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.
Highlights
Collapsin response mediator protein 2 (CRMP2)3 was initially identified in a screen for molecules involved in collapsin-1 signaling pathways in Xenopus laevis oocytes; its name [1]
CRMP2 is hyperphosphorylated at the cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3 (GSK3) sites in brain tissue from human AD patients and in some mouse models of AD [15, 16]
PP1 Antagonizes GSK3 Phosphorylation of CRMP2—To determine the class of phosphatase(s) that most efficiently removes phosphate from Ser-518/Thr-514/Thr-509 of CRMP2, recombinant CRMP2 was first phosphorylated at Ser522 by Cdk5 in the presence of unlabeled ATP (Fig. 1A)
Summary
Materials—Production of CRMP2 cDNA constructs, GSTCRMP2 fusion proteins, and CRMP2 antibodies have been described previously [11, 14]. In Vitro Phosphatase Assays—For analysis of CRMP2-Ser522 dephosphorylation, GST-CRMP2 (2 M) was phosphorylated using Cdk (2.5 milliunits/l) in the presence of radiolabeled Mg-[␥-32P]ATP in buffer containing 50 mM Tris-HCl, pH 7.5, 0.03% (v/v) Brij-35, and 0.1% (v/v) -mercaptoethanol (30 °C, 1 h). Cdk activity was inhibited by the addition of purvalanol (50 M), phosphatase was added at the indicated amounts to phosphorylated CRMP2 (1 M) for up to 1 h at 30 °C. Co-immunoprecipitation in Vitro—Equal amounts (2.5 g) of recombinant PP1 and CRMP2 (non-phosphorylated or prephosphorylated by Cdk and GSK3) were incubated in 150 l of kinase buffer containing 100 nM calyculin A for 10 min at room temperature followed by 1 h at 4 °C in the presence of 25 l of a 50% slurry of glutathione-Sepharose. These were pooled, and protein concentrations were measured before snap-freezing in liquid nitrogen
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