GSK-3 mediates the okadaic acid-induced modification of collapsin response mediator protein-2 in human SK-N-SH neuroblastoma cells.
Collapsin response mediator protein-2 (CRMP-2), a phosphoprotein involved in axonal outgrowth and microtubule dynamics, is aberrantly phosphorylated in Alzheimer's disease (AD) brain. Alteration of glycogen synthase kinase-3 (GSK-3) activity is associated with the pathogenesis of AD. Here, we show that CRMP-2 is one of the major substrates for GSK-3 in pig brain extracts. Both GSK-3alpha and 3beta phosphorylate purified pig brain CRMP-2 and significantly alter its mobility in SDS-gels, resembling the CRMP-2 modification observed in AD brain. Interestingly, this modification can be detected in SK-N-SH neuroblastoma cells treated with a phosphatase inhibitor, okadaic acid (OA), and GSK-3 inhibitors completely block this OA-induced event. Knockdown of both GSK-3alpha and 3beta, but not either kinase alone, impairs OA-induced modification of CRMP-2. Mutation of Ser-518 or Ser-522 of CRMP-2, which are highly phosphorylated in AD brain, to Ala blocks the OA-induced modification of CRMP-2 in SK-N-SH cells. Ser-522 prephosphorylated by Cdk5 is required for subsequent GSK-3alpha-mediated phosphorylation of CRMP-2 in vitro. Collectively, our results demonstrate for the first time that OA can induce phosphorylation of CRMP-2 in SK-N-SH cells at sites aberrantly phosphorylated in AD brain, and both GSK-3alpha and 3beta and Ser-522 kinase(s) are involved in this process.
Ni
Mei-Hui
MH
Graduate Institute of Basic Medical Sciences, Medical College of Chang Gung University, Tao-Yuan, Taiwan, Republic of China.
Wu
Chih-Ching
CC
Chan
Wen-Hsiung
WH
Chien
Kun-Yi
KY
Yu
Jau-Song
JS
D000544:Alzheimer Disease / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism; D045744:Cell Line, Tumor; D051360:Cyclin-Dependent Kinase 5 / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000502:physiology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000502:physiology*; D009447:Neuroblastoma; D019319:Okadaic Acid / Q000494:pharmacology*; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D015398:Signal Transduction; D013552:Swine; D014020:Tissue Extracts / Q000378:metabolism
D036341:Intercellular Signaling Peptides and Proteins; D007527:Isoenzymes; D009419:Nerve Tissue Proteins; D014020:Tissue Extracts; C095114:collapsin response mediator protein-2; D019319:Okadaic Acid; D051360:Cyclin-Dependent Kinase 5; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3; C448021:glycogen synthase kinase 3 alpha; D010744:Phosphoric Monoester Hydrolases