DIFFERENT PHOSPHORYLATED FORMS OF INOSITOLPHOSPHATE GLYCAN COULD BE INVOLVED IN THE TRANSFORMING GROWTH-FACTOR-BETA-1 (TGF-BETA-1) SIGNALING PATHWAY

Labelling with [H-3]glucosamine was used to prepare a transforming gro wth factor-beta 1 (TGF-beta 1)-sensitive glycosylphosphatidylinositol (GPI) from monolayer cultures of rabbit articular chondrocytes (RAC), which may be involved in control of the cell cycle. The polar headgrou p of this glycosylphosphatidylinositol was generated by both phosphati dylinositol-specific phospholipase C (PI-PLC) and pronase E digestion. The molecule emerged in only one peak on a Dowex AG1-X8 chromatogram, eluted at 0.1 N ammonium formate. In contrast, similar experiments pe rformed on cellular extract from cultures previously labelled with [H- 3]glucosamine displayed four radioactive peaks eluting at 0.1, 0.2, 0. 5 and 1 N ammonium formate, respectively. Evidence that the eluting po sition of these peaks was dependent on the number of phosphate residue s present in each fraction was demonstrated by both [P-32]phosphorus l abelling and change in the position of alkaline phosphatase-induced sh ift in elution volume. We also demonstrated that the GPI-derived inosi tolphosphate glycan (IPG) could be hyperphosphorylated into the cell u nder the action of a kinase whose activity was enhanced by TGF-beta 1 itself. We have also shown that all of these IPG forms could mimic the TGF-beta-induced increase of DNA replication rate of RAC, with a high er activity for peaks m and IV than peaks I and II.