP 3 BP) revealed that PIP 3 BP was targeted to the nucleus. Targeting was dependent on a putative nuclear localization signal in PIP 3 BP. Generation of PIP 3 in the nucleus was detected in H 2 O 2 -treated 293T cells, nerve growth factor (NGF)-treated PC12 cells, and platelet-derived growth factor (PDGF)-treated NIH 3T3 cells. Translocation of phosphatidylinositol 3-kinase (PI 3-kinase) to the nucleus and enhanced activity of PI 3-kinase in the nucleus fraction were observed after H 2 O 2 treatment of 293T cells, suggesting that PI 3-kinase can be activated in the nucleus as well as in the membrane after appropriate stimulation of the cells. Co-expression of the constitutively active PI 3-kinase with PIP 3 BP resulted in exportation of the protein from the nucleus to the cytoplasm, suggesting that PIP 3 BP can function as a PIP 3 -binding protein in the intact cells. These results imply that there may be an unknown function of PI 3-kinase in the nucleus.Phosphatidylinositol 3-kinase 1 is an enzyme that is activated immediately after growth factor or differentiation factor stimulation of the cells (1) and that generates second messengers, phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) and phosphatidylinositol 3,4-bisphosphate (PI 3,4-P 2 ) (2-5). These 3Ј-phosphorylated phosphoinositides can activate serine, threonine kinases such as PKB/Akt, PKCs, and PDKs (6 -9). They are also suggested to be involved in other events such as rearrangement of cytoskeleton and vesicle transport because these phenomena are sensitive to the PI 3-kinase inhibitors and dominant negative mutants of PI 3-kinase (10). Recently, it was reported that the 3Ј-phosphorylated phosphoinositides can activate guanine nucleotide exchanging factors of Rac and Arf, small G proteins involved in actin rearrangement and vesicle transport, respectively (11,12). Therefore, G proteins as well as kinases are downstream of PI 3-kinase.We have identified PIP 3 BP as a PIP 3 -binding protein, using a PIP 3 analogue column (13). It is abundant in brain, implying that it may be involved in the function of nerve systems. PIP 3 BP binds to PIP 3 but not to PI 3,4-P 2 or phosphatidylinositol 4,5-bisphosphate (PI 4,5-P 2 ). It has a zinc finger motif homologous to that of Arf-GTPase activating protein (GAP) and two PH domains. Both PH domains are shown to be involved in binding to PIP 3 . Another PIP 3 -binding protein, centaurin ␣, is highly homologous to PIP 3 BP (14). No GAP activity to Arf has been detected in either protein. Although the binding of centaurin ␣ and PIP 3 BP to PIP 3 was specific, the role of the protein is unclear. To address this question, we determined the intracellular localization by immunological techniques, using mono...
It has been proposed that the cortical actin filament networks act as a cortical barrier that must be reorganized to enable docking and fusion of the synaptic vesicles with the plasma membranes. We identified a novel neuron-associated developmentally regulated protein, designated as Nadrin. Expression of Nadrin is restricted to neurons and correlates well with the differentiation of neurons. Nadrin has a unique structure; it contains a GTPase-activating protein (GAP) domain for Rho family GTPases, a potential coiled-coil domain, and a succession of 29 glutamines. In vitro the GAP domain activates RhoA, Rac1, and Cdc42 GTPases. Expression of Nadrin in NIH3T3 cells markedly reduced the number of the actin stress fibers and the formation of the ruffled membranes, suggesting that Nadrin regulates actin filament reorganization. In PC12 cells, Nadrin colocalized with synaptotagmin in the neurite termini and also with cortical actin filaments in the subplasmalemmal regions. Expression of Nadrin or its mutant composed of the coiled-coil and GAP domain enhanced Ca 2؉-dependent exocytosis of PC12 cells, but a mutant lacking the GAP domain inhibited exocytosis. These results suggest that Nadrin plays a role in regulating Ca 2؉ -dependent exocytosis, most likely by catalyzing GTPase activity of Rho family proteins and by inducing the reorganization of the cortical actin filaments.
The specific rates of solvolysis in 80% ethanol and 97% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) for various tertiary alkyl chlorides having different steric requirement and experimental (FT ICR) gas-phase stabilities of the corresponding carbocations were determined. The experimental gas-phase stabilities were in good agreement with theoretical values computed at the MP2/6-311G(d,p) or the MP2/6-311G(d,p)//MP2/ 6-31G(d) level. The relation of differential activation Gibbs energy changes for solvolysis [δ∆G ‡ ) -RT ln(k/k o )] (relative to 1-chloroadamantane) vs the experimental gas-phase cation-stabilities ∆G°(relative to 1-adamantyl cation) was compared with the previously established similar relation for bridgehead systems. It was revealed that the solvolysis of tert-butyl chloride in 80% ethanol is nucleophilically assisted by 4-8 kcal mol -1 . The δ∆G ‡ vs ∆G°relation for heavily crowded 4-chloro-2,2,4,6,6-pentamethylheptane was found to be comparable to that of bridgehead compounds. The reversal of the ranking of stabilities of the tert-butyl cation and 1-adamantyl cation on going from the gas phase to aqueous solution was computationally assessed. The results agree with the fact that larger substituents around a cationic center increase the stability of the ion in the gas phase thanks to their larger polarizability, and that this effect is either offset or even dramatically reversed in solution.
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