FIGURE 3.

Dyrk1A is proteolyzed by calcium-mediated truncation/activation of calpain I. A, calcium-activated proteolysis of calpain I and Dyrk1A in the human brain extract. Normal human brain frontal cortical extract was incubated at 30 °C for 10 min in the presence of 2.0 mm EDTA and various concentrations (0.00–2.14 mm) of CaCl₂. Then the incubated tissue extract was analyzed by Western blots developed with anti-calpain I or anti-Dyrk1A. B, selective inhibition of calcium-activated proteolysis of calpain I and Dyrk1A. Normal human frontal cortical extract was incubated at 30 °C for 10 min in the presence of 2.0 mm each of EDTA and CaCl₂ plus various selective protease inhibitors, as indicated in the figure, followed by Western blots probed with anti-calpain I or anti-Dyrk1A (8D9, against aa 91–151) to detect the proteolysis. An arrow indicates the full-length calpain I (top) or Dyrk1A (bottom); vertical lines indicate the truncated calpain I or Dyrk1A. Apr, aprotinin; Pep, pepstatin; Leu, leupeptin; Calp, calpestatin peptide. C, proteolysis of Dyrk1A by calpain I. Dyrk1A was immunoprecipitated by anti-His from HEK-293FT cells that overexpressed Dyrk1A and incubated with various concentration of calpain I in the presence of CaCl₂ for 10 min at 30 °C, followed by Western blot analyses. AD, AD brain; Con, control brain; HC, heavy chain of antibody; LC, light chain of antibody. D, depletion of the full-length but not truncated Dyrk1A from human brain extract by anti-His antibody. Human brain frontal cortical extracts from AD and control cases were incubated with anti-His precross-linked protein G-agarose beads overnight at 4 °C. The same volumes of the original brain extracts and of those after immunodepletion were subjected to Western blots developed with anti-Dyrk1A (8D9) and anti-GAPDH. The depletion of full-length Dyrk1A by anti-His from AD brain indicated cleavage of Dyrk1A upstream of the histidine domain.