Browsing by Subject "Phosphotransferases (Alcohol Group Acceptor)"
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Item Functions of Phosphatidylinositol 4-Phosphate 5 Kinases in Actin Cytoskeletal Regulation during Phagocytosis(2009-06-18) Mao, Yuntao; Yin, Helen L.Phosphatidylinositol (4,5)-bisphosphate (PIP2) is a crucial signaling phosphoinositide at the plasma membrane (PM) which mediates a variety of biochemical activities and cellular functions. It is primarily synthesized by type I phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) through the phosphorylation on the D-5 position of the inositol ring of phatidylinositol 4-phosphate [PI(4)P]. Mammals have three PIP5K isoforms named a, b, and g (human isoform designation) which have a highly conserved central kinase homology domain and divergent amino and carboxyl terminal extensions. There is now extensive evidence suggesting that PIP5Ks have unique functions and regulations in many cellular processes which provide the key to understand how functionally, and possibly physically, segregated PIP2 pools are generated. The actin cytoskeleton is dynamically remodeled during Fcg receptor (FcgR)-mediated phagocytosis in a PIP2-dependent manner. I investigated the role of PIP5Kg and a isoforms, which synthesize PIP2, during phagocytosis. PIP5Kg-/- bone marrow-derived macrophages (BMM) have a highly polymerized actin cytoskeleton and are defective in attachment to IgG-opsonized particles and FcgR clustering. Delivery of exogenous PIP2 rescued these defects. PIP5Kg knockout BMM also have more RhoA and less Rac1 activation and pharmacological manipulations establish that they contribute to the abnormal phenotype. Likewise, depletion of PIP5Kg by RNA interference (RNAi) inhibits particle attachment. In contrast, PIP5Ka knockout or silencing has no effect on attachment but inhibits ingestion by decreasing Wiskott-Aldrich syndrome protein (WASP) activation and hence actin polymerization, in the nascent phagocytic cup. In addition, PIP5Kg but not a is transiently activated by spleen tyrosine kinase (Syk)-mediated phosphorylation. I propose that PIP5Kg acts upstream of Rac/Rho and that the differential regulation of PIP5Kg and a allows them to work in tandem to modulate the actin cytoskeleton during the attachment and ingestion phases of phagocytosis.Item Structural and Functional Study of the Type III Pantothenate Kinase from Thermotoga Maritima(2007-08-08) Yang, Kun; Zhang, HongCoenzyme A (CoA) is one of the most ubiquitous and essential cofactors in all living organisms. Pantothenate kinase (PanK) catalyzes the first step in the five-step universal pathway of CoA biosynthesis. Three types of PanK have been characterized so far. Prokaryotic PanK (PanK-I) and eukaryotic PanK (PanK-II) were identified previously. A third type of PanK (encoded by coaX gene) was identified by genetic complementation in 2005. PanK-III has a wider phylogenetic distribution than the long known PanK-I, and is nearly universally present in most of the major bacteria divisions, including many pathogenic bacteria. Different from the type I and type II PanKs, PanK-III is not feedback inhibited by CoA, and can not use pantothenamide antibiotics as substrate. In addition, PanK-III has a high Km for ATP (in the mM range) and requires a monovalent cation to have activity. The focus of my research is to unravel the underlying molecular basis for the unique enzymatic properties of PanK-III through crystallographic and other biochemical methods. I have solved the first crystal structure of PanK-III from Thermotoga maritima (TmCoaX). As the structure reveals, PanK-III belong to the acetate and sugar kinase/heat shocks protein 70/actin (ASKHA) protein superfamily, same as PanK-II, whereas PanK-I belongs to P-loop kinase superfamily. Recently, I also solved the crystal structures of two binary complexes of PanK-III with substrate pantothenate and product phospho-pantothenate, respectively, as well as a ternary complex of PanK-III with pantothenate and ADP. Combined with isothermal titration calorimetry, we present a detailed structural and thermodynamic characterization of the interactions between PanK-III and its substrates ATP and pantothenate. Comparison of substrate binding and catalytic sites of PanK-III with that of eukaryotic PanK-II revealed drastic differences in the binding modes of both ATP and pantothenate, even though both PanK-II and PanK-III belong to the same ASKHA superfamily and may share a common catalytic mechanism. In conclusion, our studies not only are important for understanding the fundamental metabolic pathways in PanK-III-harboring pathogenic bacteria, but also provide a structural basis for designing specific inhibitors.