Mobilization of the A-kinase N-myristate through an isoform-specific intermolecular switch

Although the catalytic (C) subunit of cAMP-dependent protein kinase is N-myristylated, it is a soluble protein, and no physiological role has been identified for its myristyl moiety. To determine whether the interaction of the two regulatory (R) subunit isoforms (RI and RII) with the N-myristylated C subunit affects its ability to target membranes, the effect of N-myristylation and the RI and RII subunit isoforms on C subunit binding to phosphatidylcholine/phosphatidylserine liposomes was examined. Only the combination of N-myristylation and RII subunit interaction produced a dramatic increase in the rate of liposomal binding. To assess whether the RII subunit also increased the conformational flexibility of the C subunit N terminus, the effect of N-myristylation and the RI and RII subunits on the rotational freedom of the C subunit N terminus was measured. Specifically, fluorescein maleimide was conjugated to Cys-16 in the N-terminal domain of a K16C mutant of the C subunit, and the time-resolved emission anisotropy was determined. The interaction of the RII subunit, but not the RI subunit, significantly increased the backbone flexibility around the site of mutation and labeling, strongly suggesting that RII subunit binding to the myristylated C subunit induced a unique conformation of the C subunit that is associated with an increase in both the N-terminal flexibility and the exposure of the N-myristate. RII subunit thus appears to serve as an intermolecular switch that disrupts of the link between the N-terminal and core catalytic domains of the C subunit to expose the N-myristate and poise the holoenzyme for interaction with membranes.

Ca2+-independent inhibition of inositol trisphosphate receptors by calmodulin: Redistribution of calmodulin as a possible means of regulating Ca2+ mobilization

The interactions between calmodulin, inositol 1,4,5-trisphosphate (InsP3), and pure cerebellar InsP3 receptors were characterized by using a scintillation proximity assay. In the absence of Ca2+, 125I-labeled calmodulin reversibly bound to multiple sites on InsP3 receptors and Ca2+ increased the binding by 190% ± 10%; the half-maximal effect occurred when the Ca2+ concentration was 184 ± 14 nM. In the absence of Ca2+, calmodulin caused a reversible, concentration-dependent (IC50 = 3.1 ± 0.2 μM) inhibition of [3H]InsP3 binding by decreasing the affinity of the receptor for InsP3. This effect was similar at all Ca2+ concentrations, indicating that the site through which calmodulin inhibits InsP3 binding has similar affinities for calmodulin and Ca2+-calmodulin. Calmodulin (10 μM) inhibited the Ca2+ release from cerebellar microsomes evoked by submaximal, but not by maximal, concentrations of InsP3. Tonic inhibition of InsP3 receptors by the high concentrations of calmodulin within cerebellar Purkinje cells may account for their relative insensitivity to InsP3 and limit spontaneous activation of InsP3 receptors in the dendritic spines. Inhibition of InsP3 receptors by calmodulin at all cytosolic Ca2+ concentrations, together with the known redistribution of neuronal calmodulin evoked by protein kinases and Ca2+, suggests that calmodulin may also allow both feedback control of InsP3 receptors and integration of inputs from other signaling pathways.

Division of Labor among the α6β4 Integrin, β1 Integrins, and an E3 Laminin Receptor to Signal Morphogenesis and β-Casein Expression in Mammary Epithelial Cells

Contact of cultured mammary epithelial cells with the basement membrane protein laminin induces multiple responses, including cell shape changes, growth arrest, and, in the presence of prolactin, transcription of the milk protein β-casein. We sought to identify the specific laminin receptor(s) mediating the multiple cell responses to laminin. Using assays with clonal mammary epithelial cells, we reveal distinct functions for the α6β4 integrin, β1 integrins, and an E3 laminin receptor. Signals from laminin for β-casein expression were inhibited in the presence of function-blocking antibodies against both the α6 and β1 integrin subunits and by the laminin E3 fragment. The α6-blocking antibody perturbed signals mediated by the α6β4 integrin, and the β1-blocking antibody perturbed signals mediated by another integrin, the α subunit(s) of which remains to be determined. Neither α6- nor β1-blocking antibodies perturbed the cell shape changes resulting from cell exposure to laminin. However, the E3 laminin fragment and heparin both inhibited cell shape changes induced by laminin, thereby implicating an E3 laminin receptor in this function. These results elucidate the multiplicity of cell-extracellular matrix interactions required to integrate cell structure and signaling and ultimately permit normal cell function.

Expression of a Hox gene, Cnox-2, and the division of labor in a colonial hydroid

We report the isolation and expression of the Hox gene, Cnox-2, in Hydractinia symbiolongicarpus, a hydrozoan displaying division of labor. We found different patterns of aboral-to-oral Cnox-2 expression among polyp polymorphs, and we show that experimental conversion of one polyp type to another is accompanied by concordant alteration in Cnox-2 expression. Our results are consistent with the suggestion that polyp polymorphism, characteristic of hydractiniid hydroids, arose via evolutionary modification of proportioning of head to body column.

Mobilization of MHC class I molecules from late endosomes to the cell surface following activation of CD34-derived human Langerhans cells

Langerhans cells are a subset of dendritic cells (DCs) found in the human epidermis with unique morphological and molecular properties that enable their function as “sentinels” of the immune system. DCs are pivotal in the initiation and regulation of primary MHC class I restricted T lymphocyte immune responses and are able to present both endogenous and exogenous antigen onto class I molecules. Here, we study the MHC class I presentation pathway following activation of immature, CD34-derived human Langerhans cells by lipopolysaccharide (LPS). LPS induces an increase in all components of the MHC class I pathway including the transporter for antigen presentation (TAP), tapasin and ERp57, and the immunoproteasome subunits LMP2 and LMP7. Moreover, in CD34-derived Langerhans cells, the rapid increase in expression of MHC class I molecules seen at the cell surface following LPS activation is because of mobilization of MHC class I molecules from HLA-DM positive endosomal compartments, a pathway not seen in monocyte-derived DCs. Mobilization of class I from this compartment is primaquine sensitive and brefeldin A insensitive. These data demonstrate the regulation of the class I pathway in concert with the maturation of the CD34-derived Langerhans cells and suggest potential sites for antigen loading of class I proteins.

Rac and Cdc42 GTPases control hematopoietic stem cell shape, adhesion, migration, and mobilization

Critical to homeostasis of blood cell production by hematopoietic stem/progenitor (HSC/P) cells is the regulation of HSC/P retention within the bone marrow microenvironment and migration between the bone marrow and the blood. Key extracellular regulatory elements for this process have been defined (cell–cell adhesion, growth factors, chemokines), but the mechanism by which HSC/P cells reconcile multiple external signals has not been elucidated. Rac and related small GTPases are candidates for this role and were studied in HSC/P deficient in Rac2, a hematopoietic cell-specific family member. Rac2 appears to be critical for HSC/P adhesion both in vitro and in vivo, whereas a compensatory increase in Cdc42 activation regulates HSC/P migration. This genetic analysis provides physiological evidence of cross-talk between GTPase proteins and suggests that a balance of these two GTPases controls HSC/P adhesion and mobilization in vivo.

Metabolic Bypass of the Tricarboxylic Acid Cycle during Lipid Mobilization in Germinating Oilseeds1 : Regulation of NAD+-Dependent Isocitrate Dehydrogenase Versus Fumarase

Biosynthesis of sucrose from triacylglycerol requires the bypass of the CO2-evolving reactions of the tricarboxylic acid (TCA) cycle. The regulation of the TCA cycle bypass during lipid mobilization was examined. Lipid mobilization in Brassica napus was initiated shortly after imbibition of the seed and proceeded until 2 d postimbibition, as measured by in vivo [1-14C]acetate feeding to whole seedlings. The activity of NAD+-isocitrate dehydrogenase (a decarboxylative enzyme) was not detected until 2 d postimbibition. RNA-blot analysis of B. napus seedlings demonstrated that the mRNA for NAD+-isocitrate dehydrogenase was present in dry seeds and that its level increased through the 4 d of the experiment. This suggested that NAD+-isocitrate dehydrogenase activity was regulated by posttranscriptional mechanisms during early seedling development but was controlled by mRNA level after the 2nd or 3rd d. The activity of fumarase (a component of the nonbypassed section of the TCA cycle) was low but detectable in B. napus seedlings at 12 h postimbibition, coincident with germination, and increased for the next 4 d. RNA-blot analysis suggested that fumarase activity was regulated primarily by the level of its mRNA during germination and early seedling development. It is concluded that posttranscriptional regulation of NAD+-isocitrate dehydrogenase activity is one mechanism of restricting carbon flux through the decarboxylative section of the TCA cycle during lipid mobilization in germinating oilseeds.

Common endocrine and genetic mechanisms of behavioral development in male and worker honey bees and the evolution of division of labor.

Temporal polyethism is a highly derived form of behavioral development displayed by social insects. Hormonal and genetic mechanisms regulating temporal polyethism in worker honey bees have been identified, but the evolution of these mechanisms is not well understood. We performed three experiments with male honey bees (drones) to investigate how mechanisms regulating temporal polyethism may have evolved because, relative to workers, drones display an intriguing combination of similarities and differences in behavioral development. We report that behavioral development in drones is regulated by mechanisms common to workers. In experiment 1, drones treated with the juvenile hormone (JH) analog methoprene started flying at significantly younger ages than did control drones, as is the case for workers. In experiment 2, there was an age-related increase in JH associated with the onset of drone flight, as in workers. In experiment 3, drones derived from workers with fast rates of behavioral development themselves started flying at younger ages than drones derived from workers with slower rates of behavioral development. These results suggest that endocrine and genetic mechanisms associated with temporal polyethism did not evolve strictly within the context of worker social behavior.

Lipoxygenase-catalyzed oxygenation of storage lipids is implicated in lipid mobilization during germination.

The etiolated germination process of oilseed plants is characterized by the mobilization of storage lipids, which serve as a major carbon source for the seedling. We found that during early stages of germination in cucumber, a lipoxygenase (linoleate: oxygen oxidoreductase, EC 1.13.11.12) form is induced that is capable of oxygenating the esterified fatty acids located in the lipid-storage organelles, the so-called lipid bodies. Large amounts of esterified (13S)-hydroxy-(9Z,11E)-octadecadienoic acid were detected in the lipid bodies, whereas only traces of other oxygenated fatty acid isomers were found. This specific product pattern confirms the in vivo action of this lipoxygenase form during germination. Lipid fractionation studies of lipid bodies indicated the presence of lipoxygenase products both in the storage triacylglycerols and, to a higher extent, in the phospholipids surrounding the lipid stores as a monolayer. The degree of oxygenation of the storage lipids increased drastically during the time course of germination. We show that oxygenated fatty acids are preferentially cleaved from the lipid bodies and are subsequently released into the cytoplasm. We suggest that they may serve as substrate for beta-oxidation. These data suggest that during the etiolated germination, a lipoxygenase initiates the mobilization of storage lipids. The possible mechanisms of this implication are discussed.

Pericellular mobilization of the tissue-destructive cysteine proteinases, cathepsins B, L, and S, by human monocyte-derived macrophages.

Human macrophages are believed to damage host tissues in chronic inflammatory disease states, but these cells have been reported to express only modest degradative activity in vitro. However, while examining the ability of human monocytes to degrade the extracellular matrix component elastin, we identified culture conditions under which the cells matured into a macrophage population that displayed a degradative phenotype hundreds of times more destructive than that previously ascribed to any other cell population. The monocyte-derived macrophages synthesized elastinolytic matrix metalloproteinases (i.e., gelatinase B and matrilysin) as well as cysteine proteinases (i.e., cathepsins B, L, and S), but only the cathepsins were detected in the extracellular milieu as fully processed, mature enzymes by either vital fluorescence or active-site labeling. Consistent with these observations, macrophage-mediated elastinolytic activity was not affected by matrix metalloproteinase inhibitors but could be almost completely abrogated by inhibiting cathepsins L and S. These data demonstrate that human macrophages mobilize cysteine proteinases to arm themselves with a powerful effector mechanism that can participate in the pathophysiologic remodeling of the extracellular matrix.