Phosphorylation of lipocortins in vitro by protein kinase C

Protein kinase C catalyzes the incorporation of about 1.1, 0.7 and 0.4 mole of phosphate per mole of Lipocortin-I (P35), Lipocortin-II (P36) and Lipocortin-85 (P36 oligomer) respectively. The phosphorylation is specific for protein kinase C and is dependent on the presence of both calcium and phospholipids. While Lipocortin-I is phosphorylated on threonine residues, Lipocortin-II and Lipocortin-85 are phosphorylated on serine residues. The substoichiometric phosphorylation of Lipocortin-85 appears to preclude the potential regulation of this protein by protein kinase C. The phosphorylation of Lipocortin-I on threonine residues and Lipocortin-II on serine residues suggests these proteins may be regulated by distinct phosphorylation-dephosphorylation reactions.     

THE INFLUENCE OF EXCESS METHIONINE ON THE FREE AMINO ACIDS OF BRAIN AND LIVER OF THE WEANLING RAT*

Abstract— —High circulating levels of l -methionine produced by inclusion in the diet or parenteral injection of the amino acid caused alterations in the free amino acid pattern of liver and brain tissues. Acute effects following l -methionine injection were more pronounced than those following long term feeding where adaptation played a role. The net effect following parenteral injection was to increase the total free amino acids of liver while decreasing those of brain. Individually, hepatic levels of aspartic acid, threonine, serine, glutamine, glutamic acid, glycine, and alanine were depressed while levels of taurine, cystathionine, methionine, lysine, and ornithine were markedly elevated. Brain levels of aspartic acid, threonine, serine, glutamic acid, glycine, alanine, and γ-aminobutyric acid were markedly depressed and increased levels of cystathionine, methionine, lysine, and glutamine were observed. A generalized aminoaciduria occurred shortly after excessive methionine intake. Disruption of the free amino acid pools was of two kinds. The first depended on the continued presence of excess l -methionine, the second did not.     

Calcium-dependent regulation of actin filament bundling by lipocortin-85

Lipocortin-85 (L-85, calpactin-I/lipocortin-II heterotetramer) binds to F-actin in the presence of calcium with high affinity and in a cooperative manner. Quantitative analysis of binding curves indicate an apparent Kd (L-85) of 0.226 microM +/- 0.153 (2 S.D., n = 3), a stoichiometry of L-85/actin of 1:1.9 and a Hill coefficient of 1.37 +/- 0.14 (2 S.D., n = 3). Large anisotropic bundles were visualized by electron microscopy under these conditions, and quantitation of bundling by both low speed sedimentation and light scattering yielded apparent Kd values between 0.12 and 0.27 microM L-85. Filament bundling was dependent upon calcium, and the calcium sensitivity was increased by raising the molar ratio of lipocortin-85/F-actin. At saturating levels of L-85, apparent K0.5 values of 0.1-2 microM Ca2+f were obtained. The monomeric heavy chain, lipocortin-II, bundled F-actin to a much lesser extent and at much higher concentrations than for lipocortin-85. Bundling of F-actin by lipocortin-I was not detected at molar ratios of lipocortin-I to actin as high as 2.5 mol/mol (lipocortin-I/actin). At 5-10 microM Ca2+f and saturating levels of L-85, F-actin bundling progressed very rapidly with a t0.5 of 6 s. The process was quickly reversed by the addition of excess EGTA, and bundles could be reformed by the addition of a second burst of 5-10 microM Ca2+f. Thus, our data suggest that lipocortin-85 can rapidly regulate F-actin bundling in a calcium-dependent manner at physiologically relevant calcium levels.     

Studies of the Ca2+ transport mechanism of human erythrocyte inside-out plasma membrane vesicles. I. Regulation of the Ca2+ pump by calmodulin

Calcium accumulation by human erythrocyte inside-out vesicles was linear for at least 30 min in the presence of ATP. In untreated inside-out vesicles, 3.76 +/- 1.44 nmol of calcium/min/unit of acetylcholinesterase were transported, compared with 10.57 +/- 2.05 (+/- S.D.; n = 11) in those treated with calmodulin. The amount of calmodulin necessary for 50% activation of Ca2+ accumulation was 60 +/- 22 ng/ml (+/- S.D.; n = 4). The Km (Ca2+) for calmodulin-stimulated accumulation was 0.8 +/- 0.05 microM (+/- S.D.; n = 5) using Ca2+ /ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) buffers, or 25 microM with direct addition of unbuffered calcium. In the absence of calmodulin, these values were 0.4 and 60 microM, respectively, Km (ATP) values of 90 and 60 microM in the presence and absence of calmodulin, respectively, were measured at constant magnesium concentration (3 mM). In the presence of calmodulin, a broad pH profile is exhibited from pH 6.6 to 8.2. Maximal calcium accumulation occurs at pH 7.8. In the absence of calmodulin, the pH profile exhibits a linear upward increase from pH 7.0 to 8.2. The (Ca2+-Mg2+)-ATPase activity, measured under identical conditions, was 2.40 +/- 0.72 nmol of Pi/min/unit of acetylcholinesterase in the untreated vesicles and 11.29 +/- 2.87 nmol of Pi/min/unit of acetylcholinesterase (+/- S.D.; n = 4) in calmodulin-treated vesicles. A stoichiometry of 1.6 Ca2+/ATP hydrolyzed was determined in the absence of calmodulin; in the presence of calmodulin, this ratio was decreased to 0.94 Ca2+/ATP hydrolyzed.     

Neurogenin 3+ cells contribute to β-cell neogenesis and proliferation in injured adult mouse pancreas

We previously showed that injury by partial duct ligation (PDL) in adult mouse pancreas activates Neurogenin 3 (Ngn3)⁺ progenitor cells that can differentiate to β cells ex vivo. Here we evaluate the role of Ngn3⁺ cells in β cell expansion in situ. PDL not only induced doubling of the β cell volume but also increased the total number of islets. β cells proliferated without extended delay (the so-called ‘refractory'' period), their proliferation potential was highest in small islets, and 86% of the β cell expansion was attributable to proliferation of pre-existing β cells. At sufficiently high Ngn3 expression level, upto 14% of all β cells and 40% of small islet β cells derived from non-β cells. Moreover, β cell proliferation was blunted by a selective ablation of Ngn3⁺ cells but not by conditional knockout of Ngn3 in pre-existing β cells supporting a key role for Ngn3⁺ insulin⁻ cells in β cell proliferation and expansion. We conclude that Ngn3⁺ cell-dependent proliferation of pre-existing and newly-formed β cells as well as reprogramming of non-β cells contribute to in vivo β cell expansion in the injured pancreas of adult mice.     

The Toxoplasma gondii rhoptry protein ROP18 is an Irga6‐specific kinase and regulated by the dense granule protein GRA7

In mice, avirulent strains (e.g. types II and III) of the protozoan parasite Toxoplasma gondii are restricted by the immunity‐related GTPase (IRG) resistance system. Loading of IRG proteins onto the parasitophorous vacuolar membrane (PVM) is required for vacuolar rupture resulting in parasite clearance. In virulent strain (e.g. type I) infections, polymorphic effector proteins ROP5 and ROP18 cooperate to phosphorylate and thereby inactivate mouse IRG proteins to preserve PVM integrity. In this study, we confirmed the dense granule protein GRA7 as an additional component of the ROP5/ROP18 kinase complex and identified GRA7 association with the PVM by direct binding to ROP5. The absence of GRA7 results in reduced phosphorylation of Irga6 correlated with increased vacuolar IRG protein amounts and attenuated virulence. Earlier work identified additional IRG proteins as targets of T. gondii ROP18 kinase. We show that the only specific target of ROP18 among IRG proteins is in fact Irga6. Similarly, we demonstrate that GRA7 is strictly an Irga6‐specific virulence effector. This identifies T. gondii GRA7 as a regulator for ROP18‐specific inactivation of Irga6. The structural diversity of the IRG proteins implies that certain family members constitute additional specific targets for other yet unknown T. gondii virulence effectors.