A novel CDK5-dependent pathway for regulating GSK3 activity and kinesin-driven motility in neurons - PubMed (original) (raw)

A novel CDK5-dependent pathway for regulating GSK3 activity and kinesin-driven motility in neurons

Gerardo Morfini et al. EMBO J. 2004.

Abstract

Neuronal transmission of information requires polarized distribution of membrane proteins within axonal compartments. Membrane proteins are synthesized and packaged in membrane-bounded organelles (MBOs) in neuronal cell bodies and later transported to axons by microtubule-dependent motor proteins. Molecular mechanisms underlying targeted delivery of MBOs to discrete axonal subdomains (i.e. nodes of Ranvier or presynaptic terminals) are poorly understood, but regulatory pathways for microtubule motors may be an essential step. In this work, pharmacological, biochemical and in vivo experiments define a novel regulatory pathway for kinesin-driven motility in axons. This pathway involves enzymatic activities of cyclin-dependent kinase 5 (CDK5), protein phosphatase 1 (PP1) and glycogen synthase kinase-3 (GSK3). Inhibition of CDK5 activity in axons leads to activation of GSK3 by PP1, phosphorylation of kinesin light chains by GSK3 and detachment of kinesin from transported cargoes. We propose that regulating the activity and localization of components in this pathway allows nerve cells to target organelle delivery to specific subcellular compartments. Implications of these findings for pathogenesis of neurodegenerative diseases such as Alzheimer's disease are discussed.

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Figures

Figure 1

Sustained CDK5 activity is required for maintenance of kinesin-driven motility in axons. (A) Perfusion of active CDK5/p25 (1 μM) into axoplasm had no effect on either anterograde (dark) or retrograde (gray) FAT rates. (B) Perfusion of the CDK5 inhibitor Olo (5 μM) decreases anterograde, but not retrograde, FAT. (C) Average transport rates for anterograde and retrograde FAT in standard perfusion buffer (buf) or CDK5 inhibitors roscovitine (0.7 μM, ros), and Olo (5 μM, olo). Note inhibitory effect of CDK5 inhibitors on anterograde, but not retrograde, FAT. Differences (*) are significant at _P_⩽0.0001. (D) Anti-CDK5 detects endogenous CDK5 in squid optic lobe (OL) and isolated axoplasm (Ax). Rat brain (R) lysate is a positive control. (E) Kinesin is not directly phosphorylated by CDK5. Purified rat brain kinesin was incubated with recombinant casein kinase 2 (CK2 (1)) or CDK5/p25 (3). CDK5/p25 failed to phosphorylate kinesin despite displaying strong kinase activity toward H1 histone (5). CK2 phosphorylates both kinesin heavy (KHC) and light (KLC) chains (Donelan et al, 2002). Control reactions with CK2 (2) or GST-CDK5/P25 (4) alone are also shown. Longer exposures show P25 autophosphorylation.

Figure 2

Inhibiting CDK5 increases KLC phosphorylation. (A) Representative autoradiogram showing immunoprecipitated–radiolabeled kinesin from wild-type (Ctrl), Olo-treated (Olo) or p35/p39 KO primary cultures of cortical neurons. (B) Phosphoimager quantitation showed increased KLC phosphorylation in neurons derived from p35−/− and p39−/− double KO and wild-type mouse embryos treated with Olo. Differences were significant at _P_⩽0.01.

Figure 3

Inhibiting CDK5 activates GSK3. (A) Axoplasms were treated with DMSO (Ctrl) or 5 μM Olo and radiolabeled ATP using histone H1 (H1) as a phosphate acceptor. Autoradiogram shows that Olo increases H1 phosphorylation. Neurofilament heavy chain (NF220) and HMW neurofilament also exhibit increased phosphorylation. (B). Control (1) or Olo-treated (2–5) axoplasms prepared as in (A) were incubated with no peptide (1, 2), ERK peptide (3), CK1 peptide (4) or CREBp (5). Only CREBp prevented Olo-induced increases in histone H1 phosphorylation. (C) GSK3 kinase activity was measured in axoplasm extracts using CREBp as substrate. CREBp phosphorylation increased relative to control axoplasms (Axo) with Olo (Axo+Olo). Increase is significant (_P_=0.0017; pooled _t_-test (*)). (D) Effect of 50 μM Iso-Olo (Iso), 50 μM Olo (Olo), 3 μM roscovitine (Ros) and 100 mM LiCl on GSK3 phosphorylation of CREBp in vitro. Values are expressed as percent of GSK3 activity without inhibitors. Vesicle motility assays in isolated axoplasm show effects of PAK (E), ERK2 (F) and GSK3 (G) kinase activities on FAT. Note the specific inhibitory effect on anterograde FAT of GSK3, but not PAK or ERK2, similar to that of CDK5 inhibition. (H) Autoradiogram (P32) shows that GSK3, but not PAK or ERK2, phosphorylates KLCs. Immunoblot (WB) shows position of kinesin heavy (HCs) and light chains (LCs). Asterisk (*) indicates autophosphorylated GSK3.

Figure 4

GSK3 mediates reduced kinesin-driven motility due to CDK5 inhibition. (A) Co-perfusion of 0.5 mM CREBp and 5 μM Olo abrogates the effects of Olo on FAT. (B) Average transport rates are shown for anterograde and retrograde FAT with control buffer (buf), 1 mM CREBp (cr), 5 μM Olo (ol) and 5 μM Olo plus CREBp (crol). Note that CREBp blocks the effect of Olo on anterograde FAT. CREBp alone did not affect either direction of transport. Asterisk denotes P<0.001. (C) Inhibiting CDK5 in cortical neurons reduces the amount of kinesin associated with microsomes (MBOs) but not total kinesin (Total). Representative immunoblots of KHC with H2 and fluorescent secondary antibodies are shown. The _Y_-axis shows pixel values from Typhoon scans.

Figure 5

Inhibition of CDK5 leads to GSK3β Ser9 dephosphorylation/activation. (A) PC12 cells or primary cortical neurons were treated with iso-Olo (Iso) or Olo (Olo) for the indicated times (min). Samples were immunoblotted with total GSK3β or GSK3β Ser9p antibodies. (B) Immunoblots with PHF-1 and pS396 antibodies show increased tau phosphorylation at Ser396 in Olo-treated (Olo) cortical neurons relative to vehicle-treated (Ctrl) ones. These epitopes are GSK3 sites in vitro and in vivo. Similar levels of total tau (Tau-5) indicate equal protein loading. (C) CDK5 does not phosphorylate GSK3 Ser9. Histone H1 (lane 1), GSK3β wild type (lanes 2 and 4) or GSK3β kinase-dead (lanes 3 and 5) were incubated with (lanes 1–3) or without CDK5/25 (lanes 4–5). Autoradiogram shows that CDK5/p25 phosphorylated histone (H1) but not GSK3 (GSK3β). Note: Wild-type GSK3β autophosphorylation in lanes 2 and 4 does not increase in the presence of CDK5 (lane 2).

Figure 6

Reduced kinesin-driven motility due to inhibition of CDK5 depends on PP1 and PP1 activates GSK3. (A) Co-perfusion of Olo and 20 nM okadaic acid prevented inhibition of FAT by Olo. (B) Co-perfusion with 8 nM I2 also blocked Olo effects, suggesting that PP1 mediates Olo-induced inhibition of kinesin-driven motility. (C) Incubation of cortical neurons with 0, 5, 20 and 50 nM okadaic acid (lanes 1–4) shows dose-dependent increases in GSK3β Ser9 phosphorylation, suggesting a role for serine–threonine protein phosphatases in GSK3β regulation. (D) Recombinant PP1 can dephosphorylate Ser9 of GSK3β. Recombinant GSK3β was incubated for 30 min with 100 μM ATP to allow autophosphorylation. PP1 catalytic subunit (lanes 1 and 2) or vehicle (lanes 3 and 4) was added to autophosphorylated GSK3β and immunoblotted with total GSK3β or GSK3β pSer9 antibodies. (E) Immunoblots show that microcystin–Sepharose (Micr), but not control Sepharose (Ctrl), pulls down GSK3, suggesting association between GSK3 and phosphatases. Note an increase in the PP1/GSK3β ratio between rat brain lysate (Lys) and microcystin–Sepharose lanes.

Figure 7

CDK5, GSK3 and PP1 colocalize in the centers of axonal growth cones. (A) Double immunostainings for CDK5 and PP1 (left) and total GSK3 and PP1 (right) show co-enrichment of CDK5, PP1 and GSK3 in growth cones of cultured neurons. All three enzymes are abundant in cell bodies and found all along the neurites. (B) Purified growth cone particles (GCPs) and rat brain homogenates (Hom) were analyzed by immunoblot. High levels of GAP43 show enrichment in GCP components. GSK3, CDK5 and PP1 are all present at significant levels. In contrast, PP2A is not enriched relative to homogenates. (C) Higher magnification of CDK5/PP1 and GSK3/PP1 shows colocalization in the centers of actively extending growth cones.

Figure 8

Schematic of a pathway for regulation of GSK3 activity and kinesin function in axons by CDK5 and PP1. KLCs on MBOs have a priming phosphorylation making them substrates for GSK3, but GSK3 is inactive in axons with active CDK5 and suppressed PP1 activity. CDK5 is normally active in axons, but local inhibition of CDK5 activity (Olo) (Ratner et al, 1998) allows local activation of PP1 that dephosphorylates and activates GSK3. PP1 inhibitors (okadaic acid, I2) blocked activation of GSK3. Further phosphorylation of KLCs by GSK3 makes kinesin subject to removal from MBOs by hsc70 and effects delivery of that cargo to an axonal domain. Kinesin phosphorylation is blocked by the addition of an inhibitor of GSK3 (CREBpp). Released kinesin is apparently degraded (Li et al, 1999). Consistent with this model, local changes in kinase/phosphatase activity were implicated in targeting Na channels to nodes of Ranvier (de Waegh et al, 1992).

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A novel CDK5-dependent pathway for regulating GSK3 activity and kinesin-driven motility in neurons - PubMed (original) (raw)