Phosphorylation, calpain proteolysis and tubulin binding of recombinant human tau isoforms

Brain Res. 1993 Feb 26;604(1-2):32-40. doi: 10.1016/0006-8993(93)90349-r.

Abstract

In this study, the phosphorylation, calpain hydrolysis and tubulin binding of three recombinant human tau isoforms were examined. The three isoforms used in these studies were tau with three (T3) or four (T4) tandemly repeated tubulin binding domains located in the carboxy-terminal half of the molecule; and tau with four-tandem repeats and a 58-amino acid insert in the amino terminus (T4L). Both cAMP-dependent protein kinase (cAMP-PK) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) readily phosphorylated the three human tau isoforms, although cAMP-PK phosphorylated them to a significantly greater extent than CaMKII. Phosphorylation of T3, T4 and T4L by cAMP-PK or CaMKII resulted in the slowed migration of the protein bands on sodium dodecyl sulfate-polyacrylamide gels and a shift of the isoelectric variants to more acidic positions on two-dimensional non-equilibrium pH gradient electrophoresis gels compared with controls. However, the phosphorylation-induced changes in the electrophoretic migration of the tau isoforms were unique for each kinase. Two-dimensional phosphopeptide maps and sequential phosphorylation experiments indicate that cAMP-PK phosphorylates sites in the human tau isoforms that are phosphorylated by CaMKII, as well as unique sites that are not phosphorylated by CaMKII. T3, T4 and T4L were hydrolyzed similarly by calpain; however, the calpain proteolysis of the recombinant tau isoforms was significantly faster than the proteolysis of human or bovine tau. Phosphorylation of the isoforms by either cAMP-PK or CaMKII did not alter the rate or extent of calpain proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Calcium-Calmodulin-Dependent Protein Kinases
  • Calpain / metabolism*
  • Electrophoresis, Polyacrylamide Gel
  • Humans
  • Immunoblotting
  • Kinetics
  • Phosphorylation
  • Protein Binding
  • Protein Kinases / metabolism*
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Tubulin / metabolism*
  • tau Proteins / genetics
  • tau Proteins / isolation & purification
  • tau Proteins / metabolism*

Substances

  • Recombinant Proteins
  • Tubulin
  • tau Proteins
  • Protein Kinases
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Calpain