Expression of autocrine motility factor/phosphohexose isomerase in Cos7 cells

Autocrine motility factor (AMF) is identical to the glycolytic enzyme phosp hohexose isomerase (PHI) and overexpression of AMF/PHI is associated with t umor malignancy. In order to study the overexpression of AMF/PHI, an HA-tag ged AMF construct was transiently transfected into Cos7 cells. Expression o f a tagged AMF-HA allowed us to determine that over a period of 16 hours on ly a small amount (0.1-1%) of total cellular AMF-HA was secreted into the c ell medium. Cell-associated AMF-HA was exclusively cytosolic as it could be completely extracted with Triton X-100 and concentrated within actin rich pseudopodial domains. Treatment of the cells with the glycolysis inhibitor oxamate disrupted the association of AMF-HA with actin concentrations demon strating that glycolysis regulates the formation of these AMF/PHI-associate d actin-rich protrusions. AMF/PHI is a well-characterized tumor cell secret ed cytokine and we identify here an alternate intracellular function for th is glycolytic enzyme/cytokine in cell motility, (C) 2000 Academic Press.