A RNA Interference Screen Identifies the Protein Phosphatase 2A Subunit PR55γ as a Stress-Sensitive Inhibitor of c-SRC

Protein Phosphatase type 2A (PP2A) represents a family of holoenzyme complexes with diverse biological activities. Specific holoenzyme complexes are thought to be deregulated during oncogenic transformation and oncogene-induced signaling. Since most studies on the role of this phosphatase family have relied on the use of generic PP2A inhibitors, the contribution of individual PP2A holoenzyme complexes in PP2A-controlled signaling pathways is largely unclear. To gain insight into this, we have constructed a set of shRNA vectors targeting the individual PP2A regulatory subunits for suppression by RNA interference. Here, we identify PR55γ and PR55δ as inhibitors of c-Jun NH₂-terminal kinase (JNK) activation by UV irradiation. We show that PR55γ binds c-SRC and modulates the phosphorylation of serine 12 of c-SRC, a residue we demonstrate to be required for JNK activation by c-SRC. We also find that the physical interaction between PR55γ and c-SRC is sensitive to UV irradiation. Our data reveal a novel mechanism of c-SRC regulation whereby in response to stress c-SRC activity is regulated, at least in part, through loss of the interaction with its inhibitor, PR55γ.     

Endocytic down-regulation of ErbB2 is stimulated by cleavage of its C-terminus

High ErbB2 levels are associated with cancer, and impaired endocytosis of ErbB2 could contribute to its overexpression. Therefore, knowledge about the mechanisms underlying endocytic down-regulation of ErbB2 is warranted. The C-terminus of ErbB2 can be cleaved after various stimuli, and after inhibition of HSP90 with geldanamycin this cleavage is accompanied by proteasome-dependent endocytosis of ErbB2. However, it is unknown whether C-terminal cleavage is linked to endocytosis. To study ErbB2 cleavage and endocytic trafficking, we fused yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) to the N- and C-terminus of ErbB2, respectively (YFP-ErbB2-CFP). After geldanamycin stimulation YFP-ErbB2-CFP became cleaved in nonapoptotic cells in a proteasome-dependent manner, and a markedly larger relative amount of cleaved YFP-ErbB2-CFP was observed in early endosomes than in the plasma membrane. Furthermore, cleavage took place at the plasma membrane, and cleaved ErbB2 was internalized and degraded far more efficiently than full-length ErbB2. Concordantly, a C-terminally truncated ErbB2 was also readily endocytosed and degraded in lysosomes compared with full-length ErbB2. Altogether, we suggest that geldanamycin leads to C-terminal cleavage of ErbB2, which releases the receptor from a retention mechanism and causes endocytosis and lysosomal degradation of ErbB2.     

Hyperglycemia and hyperlipidemia are associated with endothelial dysfunction during the development of type 2 diabetes

Diabetes mellitus impairs endothelial function, which can be considered as the hallmark in the development of cardiovascular diseases. Hyperglycemia, hyperinsulinemia, and hyperlipidemia are believed to contribute to endothelial dysfunction. In the present study, we investigated the possible links among these plasma metabolic markers and endothelial function in a mouse model during the development of type 2 diabetes. C57BL/6J-Lepob/ob mice at 8, 12, and 16 weeks were used to study endothelial function during the establishment of type 2 diabetes. Endothelial function was accessed in vitro in the thoracic aorta by measuring acetylcholine (ACh)-stimulated vasodilatation. Blood plasma was obtained for the measurements of glucose, insulin, triglycerides, and cholesterol levels. Correlation and multiple regression analysis revealed strong negative associations between the ACh responsiveness and the plasma levels of glucose, insulin, and lipid profiles at the age of 8 weeks. Associations were observed at neither older age nor in C57BL/6J mice. In conclusion, the increase in plasma levels of glucose, insulin, and lipids is associated with the impairment of the endothelial function during the early stage of the development of type 2 diabetes. The loss of correlation at an older age suggests multifactorial regulation of endothelial function and cardiovascular complications at later stages of the disease.     

Endogenous phosphotyrosine signaling in zebrafish embryos

In the developing embryo, cell growth, differentiation, and migration are strictly regulated by complex signaling pathways. One of the most important cell signaling mechanisms is protein phosphorylation on tyrosine residues, which is tightly controlled by protein-tyrosine kinases and protein-tyrosine phosphatases. Here we investigated endogenous phosphotyrosine signaling in developing zebrafish embryos. Tyrosine phosphorylated proteins were immunoaffinity-purified from zebrafish embryos at 3 and 5 days postfertilization and identified by multidimensional LC-MS. Among the identified proteins were tyrosine kinases, including Src family kinases, Eph receptor kinases, and focal adhesion kinases, as well as the adaptor proteins paxillin, p130Cas, and Crk. We identified several known and some unknown in vivo tyrosine phosphorylation sites in these proteins. Whereas most immunoaffinity-purified proteins were detected at both developmental stages, significant differences in abundance and/or phosphorylation state were also observed. In addition, multiplex in vitro kinase assays were performed by incubating a microarray of peptide substrates with the lysates of the two developmental stages. Many of the in vivo observations were confirmed by this on-chip in vitro kinase assay. Our experiments are the first to show that global tyrosine phosphorylation-mediated signaling can be studied at endogenous levels in complex multicellular organisms.     

The melanin-concentrating hormone (MCH) system modulates behaviors associated with psychiatric disorders

Deficits in sensorimotor gating measured by prepulse inhibition (PPI) of the startle have been known as characteristics of patients with schizophrenia and related neuropsychiatric disorders. PPI disruption is thought to rely on the activity of the mesocorticolimbic dopaminergic system and is inhibited by most antipsychotic drugs. These drugs however act also at the nigrostriatal dopaminergic pathway and exert adverse locomotor responses. Finding a way to inhibit the mesocorticolimbic- without affecting the nigrostriatal-dopaminergic pathway may thus be beneficial to antipsychotic therapies. The melanin-concentrating hormone (MCH) system has been shown to modulate dopamine-related responses. Its receptor (MCH1R) is expressed at high levels in the mesocorticolimbic and not in the nigrostriatal dopaminergic pathways. Interestingly a genomic linkage study revealed significant associations between schizophrenia and markers located in the MCH1R gene locus. We hypothesize that the MCH system can selectively modulate the behavior associated with the mesocorticolimbic dopamine pathway. Using mice, we found that central administration of MCH potentiates apomorphine-induced PPI deficits. Using congenic rat lines that differ in their responses to PPI, we found that the rats that are susceptible to apomorphine (APO-SUS rats) and exhibit PPI deficits display higher MCH mRNA expression in the lateral hypothalamic region and that blocking the MCH system reverses their PPI deficits. On the other hand, in mice and rats, activation or inactivation of the MCH system does not affect stereotyped behaviors, dopamine-related responses that depend on the activity of the nigrostriatal pathway. Furthermore MCH does not affect dizocilpine-induced PPI deficit, a glutamate related response. Thus, our data present the MCH system as a regulator of sensorimotor gating, and provide a new rationale to understand the etiologies of schizophrenia and related psychiatric disorders.     

Glycogen synthase kinase-3β is required for the induction of skeletal muscle atrophy

Skeletal muscle atrophy commonly occurs in acute and chronic disease. The expression of the muscle-specific E3 ligases atrogin-1 (MAFbx) and muscle RING finger 1 (MuRF1) is induced by atrophy stimuli such as glucocorticoids or absence of IGF-I/insulin and subsequent Akt signaling. We investigated whether glycogen synthase kinase-3β (GSK-3β), a downstream molecule in IGF-I/Akt signaling, is required for basal and atrophy stimulus-induced expression of atrogin-1 and MuRF1, and myofibrillar protein loss in C(2)C(12) skeletal myotubes. Abrogation of basal IGF-I signaling, using LY294002, resulted in a prominent induction of atrogin-1 and MuRF1 mRNA and was accompanied by a loss of myosin heavy chain fast (MyHC-f) and myosin light chains 1 (MyLC-1) and -3 (MyLC-3). The synthetic glucocorticoid dexamethasone (Dex) also induced the expression of both atrogenes and likewise resulted in the loss of myosin protein abundance. Genetic ablation of GSK-3β using small interfering RNA resulted in specific sparing of MyHC-f, MyLC-1, and MyLC-3 protein levels after Dex treatment or impaired IGF-I/Akt signaling. Interestingly, loss of endogenous GSK-3β suppressed both basal and atrophy stimulus-induced atrogin-1 and MuRF1 expression, whereas pharmacological GSK-3β inhibition, using CHIR99021 or LiCl, only reduced atrogin-1 mRNA levels in response to LY294002 or Dex. In conclusion, our data reveal that myotube atrophy and myofibrillar protein loss are GSK-3β dependent, and demonstrate for the first time that basal and atrophy stimulus-induced atrogin-1 mRNA expression requires GSK-3β enzymatic activity, whereas MuRF1 expression depends solely on the physical presence of GSK-3β.     

Trained immunity: a memory for innate host defense

Immune responses in vertebrates are classically divided into innate and adaptive, with only the latter being able to build up immunological memory. However, although lacking adaptive immune responses, plants and invertebrates are protected against reinfection with pathogens, and invertebrates even display transplant rejection. In mammals, past "forgotten" studies demonstrate cross-protection between infections independently of T and B cells, and more recently memory properties for NK cells and macrophages, prototypical cells of innate immunity, have been described. We now posit that mammalian innate immunity also exhibits an immunological memory of past insults, for which we propose the term "trained immunity." Understanding trained immunity will revolutionize our view of host defense and immunological memory, and could lead to defining a new class of vaccines and immunotherapies.     

The effect of potassium chloride on the Bohr effect of human hemoglobin.

The normal and differential titration curves of liganded and unliganded hemoglobin were measured at various KCl concentrations (0.1 to 2.0 M). In this range of KCl concentrations, the curves for deoxyhemoglobin showed no salt-induced pK changes of titratable groups. In the same salt concentration range oxyhemoglobin showed a marked change in titration behavior which could only be accounted for by a salt-induced increase in pK of some titratable groups. These results show that the suppression of the alkaline Bohr effect by high concentrations of neutral univalent salt is not caused by a weakening of the salt bridges in deoxyhemoglobin but is due to an interaction of chloride ions with oxyhemoglobin. Measurements of the Bohr effect at various KCl concentrations showed that at low chloride ion concentration (5 times 10-3 M) the alkaline Bohr effect is smaller than at a concentration of 0.1 M. This observation indicates that at a chloride ion concentration of 0.1 M, part of the alkaline Bohr effect is due to an interaction of chloride ions with hemoglobin. Furthermore, at low concentrations of chloride ions the acid Bohr effect has almost vanished. This result suggests that part of the acid Bohr effect arises from an interaction of chloride ions with oxyhemoglobin. The dependence of the Bohr effect upon the chloride ion concentration can be explained by assuming specific binding of chloride ions to both oxy- and deoxyhemoglobin, with deoxyhemoglobin having the highest affinity.     

UDP-N-acetylglucosamine 4'-epimerase from the intestinal protozoan Giardia intestinalis lacks UDP-glucose 4'-epimerase activity

The protozoan parasite Giardia intestinalis has a simple life cycle consisting of an intestinal trophozoite stage and an environmentally resistant cyst stage. The cyst is formed when a trophozoite encases itself within an external filamentous covering, the cyst wall, which is crucial to the cyst's survival outside of the host. The filaments in the cyst wall consist mainly of a beta (1-3) polymer of N-acetylgalactosamine. Its precursor, UDP-N-acetylgalactosamine, is synthesized from fructose 6-phosphate by a pathway of five inducible enzymes. The fifth, UDP-N-acetylglucosamine 4'-epimerase, epimerizes UDP-N-acetylglucosamine to UDP-N-acetylgalactosamine reversibly. The epimerase of G. intestinalis lacks UDP-glucose/UDP-galactose 4'-epimerase activity and shows characteristic amino acyl residues to allow binding of only the larger UDP-N-acetylhexosamines. While the Giardia epimerase catalyzes the reversible epimerization of UDP-N-acetylglucosamine to UDP-N-acetylgalactosamine, the reverse reaction apparently is favored. The enzyme has a higher Vmax and a smaller Km in this direction. Therefore, an excess of UDP-N-acetylglucosamine is required to drive the reaction towards the synthesis of UDP-N-acetylgalactosamine, when it is needed for cyst wall formation. This forms the ultimate regulatory step in cyst wall biosynthesis.