A novel CDK5-dependent pathway for regulating GSK3 activity and kinesin-driven motility in neurons
2004; Springer Nature; Volume: 23; Issue: 11 Linguagem: Inglês
10.1038/sj.emboj.7600237
ISSN1460-2075
AutoresGerardo Morfini, Györgyi Szebenyi, Hannah J. Brown, Harish C. Pant, Gustavo Pigino, Scott R. DeBoer, Uwe Beffert, Scott T. Brady,
Tópico(s)Cancer-related Molecular Pathways
ResumoArticle20 May 2004free access A novel CDK5-dependent pathway for regulating GSK3 activity and kinesin-driven motility in neurons Gerardo Morfini Gerardo Morfini Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, IL, USA Marine Biological Laboratory, Woods Hole, MA, USA Search for more papers by this author Györgyi Szebenyi Györgyi Szebenyi Department of Cell Biology and Center for Basic Neuroscience, UT Southwestern, Dallas, TX, USA Search for more papers by this author Hannah Brown Hannah Brown Marine Biological Laboratory, Woods Hole, MA, USA Search for more papers by this author Harish C Pant Harish C Pant Marine Biological Laboratory, Woods Hole, MA, USA Laboratory of Neurochemistry, NINDS, Bethesda, MD, USA Search for more papers by this author Gustavo Pigino Gustavo Pigino Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, IL, USA Search for more papers by this author Scott DeBoer Scott DeBoer Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, IL, USA Search for more papers by this author Uwe Beffert Uwe Beffert Department of Molecular Genetics, UT Southwestern, Dallas, TX, USA Search for more papers by this author Scott T Brady Corresponding Author Scott T Brady Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, IL, USA Marine Biological Laboratory, Woods Hole, MA, USA Search for more papers by this author Gerardo Morfini Gerardo Morfini Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, IL, USA Marine Biological Laboratory, Woods Hole, MA, USA Search for more papers by this author Györgyi Szebenyi Györgyi Szebenyi Department of Cell Biology and Center for Basic Neuroscience, UT Southwestern, Dallas, TX, USA Search for more papers by this author Hannah Brown Hannah Brown Marine Biological Laboratory, Woods Hole, MA, USA Search for more papers by this author Harish C Pant Harish C Pant Marine Biological Laboratory, Woods Hole, MA, USA Laboratory of Neurochemistry, NINDS, Bethesda, MD, USA Search for more papers by this author Gustavo Pigino Gustavo Pigino Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, IL, USA Search for more papers by this author Scott DeBoer Scott DeBoer Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, IL, USA Search for more papers by this author Uwe Beffert Uwe Beffert Department of Molecular Genetics, UT Southwestern, Dallas, TX, USA Search for more papers by this author Scott T Brady Corresponding Author Scott T Brady Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, IL, USA Marine Biological Laboratory, Woods Hole, MA, USA Search for more papers by this author Author Information Gerardo Morfini1,2, Györgyi Szebenyi3, Hannah Brown2, Harish C Pant2,4, Gustavo Pigino1, Scott DeBoer1, Uwe Beffert5 and Scott T Brady 1,2 1Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, IL, USA 2Marine Biological Laboratory, Woods Hole, MA, USA 3Department of Cell Biology and Center for Basic Neuroscience, UT Southwestern, Dallas, TX, USA 4Laboratory of Neurochemistry, NINDS, Bethesda, MD, USA 5Department of Molecular Genetics, UT Southwestern, Dallas, TX, USA *Corresponding author. Anatomy and Cell Biology M/C 512, 808 S Wood St, University of Illinois at Chicago, Chicago, IL 60612, USA. Tel.: +1 312 996 6791; Fax: +1 312 413 0354; E-mail: [email protected] The EMBO Journal (2004)23:2235-2245https://doi.org/10.1038/sj.emboj.7600237 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction Mature axons lack protein synthesis machinery, so proteins and membrane components necessary for maintaining neuronal functions need to be transported from the cell body into axons by axonal transport (Brady, 1993). Fast anterograde axonal transport (FAT) delivers many different membrane proteins packaged in membrane-bounded organelles (MBOs) to specific subdomains within axons. For example, sodium channels are delivered to nodes of Ranvier, whereas synaptic vesicles are provided to presynaptic terminals. Specialized delivery of MBOs at biochemically and structurally distinct domains within neurons implies the existence of regulated targeting mechanisms (Morfini et al, 2001). The molecular motor kinesin is essential for fast anterograde transport of a variety of MBOs in neurons (Brady, 1995). Kinesin is a heterotetramer with two heavy and two light chains. Heavy chains (KHCs) are responsible for generation of force, comprising ATPase and microtubule (MT)-binding domains. Kinesin light-chain subunits (KLCs) are essential for kinesin binding to transported cargoes and are implicated in both cargo specificity (Cyr et al, 1991; Stenoien and Brady, 1997; Khodjakov et al, 1998; Tsai et al, 2000) and regulation of motility (Tsai et al, 2000; Morfini et al, 2001, 2002b). Molecular mechanisms for regulating kinesin-based motility have begun to emerge. Kinesin is a phosphoprotein in vivo (Hollenbeck, 1993; Morfini et al, 2001), so regulation of kinesin activity may involve specific protein kinases and phosphatases. Different kinesin functions may be affected by phosphorylating specific sites within kinesin (Donelan et al, 2002; Morfini et al, 2002b). One property of kinesin regulated by kinases in vivo is association with cargoes, and specific phosphorylation of KLCs leads to release of kinesin from MBOs (Morfini et al, 2002b). Glycogen synthase kinase 3 (GSK3) phosphorylates KLCs and promotes kinesin release from MBOs (Morfini et al, 2002b). GSK3 is a ubiquitous kinase with roles in the Wnt pathway, intracellular trafficking, adhesion and apoptosis (Frame and Cohen, 2001). Multiple mechanisms for inactivating GSK3 are known (Woodgett, 2001), but less is known about pathways for activation. GSK3 can interact with kinesin and inhibits fast anterograde (kinesin-based), but not retrograde (dynein-based), axonal transport in extruded squid axoplasm (Morfini et al, 2002b). This finding suggested that GSK3 must be inactive in much of the axon and that local pathways for modulating its activity must exist. Selective activation of GSK3 in specific neuronal compartments may allow delivery of specific cargo to discrete subcellular functional domains in neurons, but relatively little is known about localized regulation of GSK3 kinase activity. As with GSK3 activation, inhibiting CDK5 kinase in axons specifically reduced transport rates for anterograde, but not retrograde, FAT (Ratner et al, 1998). This was shown by pharmacological and biochemical manipulations of CDK5 activity. The targets and molecular basis for CDK5-mediated regulation of kinesin-driven motility were not defined, because kinesin lacks CDK5 consensus sites. One possibility was a functional link between CDK5 and GSK3. The present work identifies a novel mode of GSK3 activation in neurons within axonal compartments. A pathway is defined that links activities of CDK5 and protein phosphatase 1 (PP1) to GSK3. Inhibition of CDK5 leads to activation of PP1 and GSK3, resulting in the release of kinesin from MBOs. We propose that regulating the distribution and activities of one or more elements in this pathway allows neurons to deliver kinesin cargoes selectively to different subcellular domains. Results Inhibiting CDK5 reduces kinesin-based motility FAT was analyzed in extruded axoplasm from squid giant axons, an in vivo model for the study of FAT that was instrumental in the discovery of kinesins (Brady, 1985; Vale et al, 1985). Extruded axoplasms continue bidirectional MBO transport with properties essentially unchanged from intact axons (Brady et al, 1985). Video-enhanced microscopic techniques allow quantitative analysis of MBO movement for both directions of FAT. Typically, anterograde rates are 1.5–2.0 μm/s and retrograde rates are 1–1.2 μm/s in perfused axoplasms (see Figure 1A–C). These rates are maintained with little or no reduction for >1 h with control buffers (Brady et al, 1985). 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. Download figure Download PowerPoint Conventional kinesin is the most abundant kinesin family member in mammalian brain and squid axoplasm (Wagner et al, 1989) and is essential for MBO movement in axoplasm (Brady et al, 1990; Stenoien and Brady, 1997). Studies with olomoucine (Olo) (Ratner et al, 1998) first provided evidence that kinases were important for FAT regulation. Olo and roscovitine are two well-characterized inhibitors of cdc2-like kinases, with IC50 values determined for >28 different kinases (Vesely et al, 1994; Bain et al, 2003). Cdc2 kinase family members (cdc2/cyclin A, CDK5/P35, etc.) are particularly sensitive to Olo (IC50 <10 μM) and roscovitine (IC50 ⩽0.25 μM). The major cdc2-like kinase in mature, postmitotic neurons is CDK5 (Hellmich et al, 1992; Lew and Wang, 1995). Immunoblots detect CDK5 in squid axoplasm (Figure 1D), comparable to distributions reported in mammalian axons (Tsai et al, 1993; Pigino et al, 1997). Axoplasmic CDK5 is active (Takahashi et al, 1995) and perfusion of 1 μM active GST-CDK5/p25 into axoplasm did not affect vesicle motility (Figure 1A). Perfusion of 5 μM Olo (IC50 CDK5=3 μM) (Figure 1B and C; see also Ratner et al, 1998) or 0.7 μM roscovitine (IC50 ros=0.25 μM) (Figure 1C) into axoplasm significantly reduced anterograde, but not retrograde, transport. These studies show that active CDK5 is required for kinesin-based motility in axons. CDK5 inhibition increases KLC phosphorylation Specific inhibition of anterograde, but not retrograde, FAT by CDK5 suggested that kinesin-based motility is modulated by CDK5. However, recombinant CDK5/p25 did not phosphorylate either KHC or KLC purified from rat brain (Figure 1E). CDK5/P25 also failed to phosphorylate recombinant KHC and KLC (not shown); so potential CDK5 sites were not phosphorylated in purified rat brain kinesin. This suggested that CDK5 does not directly phosphorylate kinesin. Next we determined whether kinesin phosphorylation changed in cells when CDK5 was inhibited. Primary cortical neurons were radiolabeled and kinesin was immunoprecipitated from lysates. KHCs and KLCs were both phosphorylated (Figure 2A), but Olo treatment of cultured neurons increased KLC phosphorylation by 20% (Figure 2B). CDK5 activity in mammals requires activation by one of two activator subunits, p35 and p39 (Ko et al, 2001). To rule out nonspecific effects of Olo, we did metabolic labeling of cultured cortical neurons from mice lacking both p35 and p39 subunits (i.e. lacking CDK5 activity) As with Olo, KLC phosphorylation increased in double knockout (KO) neurons relative to wild type (Figure 2B). KLCs have multiple phosphorylation sites consistent with modification by different protein kinases. Some sites appear to be constitutively phosphorylated (Hollenbeck, 1993; Morfini et al, 2002b), but at least one has increased phosphorylation when CDK5 activity is reduced by Olo or absence of CDK5 activators. Kinesin is not a substrate for CDK5, so effects of reducing CDK5 activity on kinesin-driven motility and KLC phosphorylation must be indirect. 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. Download figure Download PowerPoint CDK5 inhibition results in GSK3 activation To determine whether CDK5 activates kinases in axoplasm, isolated axoplasm±5 μM Olo were incubated with [32P]ATP and histone H1, a substrate for many protein kinases in vitro. Phosphorylation of H1 and the major neurofilament subunits increased in Olo-treated axoplasm extracts (Figure 3A), so inhibiting CDK5 activates other protein kinase(s). To identify the kinases involved, peptide substrates for specific kinases (extracellular-activated kinase (ERK), casein kinase I and GSK3) were tested for an ability to block Olo-induced increases in H1 histone phosphorylation in axoplasm (Figure 3B). Only CREB phosphopeptide (CREBp), a specific GSK3 substrate (Wang et al, 1994b), significantly reduced phosphorylation of H1 histone (lane 5, Figure 3B). To evaluate GSK3 activity in Olo-treated axoplasm, kinase assays specific for GSK3 were performed. Samples treated with 5 μM Olo (Figure 3C) showed increased GSK3 activity relative to untreated axoplasms (P=0.001 in a pooled t-test). Neither Olo nor roscovitine affected GSK3 activity in kinase assays in vitro (Figure 3D), even at 50 μM Olo. Thus, inhibition of axonal CDK5 results in GSK3 activation. 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. Download figure Download PowerPoint CDK5 inhibition may also activate ERK (Wang et al, 1994b) and P21 kinase (PAK) (Nikolic et al, 1998). These observations prompted us to compare effects of ERK2, PAK and GSK3 on vesicle motility. Neither 100 nM of active ERK2 nor 100 nM of PAK catalytic fragment (Figure 3E and F) affected FAT in extruded axoplasm, suggesting that neither is a likely mediator for inhibition of kinesin motility. As with Olo, perfusion of active GSK3 at 10 nM profoundly inhibited anterograde FAT (Figure 3G). This effect correlates with phosphorylation of KLC and release of kinesin from transported cargo (Morfini et al, 2002b). Finally, in vitro kinase assays showed that GSK3, but not PAK or ERK2, directly phosphorylates KLCs (Figure 3H). Reduced anterograde FAT by CDK5 inhibition requires GSK3 activation Given that inhibiting CDK5 increases GSK3 activity and KLC phosphorylation, CDK5 and GSK3 could be part of a common pathway for regulating FAT. Previous studies showed that CREBp at 0.5 mM blocks the action of GSK3 on FAT (Morfini et al, 2002b). Co-perfusion of 5 μM Olo and 0.5 mM CREBp into axoplasm prevented inhibition of anterograde FAT by Olo (Figure 4A and B). Perfusion of 1 mM CREBp alone had no effect on either anterograde or retrograde transport (Figure 4B). Thus, GSK3 activation is required for reduced anterograde FAT due to CDK5 inhibition. 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. Download figure Download PowerPoint KLC phosphorylation by GSK3 promotes kinesin detachment from MBOs (Morfini et al, 2002b). To evaluate effects of CDK5 inhibition on kinesin association with MBOs, primary cultured cortical neurons were treated with vehicle or Olo. Cells were harvested and MBOs were isolated by subcellular fractionation. MBO-associated kinesin levels were significantly decreased in Olo-treated cortical neurons, but overall kinesin levels remained unchanged (Figure 4C), consistent with GSK3 activation. CDK5 inhibition leads to GSK3β Ser9 dephosphorylation Phosphorylations at Ser9 of GSK3β (Ser21 of GSK3α) and Tyr216 regulate GSK3 kinase activity (Wang et al, 1994a). Phosphorylation at Ser9 inactivates GSK3 and dephosphorylation activates it. Autophosphorylation at Tyr216 (Cole et al, 2004) also enhances GSK3 activity, but only if Ser9 is dephosphorylated (Wang et al, 1994a). Thus, one can follow activation of GSK3 by measuring either dephosphorylation of Ser9 or phosphorylation at Tyr216. Effects of CDK5 inhibition on GSK3β phosphorylation at these regulatory residues were analyzed in PC12 cells and cortical neurons. Immunoblots were probed for GSK3 phosphorylation at Ser9 or Tyr216. Addition of Olo, but not vehicle or iso-Olo, to PC12 cells or cortical neurons resulted in time-dependent dephosphorylation of phosphoSer9 (Figure 5A). Corresponding increases were seen in phosphorylation at Tyr216 (not shown), consistent with increased GSK3 activity. Parallel immunoblot analysis of tau, an endogenous substrate for GSK3 in neurons (Lovestone et al, 1996), showed increased immunoreactivity with pS396 and PHF-1, but not Tau-5, in Olo-treated cells. PHF-1 and pS396 recognize tau at phosphoepitopes modified by GSK3 (Bhat et al, 2003), whereas Tau-5 is unaffected by tau phosphorylation (Figure 5B). Interestingly, phosphorylation of tau at these sites is generally associated with tau not being bound to MTs. 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). Download figure Download PowerPoint CDK5 might directly phosphorylate GSK3β at Ser9, but no 32P was incorporated into recombinant wild-type or kinase-dead GSK3β incubated with CDK5/p25 in vitro (lanes 2–5, Figure 5C). This same CDK5/P25 was strongly active against histone H1 (lane 1, Figure 5C). CDK5/P25 also failed to phosphorylate and activate recombinant PKB, a kinase that phosphorylates GSK3 in vivo. No changes in PKB phosphorylation were observed in Olo-treated cells (not shown). These experiments suggested that CDK5 keeps a pool of GSK3β in an inactive Ser9-phosphorylated form by an indirect mechanism. CDK5 activity inhibits PP1 activity CDK5 inhibition could activate a protein phosphatase that dephosphorylates Ser9 and activates GSK3β. To test this, Olo and okadaic acid were co-perfused into axoplasm (Figure 6A). Okadaic acid is a strong inhibitor of PP1 and PP2 serine–threonine phosphatases (Hardie et al, 1991). Okadaic acid (50 μM) had no effect on kinesin motility (Bloom et al, 1993), but co-perfusion of 20 nM okadaic acid and Olo prevented inhibition of anterograde FAT (Figure 6A). This suggested that Olo effects on kinesin-based motility involved activation of a Ser–Thr protein phosphatase. Consistent with this, incubating cortical neurons with okadaic acid (Figure 6C) or cantharidin (not shown) increased GSK3β Ser9 phosphorylation. 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. Download figure Download PowerPoint IC50 values of okadaic acid for PP1 and PP2 are 10 and 0.1 nM respectively. So these studies did not identify which phosphatase type was activated by CDK5 inhibition. To determine this, inhibitor-2 (I2), a small polypeptide that inhibits PP1 with an IC50 of 2 nM but not PP2 (Cohen et al, 1988; Haystead et al, 1989), was co-perfused with Olo. I2 blocked inhibition of anterograde FAT by Olo (Figure 6B). PP1 also dephosphorylates GSK3β at Ser9 in vitro (Figure 6D), suggesting that PP1 mediates Olo effects on kinesin-based motility. To see if CDK5, GSK3 and PP1 interact, protein phosphatases were affinity purified by microcystin–Sepharose (Moorhead et al, 1994). Microcystin-, but not control Sepharose, pulled down GSK3 with PP1 from mouse brain extracts (Figure 6E). PP1 to GSK3 ratios were nearly equal in microcystin pulldowns but quite different in lysates (GSK3≫PP1), suggesting a pool of GSK3 in a PP1 complex. These results indicate that CDK5 inhibition increases GSK3β activity by activating PP1 to dephosphorylate GSK3β at Ser9. CDK5, PP1 and GSK3 colocalize at sites of active vesicle delivery Distribution of GSK3, PP1 and CDK5 was evaluated in cell bodies, neurites and growth cones, a main site for membrane addition in growing neurites (Craig et al, 1995). All three were detected in cell bodies, neuritic shafts and growth cones of cultured neurons (Figure 7A). At higher magnification (Figure 7C), CDK5 and GSK3 showed extensive overlap with PP1 in central areas of growth cones. Central region localization, a site where vesicles accumulate, is distinct from both MTs and microfilaments (Dailey and Bridgman, 1991; Morfini et al, 2002b). Immunoblot analysis of isolated growth cone particles (Pfenninger et al, 1983) confirmed the presence of CDK5, PP1 and active GSK3 in growth cones (Figure 7B). In summary, CDK5, PP1 and GSK3 exhibit a similar distribution in neurites and were found in neuronal compartments where delivery of membrane proteins actively takes place. 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. Download figure Download PowerPoint Discussion Nerve cell survival and proper function require efficient delivery of proteins from cell body to axonal and dendritic processes. Neurons require accurate membrane protein compartmentalization to function, but little is known about how axonal subdomains are generated and maintained. Mechanisms must exist to allow MT-dependent motors to deliver different types of MBOs as needed to specific subcellular compartments. Phosphorylation and dephosphorylation appear to regulate kinesin-based motility (Hollenbeck, 1990; Morfini et al, 2001; Donelan et al, 2002), and kinase/phosphatase activities relevant to kinesin function have been identified. GSK3 is implicated in the regulation of MBO delivery in neurons (Morfini et al, 2002b). Phosphorylation of KLC by GSK3 promotes removal of kinesin from its cargo. As a result, pathways leading to GSK3 activation are likely to inhibit kinesin-based motility. A major mode of GSK3 inactivation is phosphorylation of Ser9 in GSK3β or Ser21 in GSK3α. Ser9 phosphorylation can occur either through autophosphorylation (Wang et al, 1994a) or via the action of kinases, such as PKB/Akt (Woodgett, 1994). In addition, proteins without intrinsic enzymatic activity (i.e. AKAP220 and axin) act as scaffolds to bind GSK3 and limit its activity to a particular compartment or substrate (Ali et al, 2001). Multiple pathways for inactivating GSK3 and localizing its activity exist, but less is known about pathways for its activation. In adipocytes or epithelial cells, GSK3 may be constitutively active (Ali et al, 2001), but increased GSK3 activity in neurons leads to neurite retraction (Munoz-Montano et al, 1999), apoptosis (Lucas et al, 2001), increased embryonic lethality (Hoeflich et al, 2000) and behavioral abnormalities (Hernandez et al, 2002). This suggests that GSK3 activation in neuronal cells must be a transient and localized event. CDK5 activity is essential for proper neuronal function (Smith et al, 2001). Lack of CDK5 is embryonic lethal (Ohshima et al, 1996), and deletion of both normal CDK5 activators (p35 and p39) has a phenotype comparable to CDK5 deletion (Ko et al, 2001). Animals survive to adulthood when only one activator is eliminated (Chae et al, 1997), but have defects in neuronal migration and axon pathfinding. Sustained CDK5 activity is required for normal kinesin-driven motility in neurons (Ratner et al, 1998), vesicle transport from Golgi to neurites (Paglini et al, 2001) and neurite outgrowth
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