title: Relative resistance of Cdk5-phosphorylated CRMP2 to dephosphorylation.
abstract: Collapsin response mediator protein 2 (CRMP2) binds to microtubules and regulates axon outgrowth in neurons. This action is regulated by sequential phosphorylation by the kinases cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3 (GSK3) at sites that are hyperphosphorylated in Alzheimer disease. The increased phosphorylation in Alzheimer disease could be due to increases in Cdk5 and/or GSK3 activity or, alternatively, through decreased activity of a CRMP phosphatase. Here we establish that dephosphorylation of CRMP2 at the residues targeted by GSK3 (Ser-518/Thr-514/Thr-509) is carried out by a protein phosphatase 1 family member in vitro, in neuroblastoma cells, and primary cortical neurons. Inhibition of GSK3 activity using insulin-like growth factor-1 or the highly selective inhibitor CT99021 causes rapid dephosphorylation of CRMP2 at these sites. In contrast, pharmacological inhibition of Cdk5 using purvalanol results in only a gradual and incomplete dephosphorylation of CRMP2 at the site targeted by Cdk5 (Ser-522), suggesting a distinct phosphatase targets this residue. A direct comparison of dephosphorylation at the Cdk5 versus GSK3 sites in vitro shows that the Cdk5 site is comparatively resistant to phosphatase treatment. The presence of the peptidyl-prolyl isomerase enzyme, Pin1, does not affect dephosphorylation of Ser-522 in vitro, in cells, or in Pin1 transgenic mice. Instead, the relatively high resistance of this site to phosphatase treatment is at least in part due to the presence of basic residues located nearby. Similar sequences in Tau are also highly resistant to phosphatase treatment. We propose that relative resistance to phosphatases might be a common feature of Cdk5 substrates and could contribute to the hyperphosphorylation of CRMP2 and Tau observed in Alzheimer disease.
authors.affiliation: Neurosciences Institute, Division of Pathology and Neuroscience, University of Dundee, Ninewells Hospital, Dundee, Scotland, UK.
authors.forename: Adam R
authors.initials: AR
authors.lastname: Cole
authors.forename: Marc P M
authors.initials: MP
authors.lastname: Soutar
authors.forename: Makoto
authors.initials: M
authors.lastname: Rembutsu
authors.forename: Lidy
authors.initials: L
authors.lastname: van Aalten
authors.forename: C James
authors.initials: CJ
authors.lastname: Hastie
authors.forename: Hilary
authors.initials: H
authors.lastname: McLauchlan
authors.forename: Mark
authors.initials: M
authors.lastname: Peggie
authors.forename: Martin
authors.initials: M
authors.lastname: Balastik
authors.forename: Kun Ping
authors.initials: KP
authors.lastname: Lu
authors.forename: Calum
authors.initials: C
authors.lastname: Sutherland
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D045744:Cell Line, Tumor; D051360:Cyclin-Dependent Kinase 5 / Q000378:metabolism*; D004351:Drug Resistance; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D019696:Peptidylprolyl Isomerase / Q000378:metabolism; D010766:Phosphorylation; D011687:Purines / Q000494:pharmacology; D051381:Rats; D016875:tau Proteins / Q000737:chemistry*
chemical_list: C471843:6-((3-chloro)anilino)-2-(isopropyl-2-hydroxyethylamino)-9-isopropylpurine; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D011687:Purines; D016875:tau Proteins; D051360:Cyclin-Dependent Kinase 5; C495905:CDK5 protein, human; D038362:Glycogen Synthase Kinase 3; C000606846:PIN1 protein, human; D019696:Peptidylprolyl Isomerase; C000606850:Pin1 protein, mouse