N-terminal cleavage of GSK-3 by calpain: a new form of GSK-3 regulation.
Although GSK-3 activity can be regulated by phosphorylation and through interaction with GSK-3-binding proteins, here we describe N-terminal proteolysis as a novel way to regulate GSK-3. When brain extracts were exposed to calcium, GSK-3 was truncated, generating two fragments of approximately 40 and 30 kDa, a proteolytic process that was inhibited by specific calpain inhibitors. Interestingly, instead of inhibiting this enzyme, GSK-3 truncation augmented its kinase activity. When we digested recombinant GSK-3 alpha and GSK-3beta protein with calpain, each isoform was cleaved differently, yet the truncated GSK-3 isoforms were still active kinases. We also found that lithium, a GSK-3 inhibitor, inhibits full-length and cleaved GSK-3 isoforms with the same IC(50) value. Calpain removed the N-terminal ends of His-tagged GSK-3 isoenzymes, and exposing cultured cortical neurons with ionomycin, glutamate, or N-methyl-d-aspartate led to the truncation of GSK-3. This truncation was blocked by the calpain inhibitor calpeptin, at the same concentration at which it inhibits calpain-mediated cleavage of NMDAR-2B and of p35 (the regulatory subunit of CDK5). Together, our data demonstrate that calpain activation produces a truncation of GSK-3 that removes an N-terminal inhibitory domain. Furthermore, we show that GSK-3 alpha and GSK-3beta isoenzymes have a different susceptibility to this cleavage, suggesting a means to specifically regulate these isoenzymes. These data provide the first direct evidence that calpain promotes GSK-3 truncation in a way that has implications in signal transduction, and probably in pathological disorders such as Alzheimer disease.
Goñi-Oliver
Paloma
P
Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientificas/Universidad Autónoma(CSIC/UAM), Fac. Ciencias, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
Lucas
José J
JJ
Avila
Jesús
J
Hernández
Félix
F
D000544:Alzheimer Disease / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism; D002154:Calpain / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D018698:Glutamic Acid / Q000737:chemistry; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D020128:Inhibitory Concentration 50; D007527:Isoenzymes / Q000737:chemistry; D051379:Mice; D008954:Models, Biological; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction
D007527:Isoenzymes; D011994:Recombinant Proteins; D018698:Glutamic Acid; D038362:Glycogen Synthase Kinase 3; D002154:Calpain