Gamma interferon (IFN-␥) induces both tyrosine and serine phosphorylation of Stat1. Stat1 serine phosphorylation is required for maximal transcriptional activity of Stat1. In this report, we present evidence that Stat1 tyrosine phosphorylation is not a prerequisite for Stat1 serine phosphorylation, although an active Jak2 kinase is required for both phosphorylation events. Stat1 serine phosphorylation occurs with a more delayed time course than tyrosine phosphorylation. The occurrence of serine phosphorylation without tyrosine phosphorylation suggests that serine phosphorylation takes place in the cytoplasm. Experiments performed with cells expressing either dominant-negative or constitutively active Ras protein indicated that the Ras-mitogenactivated protein kinase pathway is probably not involved in IFN-␥-induced Stat1 serine phosphorylation. Finally, a kinase capable of correct Stat1 serine phosphorylation was detected in partially purified cytoplasmic extracts from both IFN-␥-treated and untreated cells.Polypeptide signalling that changes gene expression often involves the STAT proteins (16). These latent cytoplasmic transcription factors become activated by phosphorylation of tyrosine residues, catalyzed either by receptor-associated Janus (Jak) kinases or by receptor tyrosine kinases. The STAT molecules then dimerize by reciprocal phosphotyrosine-SH2 interactions and enter the cell nucleus to effect transcriptional changes. It has been amply demonstrated that transcriptional activation is further amplified when the serine residues of STAT proteins are phosphorylated (22). A single serine in Stat3 and Stat1, residue 727 in both cases, appears to be the major, if not the only, serine kinase target site (21). In addition to these studies, a number of other findings suggestive of serine phosphorylation of STATs that are also tyrosine phosphorylated have been described. For example, Stat3 and Stat5 show a ligand-dependent, slower migration that is also time dependent and is inhibited by the serine kinase inhibitor H7 (2,3,24).The signalling pathway(s) involved in serine phosphorylation of the STATs is not known at present, although we do show here that Jak2 is required for STAT serine phosphorylation in response to gamma interferon (IFN-␥). In this study, we explored the time course and cellular locus of serine and tyrosine phosphorylation and the potential interdependence of the two. In addition, we examined the nature of the serine phosphorylation pathways, demonstrating the lack of evidence for participation of several prominent serine kinases. However, we did detect a kinase capable of catalyzing phosphorylation of Stat1, predominantly on residue 727. MATERIALS AND METHODSCell culture. U3A cells (13,14), ␥2A cells (12), and their derived cell lines were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% cosmic serum (HyClone Laboratories Inc.). NIH 3T3 cells and cells stably transfected with either dominant-negative Ras (dnRas) or constitutively active Ras (CARas) were grown in DMEM supple...

The glycolytic enzyme glucokinase plays an important role in the regulation of insuln secretion and recent studies have shown that mutations in the human glucokinas gene are a common cause of an autosomal domint form of non-inssulin-dependent (type 2) diabetes meilitus (NIIDDM) that has an onset often during childhood. The majority of the mutations that have been identified are missense mutations that result in the syntheis of a glucokinas molecule with an altered amino acid sequence. To characterize the effect of these mutations on the catalytic properties of human 3-cell glucokinase, we have expressed native and mutant forms of this protein in Eschenchia coli. AU of the missense mutations show chnges in enzyme activity including a decrease in V.. and/or increase in K. for glucose. Using a model for the threedimensional structure of human glucokinas based on the crystal structure of the related enzyme yeast hexokinae B, the mutations map primarily to two regions of the protein. One group of mutations is lcated in the active site cleft separating the two domains of the enzyme as wefl as in surface loops leading into this cleft. These mutations usually result in large reductions in enzyme activity. The second group of mutations is located far from the active site in a region that is predicted to undergo a substrate-induced conformational change that results in closure of the active site cleft. These mutations show a smail -2-fold reduction in V., and a 5-to 10-fold increase in K. for glucose. The characterization of mutations in glucokinase that are asted with a distinct and readily recognizable form of NIDDM has led to the identification of key amino acids involved in glucokinas catalysis and locaized functionaUly important regions of the glucokinas molecule.

Regulating the differentiation and persistence of encephalitogenic T cells is critical for the development of experimental autoimmune encephalomyelitis (EAE). We reported recently that CD5 has an engagement-dependent prosurvival activity in T cells that played a direct role in the induction and progression EAE. We predicted that CD5 regulates T cell apoptosis/survival through the activation of CK2, a prosurvival serine/threonine kinase that associates with the receptor. To test this hypothesis, we generated mice expressing CD5 with the inability to bind and activate CK2 and assessed their susceptibility to EAE. We found mice deficient in CD5-CK2 signaling pathway were mostly resistant to the development of EAE. Resistance to EAE was associated with a dramatic decrease in a population of effector infiltrating Th cells that coexpress IFN-γ and IL-17 and, to a lesser extent, cells that express IFN-γ or IL-17 in draining lymph nodes and spinal cords. We further show that T cells deficient in CD5-CK2 signaling hyperproliferate following primary stimulation; however, following restimulation, they rapidly develop nonresponsiveness and exhibit elevated activation-induced cell death. Our results provide a direct role for CD5-CK2 pathway in T cell activation and persistence of effector T cells in neuroinflammatory disease. This study predicts that targeting of IFN-γ+/IL-17+ infiltrating Th cells will be useful for the treatment of multiple sclerosis and other systemic autoimmune diseases.

Nephrotoxicity of colistin is the major factor limiting its clinical application. However, the exact mechanism of colistin-induced nephrotoxicity is still elusive. N 6 -Methyladenosine (m 6 A) modification has been implicated in many biological processes, however, its role in colistin-induced nephrotoxicity needs to be elucidated. Mouse renal tubular epithelial cells (mRTECs) were treated with 200 μM colistin with or without METTL3 overexpression. Cells injury, m 6 A assay, oxidative stress and apoptosis were examined. Levels of m 6 A are decreased after colistin treatment in mRTECs. METTL3 is the major factor involved in abnormal m 6 A modification. METTL3 overexpression plays a protective role against colistin-induced oxidative stress and apoptosis. Moreover, METTL3 interacts with the microprocessor protein DGCR8 and positively modulates miR-873-5p mature process in an m 6 A-dependent manner. Further experiments show that miR-873-5p could regulate Keap1-Nrf2 pathway against colistin-induced oxidative stress and apoptosis. These studies revealed an important role of METTL3/m 6 A in colistin-induced nephrotoxicity and provide a new insight on m 6 A modification in drug induced toxicity.

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