Phosphorylation of elF-4E and initiation of protein synthesis in P19 embryonal carcinoma cells

Mitogenic stimulation of protein synthesis is accompanied by an increase in elF-4E phosphorylation. The effect on protein synthesis by induction of differentiation is less well known. We treated P19 embryonal carcinoma cells with the differentiating agent retinoic acid and found that protein synthesis increased during the first hour of addition. However, the phosphorylation state, as well as the turnover of phosphate on elF-4E, remained unchanged. Apparently, the change in protein synthesis after RA addition is regulated by another mechanism than elF-4E phosphorylation. By using P19 cells overexpressing the EGF receptor, we show that the signal transduction pathway that leads to phosphorylation of elF-4E is present in P19 cells; the EGF-induced change in phosphorylation of elF-4E in these cells is likely to be regulated by a change in elF-4E phosphatase activity. These results suggest that the onset of retinoic acid-induced differentiation is triggered by a signal transduction pathway which involves changes in protein synthesis, but not elF-4E phosphorylation. © 1995 Wiley-Liss, Inc.     

Multifunctional roles of the autoimmune disease-associated tyrosine phosphatase PTPN22 in regulating T cell homeostasis

The non-receptor tyrosine phosphatase PTPN22 has a vital function in inhibiting antigen-receptor signaling in T cells, while polymorphisms in the PTPN22 gene are important risk alleles in human autoimmune diseases. We recently reported that a key physiological function of PTPN22 was to prevent naïve T cell activation and effector cell responses in response to low affinity antigens. PTPN22 also has a more general role in limiting T cell receptor-induced proliferation. Here we present new data emphasizing this dual function for PTPN22 in T cells. Furthermore, we show that T cell activation modulates the expression of PTPN22 and additional inhibitory phosphatases. We discuss the implication of these findings for our understanding of the roles of PTPN22 in regulating T cell responses and in autoimmunity.     

Astrocyte Resilience to Oxidative Stress Induced by Insulin-like Growth Factor I (IGF-I) Involves Preserved AKT (Protein Kinase B) Activity

Disruption of insulin-like growth factor I (IGF-I) signaling is a key step in the development of cancer or neurodegeneration. For example, interference of the prosurvival IGF-I/AKT/FOXO3 pathway by redox activation of the stress kinases p38 and JNK is instrumental in neuronal death by oxidative stress. However, in astrocytes, IGF-I retains its protective action against oxidative stress. The molecular mechanisms underlying this cell-specific protection remain obscure but may be relevant to unveil new ways to combat IGF-I/insulin resistance. Here, we describe that, in astrocytes exposed to oxidative stress by hydrogen peroxide (H₂O₂), p38 activation did not inhibit AKT (protein kinase B) activation by IGF-I, which is in contrast to our previous observations in neurons. Rather, stimulation of AKT by IGF-I was significantly higher and more sustained in astrocytes than in neurons either under normal or oxidative conditions. This may be explained by phosphorylation of the phosphatase PTEN at the plasma membrane in response to IGF-I, inducing its cytosolic translocation and preserving in this way AKT activity. Stimulation of AKT by IGF-I, mimicked also by a constitutively active AKT mutant, reduced oxidative stress levels and cell death in H₂O₂-exposed astrocytes, boosting their neuroprotective action in co-cultured neurons. These results indicate that armoring of AKT activation by IGF-I is crucial to preserve its cytoprotective effect in astrocytes and may form part of the brain defense mechanism against oxidative stress injury.     

Structure of the Human TELO2-TTI1-TTI2 Complex

Phosphatidylinositol 3-kinase-related protein kinases (PIKKs) play critical roles in various metabolic pathways related to cell proliferation and survival. The TELO2-TTI1-TTI2 (TTT) complex has been proposed to recognize newly synthesized PIKKs and to deliver them to the R2TP complex (RUVBL1-RUVBL2-RPAP3-PIH1D1) and the heat shock protein 90 chaperone, thereby supporting their folding and assembly. Here, we determined the cryo-EM structure of the TTT complex at an average resolution of 4.2 Å. We describe the full-length structures of TTI1 and TELO2, and a partial structure of TTI2. All three proteins form elongated helical repeat structures. TTI1 provides a platform on which TELO2 and TTI2 bind to its central region and C-terminal end, respectively. The TELO2 C-terminal domain (CTD) is required for the interaction with TTI1 and recruitment of Ataxia-telangiectasia mutated (ATM). The N- and C-terminal segments of TTI1 recognize the FRAP-ATM-TRRAP (FAT) domain and the N-terminal HEAT repeats of ATM, respectively. The TELO2 CTD and TTI1 N- and C-terminal segments are required for cell survival in response to ionizing radiation.     

The role of lipid second messengers in aldosterone synthesis and secretion

Second messengers are small rapidly diffusing molecules or ions that relay signals between receptors and effector proteins to produce a physiological effect. Lipid messengers constitute one of the four major classes of second messengers. The hydrolysis of two main classes of lipids, glycerophospholipids and sphingolipids, generate parallel profiles of lipid second messengers: phosphatidic acid (PA), diacylglycerol (DAG), and lysophosphatidic acid versus ceramide, ceramide-1-phosphate, sphingosine, and sphingosine-1-phosphate, respectively. In this review, we examine the mechanisms by which these lipid second messengers modulate aldosterone production at multiple levels. Aldosterone is a mineralocorticoid hormone responsible for maintaining fluid volume, electrolyte balance, and blood pressure homeostasis. Primary aldosteronism is a frequent endocrine cause of secondary hypertension. A thorough understanding of the signaling events regulating aldosterone biosynthesis may lead to the identification of novel therapeutic targets. The cumulative evidence in this literature emphasizes the critical roles of PA, DAG, and sphingolipid metabolites in aldosterone synthesis and secretion. However, it also highlights the gaps in our knowledge, such as the preference for phospholipase D-generated PA or DAG, as well as the need for further investigation to elucidate the precise mechanisms by which these lipid second messengers regulate optimal aldosterone production.     

In vivo phosphorylation of proteins in Clostridium sphenoides

Abstract Cell contents of Clostridium sphenoides , labeled with [³²P]orthophosphate under strict anaerobic conditions, were analyzed by two-dimensional gel electrophoresis. Autoradiography of these gels demonstrated the presence of at least 15 ³²P-labeled protein species, of which M _r and iso-electric point were determined. Treatment of the radioactively labeled cell contents with alkaline phosphatase and acid phosphatase showed that all these proteins were modified by phosphorylation. These findings demonstrate for the first time the presence of phosphorproteins in a strictly anaerobic bacterium.     

Phosphorylation of Synaptic Membrane Glycoproteins: The Effects of Ca2+ and Calmodulin

Synaptic membranes were incubated with [gamma-32P]ATP, and glycoproteins were isolated by affinity chromatography on concanavalin A agarose. Glycoproteins accounted for 1.5-2.5% of the total 32P incorporated into synaptic membrane proteins. Ca2+ and calmodulin enhanced the phosphorylation of synaptic membrane glycoproteins approximately threefold. In the presence of Ca2+ and calmodulin, the rate of glycoprotein dephosphorylation was also increased three- to four-fold. Gel electrophoretic analysis identified several synaptic membrane glycoproteins that incorporated 32P, with the most highly labeled glycoprotein under basal phosphorylating conditions having an apparent Mr of 205,000 (gpiii). Ca2+ and calmodulin produced a marked increase in the phosphorylation of a glycoprotein with an apparent Mr of 180,000 (gpiv) and lesser increases in the labeling of three other glycoproteins. Membranes that had been labeled with [gamma-32P]ATP were extracted with Triton X-100 under conditions that yield a detergent-insoluble residue enriched in postsynaptic structures. The Triton X-100 insoluble residue accounted for 20-25% of the 32P associated with synaptic membrane glycoproteins. Gpiv and other glycoproteins, the phosphorylation of which was stimulated by calmodulin, were located exclusively in the Triton X-100 insoluble residue, whereas gpiii and other calmodulin-insensitive glycoproteins partitioned predominantly into the Triton X-100-soluble fraction. Phosphopeptide maps and phosphoamino acid analysis of gpiv isolated from synaptic membranes and a postsynaptic glycoprotein of apparent Mr of 180,000 (gp180) isolated from synaptic junctions indicated that the former protein was identical to the previously identified postsynaptic-specific gp180. In addition to phosphoserine and phosphothreonine, gpiv also contained phosphotyrosine, identifying it as a substrate for tyrosine-protein kinase as well as for Ca2+/calmodulin-dependent protein kinase.