Elevated glycogen synthase kinase-3 (GSK-3) activity is associated with Alzheimer disease. We have found that collapsin response mediator proteins (CRMP) 2 and 4 are physiological substrates of GSK-3. The amino acids targeted by GSK-3 comprise a hyperphosphorylated epitope first identified in plaques isolated from Alzheimer brain. Expression of wild type CRMP2 in primary hippocampal neurons or SH-SY5Y neuroblastoma cells promotes axon elongation. However, a GSK-3-insensitive CRMP2 mutant has dramatically reduced ability to promote axon elongation, a similar effect to pharmacological inhibition of GSK-3. Hence, we propose that phosphorylation of CRMP proteins by GSK-3 regulates axon elongation. This work provides a direct connection between hyperphosphorylation of these residues and elevated GSK-3 activity, both of which are observed in Alzheimer brain.

Collapsin response mediator proteins (CRMPs) are a family of neuron-enriched proteins that regulate neurite outgrowth and growth cone dynamics. Here, we show that Cdk5 phosphorylates CRMP1, CRMP2, and CRMP4, priming for subsequent phosphorylation by GSK3 in vitro. In contrast, DYRK2 phosphorylates and primes CRMP4 only. The Cdk5 and DYRK2 inhibitor purvalanol decreases the phosphorylation of CRMP proteins in neurons, whereas CRMP1 and CRMP2, but not CRMP4, phosphorylation is decreased in Cdk5 ؊/؊ cortices. Stimulation of neuroblastoma cells with IGF1 or TPA decreases GSK3 activity concomitantly with CRMP2 and CRMP4 phosphorylation. Conversely, increased GSK3 activity is not sufficient to increase CRMP phosphorylation. However, the growth cone collapse-inducing protein Sema3A increases Cdk5 activity and promotes phosphorylation of CRMP2 (but not CRMP4). Therefore, inhibition of GSK3 alters phosphorylation of all CRMP isoforms; however, individual isoforms can be differentially regulated by their respective priming kinase. This is the first GSK3 substrate found to be regulated in this manner and may explain the hyperphosphorylation of CRMP2 observed in Alzheimer's disease.Glycogen synthase kinase 3 (GSK3) 4 is an evolutionarily conserved and ubiquitously expressed Ser/Thr kinase that is expressed as two closely related isoforms in mammals, GSK3␣ (51 kDa) and GSK3␤ (47 kDa) (1). GSK3 is unusual when compared with other protein kinases as it is constitutively active in cells and phosphorylation of most substrates must be preceded by phosphorylation of a nearby residue by another kinase. This process is known as priming and occurs at Ser/Thr residues located 4 or 5 residues C-terminal to the site phosphorylated by GSK3 (2, 3). GSK3 activity is inhibited by phosphorylation of an N-terminal serine residue (Ser 21 on GSK3␣ and Ser 9 on GSK3␤), which is catalyzed by members of the AGC family of protein kinases upon stimulation by growth factors (4, 5). Alternatively, GSK3 activity may be inhibited by protein-protein interactions following activation of the Wnt signaling pathway (6, 7). It is also possible that regulation of priming kinases could indirectly regulate phosphorylation of substrates by GSK3, although this has yet to be proven.We have recently discovered new brain-specific substrates for GSK3, namely collapsin response mediator protein (CRMP) 2 and 4 (3). These isoforms are members of a family of five CRMP proteins (CRMP1-5) that are expressed almost ubiquitously throughout the central nervous system (8, 9). CRMP2 is the best studied isoform of the family. Mammalian CRMP2 binds to tubulin heterodimers to promote microtubule formation and co-localizes with microtubules inside cells (10). Overexpression of CRMP2 in hippocampal neurons promotes increased axon elongation (3, 10, 11). However, mutation of the GSK3 phosphorylation sites on CRMP2 to non-phosphorylatable alanine residues alters CRMP2-induced axon elongation (3, 12). Other functions attributed to CRMP2 include regulation of cell surface receptor intern...

Phosphorylation of the endogenous GSK3alpha (glycogen synthase kinase-3alpha) at Tyr279 and GSK3beta at Tyr216 was suppressed in HEK-293 or SH-SY5Y cells by incubation with pharmacological inhibitors of GSK3, but not by an Src-family inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4- d ]pyrimidine (PP2), or a general protein tyrosine kinase inhibitor (genistein). GSK3beta transfected into HEK-293 cells or Escherichia coli became phosphorylated at Tyr216, but catalytically inactive mutants did not. GSK3beta expressed in insect Sf 21 cells or E. coli was extensively phosphorylated at Tyr216, but the few molecules lacking phosphate at this position could autophosphorylate at Tyr216 in vitro after incubation with MgATP. The rate of autophosphorylation was unaffected by dilution and was suppressed by the GSK3 inhibitor kenpaullone. Wild-type GSK3beta was unable to catalyse the tyrosine phosphorylation of catalytically inactive GSK3beta lacking phosphate at Tyr216. Our results indicate that the tyrosine phosphorylation of GSK3 is an intramolecular autophosphorylation event in the cells that we have studied and that this modification enhances the stability of the enzyme.