Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin‐based motility

Membrane‐bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin‐based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule‐stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

[1]  G. Bloom,et al.  A Role for Cyclin-Dependent Kinase(s) in the Modulation of Fast Anterograde Axonal Transport: Effects Defined by Olomoucine and the APC Tumor Suppressor Protein , 1998, The Journal of Neuroscience.

[2]  R. Miller,et al.  Calmodulin binding to and cAMP-dependent phosphorylation of kinesin light chains modulate kinesin ATPase activity. , 1993, The Journal of biological chemistry.

[3]  G. Bloom,et al.  GTP , yS Inhibits Organelle Transport along Axonal Microtubules , 1993 .

[4]  P. Cohen,et al.  Insulin activates protein kinase B, inhibits glycogen synthase kinase‐3 and activates glycogen synthase by rapamycin‐insensitive pathways in skeletal muscle and adipose tissue , 1997, FEBS letters.

[5]  D. Hackney,et al.  Characterization of α2β2 and α2 forms of kinesin , 1991 .

[6]  M. Dailey,et al.  Structure and organization of membrane organelles along distal microtubule segments in growth cones , 1991, Journal of neuroscience research.

[7]  U. Wagner,et al.  Tau phosphorylation in transgenic mice expressing glycogen synthase kinase‐3β transgenes , 1997, Neuroreport.

[8]  N. Hirokawa,et al.  The activation of protein kinase A pathway selectively inhibits anterograde axonal transport of vesicles but not mitochondria transport or retrograde transport in vivo , 1995, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[9]  M. Kirschner,et al.  Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. , 1977, The Journal of biological chemistry.

[10]  G. Banker,et al.  Culturing nerve cells , 1998 .

[11]  R. Vallee.  A taxol-dependent procedure for the isolation of microtubules and microtubule-associated proteins (MAPs) , 1982, The Journal of cell biology.

[12]  P. Hollenbeck,et al.  Phosphorylation of Neuronal Kinesin Heavy and Light Chains In Vivo , 1993, Journal of neurochemistry.

[13]  N. Hirokawa,et al.  Kinesin and dynein superfamily proteins and the mechanism of organelle transport. , 1998, Science.

[14]  Akira Kikuchi,et al.  Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK‐3β and β‐catenin and promotes GSK‐3β‐dependent phosphorylation of β‐catenin , 1998 .

[15]  S. Brady,et al.  Immunochemical analysis of kinesin light chain function. , 1997, Molecular biology of the cell.

[16]  E. Krebs,et al.  Consensus sequences as substrate specificity determinants for protein kinases and protein phosphatases. , 1991, The Journal of biological chemistry.

[17]  S. Yang,et al.  The mechanism of activation of protein kinase FA (the activator of type-1 protein phosphatase) in brain synaptosomes. , 1992, Biochemical and biophysical research communications.

[18]  S. Brady,et al.  Release of kinesin from vesicles by hsc70 and regulation of fast axonal transport. , 2000, Molecular biology of the cell.

[19]  Scott T. Brady,et al.  A novel brain ATPase with properties expected for the fast axonal transport motor , 1985, Nature.

[20]  M. Sheetz,et al.  Regulation of kinesin activity by phosphorylation of kinesin-associated proteins. , 1994, The Journal of biological chemistry.

[21]  J. Woodgett,et al.  Wingless inactivates glycogen synthase kinase‐3 via an intracellular signalling pathway which involves a protein kinase C. , 1996, The EMBO journal.

[22]  J. Howard,et al.  Kinesin’s tail domain is an inhibitory regulator of the motor domain , 1999, Nature Cell Biology.

[23]  T. Iwamoto,et al.  Lithium activates mammalian Na+/H+ exchangers: isoform specificity and inhibition by genistein , 2000, Pflügers Archiv.

[24]  E. Krebs,et al.  Increased glycogen synthase kinase-3 activity in diabetes- and obesity-prone C57BL/6J mice. , 1999, Diabetes.

[25]  J. Ávila,et al.  The inhibition of phosphatidylinositol‐3‐kinase induces neurite retraction and activates GSK3 , 2001, Journal of neurochemistry.

[26]  S. Brady,et al.  Approaches to study interactions between kinesin motors and membranes. , 2001, Methods in molecular biology.

[27]  S. Helmke,et al.  Axonal origin and purity of growth cones isolated from fetal rat brain. , 1996, Brain research. Developmental brain research.

[28]  G. Bloom,et al.  GTP gamma S inhibits organelle transport along axonal microtubules , 1993, The Journal of cell biology.

[29]  René Hen,et al.  Decreased nuclear β‐catenin, tau hyperphosphorylation and neurodegeneration in GSK‐3β conditional transgenic mice , 2001 .

[30]  J. Woodgett,et al.  cDNA cloning and properties of glycogen synthase kinase-3. , 1991, Methods in enzymology.

[31]  S. Seiler,et al.  Cargo binding and regulatory sites in the tail of fungal conventional kinesin , 2000, Nature Cell Biology.

[32]  G. Bloom,et al.  Fast axonal transport of kinesin in the rat visual system: functionality of kinesin heavy chain isoforms. , 1995, Molecular biology of the cell.

[33]  S. Somlo,et al.  Nerve growth cones isolated from fetal rat brain: Subcellular fractionation and characterization , 1983, Cell.

[34]  P. Greengard,et al.  Phosphorylation-dependent inhibition by synapsin I of organelle movement in squid axoplasm , 1989, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[35]  E. Masliah,et al.  Glycogen synthase kinase 3 alteration in Alzheimer disease is related to neurofibrillary tangle formation. , 1996, Molecular and chemical neuropathology.

[36]  P. Salinas,et al.  Dishevelled-1 Regulates Microtubule Stability: A New Function Mediated by Glycogen Synthase Kinase-3β , 2000 .

[37]  P. Cohen,et al.  Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B , 1995, Nature.

[38]  J. Habener,et al.  Phosphorylation of the cAMP response element binding protein CREB by cAMP-dependent protein kinase A and glycogen synthase kinase-3 alters DNA-binding affinity, conformation, and increases net charge. , 1998, Biochemistry.

[39]  Michael P. Sheetz,et al.  Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility , 1985, Cell.

[40]  D. M. Ferkey,et al.  GBP, an Inhibitor of GSK-3, Is Implicated in Xenopus Development and Oncogenesis , 1998, Cell.

[41]  Nikolaj Blom,et al.  PhosphoBase, a database of phosphorylation sites: release 2.0 , 1999, Nucleic Acids Res..

[42]  K. Imahori,et al.  Glycogen synthase kinase 3β is identical to tau protein kinase I generating several epitopes of paired helical filaments , 1993 .

[43]  G. Bloom,et al.  Monoclonal antibodies to kinesin heavy and light chains stain vesicle- like structures, but not microtubules, in cultured cells , 1989, The Journal of cell biology.

[44]  P. Roach,et al.  Use of a synthetic peptide as a selective substrate for glycogen synthase kinase 3. , 1994, Analytical biochemistry.

[45]  R A Roth,et al.  The Role of Glycogen Synthase Kinase 3β in Insulin-stimulated Glucose Metabolism* , 1999, The Journal of Biological Chemistry.

[46]  A. McDowall,et al.  Association of kinesin with characterized membrane-bounded organelles. , 1992, Cell motility and the cytoskeleton.

[47]  A. Craig,et al.  Preferential addition of newly synthesized membrane protein at axonal growth cones , 1995, Nature.

[48]  A. Hyman,et al.  Tumor Necrosis Factor Induces Hyperphosphorylation of Kinesin Light Chain and Inhibits Kinesin-Mediated Transport of Mitochondria , 2000, The Journal of cell biology.

[49]  D. Chuang,et al.  Lithium activates the serine/threonine kinase Akt-1 and suppresses glutamate-induced inhibition of Akt-1 activity in neurons. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[50]  N. Hirokawa,et al.  The phosphorylation of kinesin regulates its binding to synaptic vesicles. , 1992, The Journal of biological chemistry.

[51]  G. Drewes,et al.  Glycogen synthase kinase‐3 and the Alzheimer‐like state of microtubule‐associated protein tau , 1992, FEBS letters.

[52]  G. Bloom,et al.  A monoclonal antibody against kinesin inhibits both anterograde and retrograde fast axonal transport in squid axoplasm. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[53]  G. Bloom,et al.  Purification of kinesin from bovine brain and assay of microtubule-stimulated ATPase activity. , 1991, Methods in enzymology.

[54]  J. Behrens,et al.  Biochemical interactions in the wnt pathway. , 2000, Biochimica et biophysica acta.

[55]  S. Helmke,et al.  Growth cone enrichment and cytoskeletal association of non-receptor tyrosine kinases. , 1995, Cell motility and the cytoskeleton.

[56]  Scott T. Brady,et al.  Local modulation of neurofilament phosphorylation, axonal caliber, and slow axonal transport by myelinating Schwann cells , 1992, Cell.

[57]  Salinas Pc.  Wnt factors in axonal remodelling and synaptogenesis. , 1999 .

[58]  A. Depaoli-Roach,et al.  Glycogen synthase kinase-3 beta is a dual specificity kinase differentially regulated by tyrosine and serine/threonine phosphorylation. , 1994, The Journal of biological chemistry.

[59]  S. Brady,et al.  Assay of vesicle motility in squid axoplasm. , 1993, Methods in cell biology.

[60]  R. Lasek,et al.  Video microscopy of fast axonal transport in extruded axoplasm: a new model for study of molecular mechanisms. , 1985, Cell motility.

[61]  S. Brady,et al.  Regulation of Kinesin: Implications for Neuronal Development , 2001, Developmental Neuroscience.

[62]  A. Dahlström,et al.  Axonal transport and distribution of immunologically distinct kinesin heavy chains in rat neurons , 1999, Journal of neuroscience research.

[63]  P. Polakis.  Wnt signaling and cancer. , 2000, Genes & development.

[64]  T. Rapoport,et al.  Light Chain– dependent Regulation of Kinesin's Interaction with Microtubules , 1998, Journal of Cell Biology.

[65]  S. Taylor,et al.  Expression of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli. , 1989, The Journal of biological chemistry.

[66]  A. Saltiel,et al.  The Activation of Glycogen Synthase by Insulin Switches from Kinase Inhibition to Phosphatase Activation during Adipogenesis in 3T3-L1 Cells* , 1998, The Journal of Biological Chemistry.

[67]  D. Kimelman,et al.  Role of Glycogen Synthase Kinase-3β in Neuronal Apoptosis Induced by Trophic Withdrawal , 2000, The Journal of Neuroscience.

[68]  S. Brady.  Molecular motors in the nervous system , 1991, Neuron.

[69]  R. Garofalo,et al.  Insulin-like growth factor I receptors of fetal brain are enriched in nerve growth cones and contain a beta-subunit variant. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[70]  J. Woodgett.  Regulation and functions of the glycogen synthase kinase-3 subfamily. , 1994, Seminars in cancer biology.

[71]  J. Woodgett,et al.  Glycogen synthase kinase-3: functions in oncogenesis and development. , 1992, Biochimica et biophysica acta.