T-cell receptor-mediated metabolism of inositol polyphosphates in Jurkat T-lymphocytes. Identification of a D-myo-inositol 1,2,3,4,6-pentakisphosphate-2-phosphomonoesterase activity, a D-myo-inositol 1,3,4,5,6-pentakisphosphate-1/3-phosphatase activity and a D/L-myo-inositol 1,2,4,5,6-pentakisphosphate-1/3-kinase activity.
Stimulation of the human T-lymphocyte cell line Jurkat via the T-cell receptor/CD3 complex by an anti-CD3 antibody (OKT3) induced time-dependent changes in the intracellular concentrations of multiple inositol polyphosphate (InsPn) isomers. Quantitative mass analysis by anion-exchange HPLC and a recently developed postcolumn dye system (Mayr, G. W. (1988) Biochem. J. 254, 585-591) revealed basal intracellular concentrations between less than 5 pmol/10(9) cells for Ins(1,3,4,5)P4 and 6380 +/- 355 pmol/10(9) cells for InsP6. Time course analysis of samples from stimulated Jurkat T-cells showed an increase of Ins(1,3,4,5)P4 to 1125 +/- 125 pmol/10(9) cells within 10 min and remained elevated over more than 30 min. Moreover, increases of the intracellular concentrations of Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, and/or Ins(3,4,5,6)P4 (determined as the enantiomeric mixture), Ins(1,3,4,5,6)P5, Ins(1,2,3,4,6)P5 and InsP6 were observed. In contrast, the concentration of Ins(1,2,4,5,6)P5 and/or Ins(2,3,4,5,6)P5 (determined as the enantiomeric mixture) decreased after stimulation. Using cytosolic extracts from Jurkat T-lymphocytes incubated with purified Ins(1,3,4,5,6)P5, Ins(1,2,3,4,6)P5, or Ins(1,2,4,5,6)P5/Ins(2,3,4,5,6)P5 three enzyme activities were observed. Ins(1,3,4,5,6)P5 was dephosphorylated by a phosphatase removing a phosphate group at the 1 and/or 3 position resulting in the formation of Ins(1,4,5,6)P4 and/or Ins(3,4,5,6)P4 (determined as the enantiomeric mixture). Ins(1,2,3,4,6)P5 was metabolized by a specific phosphatase that cleaved the phosphate group at the 2 position, thereby generating the product Ins(1,3,4,6)P4. On the other hand, Ins(1,2,4,5,6)P5/Ins(2,3,4,5,6)P5 was phosphorylated by a 1/3-kinase activity to InsP6. Together novel receptor-mediated metabolic pathways of inositol polyphosphates were demonstrated in human T-lymphocytes, and corresponding enzyme activities for the inositol pentakisphosphate metabolism were found in cell lysates.
Guse
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AH
Max-Planck-Society, Clinical Research Unit for Rheumatology/Immunology, Institute for Clinical Immunology, Erlangen, Germany.
Emmrich
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F
D000945:Antigens, Differentiation, T-Lymphocyte / Q000378:metabolism*; D017252:CD3 Complex; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D013601:T-Lymphocytes / Q000201:enzymology / Q000378:metabolism*
D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D007295:Inositol Phosphates; D011948:Receptors, Antigen, T-Cell; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C064990:inositol pentakisphosphate kinase; C072096:inositol 1,2,3,4,6-pentakisphosphate-2-phoshomonoesterase; C072095:inositol 1,3,4,5,6-pentakisphosphate-1-3-phosphatase; D010744:Phosphoric Monoester Hydrolases