CONVERSION OF SERINE TO ASPARTATE IMITATES PHOSPHORYLATION-INDUCED CHANGES IN THE STRUCTURE AND FUNCTION OF MICROTUBULE-ASSOCIATED PROTEIN-TAU

Microtubule-associated protein tau is a neuronal phosphoprotein that p romotes microtubule assembly in vitro and has been shown to play a rol e in the development of axonal morphology. Tau can be phosphorylated i n vitro by several kinases, some of which cause a change in the confor mation and activities of tau. Here we report the consequences of conve rting two of the protein kinase A phosphorylation sites (positions 156 and 327), first to alanine to eliminate phosphorylation, and second t o aspartate, to mimic phosphorylation. We show that a serine to aspart ate mutation at position 327 results in a conformational change simila r to that caused by phosphorylation of this residue. This mutation doe s not affect the activities of tau in microtubule assembly as compared with wild-type tau. However, an additional mutation at position 156 t o aspartate drastically decreases the microtubule nucleation activity of tau but does not affect the activity of tau to promote microtubule growth. All constructs are similarly bound to microtubules and promote process formation when expressed in cytochalasin-treated PC12 cells. We conclude that serine to aspartate mutations provide a useful system for analyzing the effect of individual phosphorylation sites on the c onformation and function of tau in vitro and in cells. The results pro vide evidence that microtubule growth and nucleation can be differenti ally affected by phosphorylation of individual residues in a region am ino-terminally flanking the microtubule binding domain of tau.