Identification of cellular target proteins for signaling cyclic phosphates.

Cyclic glycerophosphates and their deoxy analogs were previously found to induce intracellular tyrosine and threonine phosphorylation in Chinese hamster ovary (CHO) cells. Further studies have indicated that these compounds induce neuronal outgrowth in PC-12 cells, as well as elevation of the state of cellular differentiation in human breast cancer cell lines. The mechanism by which these cyclic phosphates operate is not yet fully delineated. Using an affinity labeling approach we probed for possible cyclic phosphate target proteins in CHO cells. A 170 kDa protein that was labeled by an affinity cyclic phosphate reagent was identified by mass spectrometry as the largest subunit of the eukaryotic initiation factor 3 (eIF3). Using In-Gel kinase assays allowed the detection of a approximately 70 kDa target kinase directly activated by cyclic phosphates. Identification of these proteins may provide a basis for deciphering the mechanisms, by which cyclic phosphates exert their effects.