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 promotes microtubule assembly in vitro and has been shown to play a role in the development of axonal morphology. Tau can be phosphorylated in vitro by several kinases, some of which cause a change in the conformation and activities of tau. Here we report the consequences of converting two of the protein kinase A phosphorylation sites (positions 156 and 327), first to alanine to eliminate phosphorylation, and second to aspartate, to mimic phosphorylation. We show that a serine to aspartate mutation at position 327 results in a conformational change similar to that caused by phosphorylation of this residue. This mutation does not affect the activities of tau in microtubule assembly as compared with wild-type tau. However, an additional mutation at position 156 to 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 conformation and function of tau in vitro and in cells. The results provide evidence that microtubule growth and nucleation can be differentially affected by phosphorylation of individual residues in a region amino-terminally flanking the microtubule binding domain of tau.
Léger
J
J
Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Kempf
M
M
Lee
G
G
Brandt
R
R
D000818:Animals; D001224:Aspartic Acid; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003572:Cytochalasins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006493:Heparin / Q000378:metabolism; D006801:Humans; D008870:Microtubules / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D016716:PC12 Cells; D010766:Phosphorylation; D051381:Rats; D012694:Serine; D013329:Structure-Activity Relationship; D016875:tau Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D003572:Cytochalasins; D016875:tau Proteins; D001224:Aspartic Acid; D012694:Serine; D006493:Heparin; D017868:Cyclic AMP-Dependent Protein Kinases