UNC-51/ATG1 kinase regulates axonal transport by mediating motor-cargo assembly.

Axonal transport mediated by microtubule-dependent motors is vital for neuronal function and viability. Selective sets of cargoes, including macromolecules and organelles, are transported long range along axons to specific destinations. Despite intensive studies focusing on the motor machinery, the regulatory mechanisms that control motor-cargo assembly are not well understood. Here we show that UNC-51/ATG1 kinase regulates the interaction between synaptic vesicles and motor complexes during transport in Drosophila. UNC-51 binds UNC-76, a kinesin heavy chain (KHC) adaptor protein. Loss of unc-51 or unc-76 leads to severe axonal transport defects in which synaptic vesicles are segregated from the motor complexes and accumulate along axons. Genetic studies show that unc-51 and unc-76 functionally interact in vivo to regulate axonal transport. UNC-51 phosphorylates UNC-76 on Ser(143), and the phosphorylated UNC-76 binds Synaptotagmin-1, a synaptic vesicle protein, suggesting that motor-cargo interactions are regulated in a phosphorylation-dependent manner. In addition, defective axonal transport in unc-76 mutants is rescued by a phospho-mimetic UNC-76, but not a phospho-defective UNC-76, demonstrating the essential role of UNC-76 Ser(143) phosphorylation in axonal transport. Thus, our data provide insight into axonal transport regulation that depends on the phosphorylation of adaptor proteins.

[1]  A. Ahantarig,et al.  Molecular characterization of Pegarn: a Drosophila homolog of UNC-51 kinase , 2009, Molecular Biology Reports.

[2]  A. Houtsmuller,et al.  Fluorescence resonance energy transfer of GFP and YFP by spectral imaging and quantitative acceptor photobleaching , 2008, Journal of microscopy.

[3]  Barry J Dickson,et al.  Identification of an axonal kinesin-3 motor for fast anterograde vesicle transport that facilitates retrograde transport of neuropeptides. , 2008, Molecular biology of the cell.

[4]  Nobutaka Hirokawa,et al.  Disruption of KIF17–Mint1 interaction by CaMKII-dependent phosphorylation: a molecular model of kinesin–cargo release , 2008, Nature Cell Biology.

[5]  Aaron DiAntonio,et al.  Control of a Kinesin-Cargo Linkage Mechanism by JNK Pathway Kinases , 2007, Current Biology.

[6]  D. Dickman,et al.  A Drosophila kinesin required for synaptic bouton formation and synaptic vesicle transport , 2007, Nature Neuroscience.

[7]  P. Hiesinger,et al.  Thirty-One Flavors of Drosophila Rab Proteins , 2007, Genetics.

[8]  T. Oliver,et al.  Unc-51-like kinase 1/2-mediated endocytic processes regulate filopodia extension and branching of sensory axons , 2007, Proceedings of the National Academy of Sciences.

[9]  T. P. Neufeld,et al.  Direct Induction of Autophagy by Atg1 Inhibits Cell Growth and Induces Apoptotic Cell Death , 2007, Current Biology.

[10]  Dawen Cai,et al.  Two binding partners cooperate to activate the molecular motor Kinesin-1 , 2007, The Journal of cell biology.

[11]  Y. Goshima,et al.  The autophagy-related kinase UNC-51 and its binding partner UNC-14 regulate the subcellular localization of the Netrin receptor UNC-5 in Caenorhabditis elegans , 2006, Development.

[12]  Masaaki Komatsu,et al.  Loss of autophagy in the central nervous system causes neurodegeneration in mice , 2006, Nature.

[13]  Hideyuki Okano,et al.  Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice , 2006, Nature.

[14]  W. Saxton,et al.  APLIP1, a Kinesin Binding JIP-1/JNK Scaffold Protein, Influences the Axonal Transport of Both Vesicles and Mitochondria in Drosophila , 2005, Current Biology.

[15]  Nobutaka Hirokawa,et al.  Analysis of the kinesin superfamily: insights into structure and function. , 2005, Trends in cell biology.

[16]  J. Rohrer,et al.  Drosophila Vps16A is required for trafficking to lysosomes and biogenesis of pigment granules , 2005, Journal of Cell Science.

[17]  E. V. van Munster,et al.  Fluorescence resonance energy transfer (FRET) measurement by gradual acceptor photobleaching , 2005, Journal of microscopy.

[18]  D. Price,et al.  Axonal Transport, Amyloid Precursor Protein, Kinesin-1, and the Processing Apparatus: Revisited , 2005, The Journal of Neuroscience.

[19]  Nobutaka Hirokawa,et al.  Molecular motors and mechanisms of directional transport in neurons , 2005, Nature Reviews Neuroscience.

[20]  M. Mann,et al.  Quantitative Phosphoproteomics Applied to the Yeast Pheromone Signaling Pathway*S , 2005, Molecular & Cellular Proteomics.

[21]  Kunihiro Matsumoto,et al.  The Caenorhabditis elegans UNC-14 RUN domain protein binds to the kinesin-1 and UNC-16 complex and regulates synaptic vesicle localization. , 2004, Molecular biology of the cell.

[22]  G. Garriga,et al.  The conserved kinase UNC-51 acts with VAB-8 and UNC-14 to regulate axon outgrowth in C. elegans , 2004, Development.

[23]  L. Goldstein,et al.  Microtubule-dependent transport in neurons: steps towards an understanding of regulation, function and dysfunction. , 2004, Current opinion in cell biology.

[24]  T. P. Neufeld,et al.  Role and regulation of starvation-induced autophagy in the Drosophila fat body. , 2004, Developmental cell.

[25]  T. Südhof.  The synaptic vesicle cycle , 2004 .

[26]  M. Hatten,et al.  Role of Unc51.1 and its binding partners in CNS axon outgrowth. , 2004, Genes & development.

[27]  Jinyun Chen,et al.  The kinesin-associated protein UNC-76 is required for axonal transport in the Drosophila nervous system. , 2003, Molecular biology of the cell.

[28]  Ronald D Vale,et al.  The Molecular Motor Toolbox for Intracellular Transport , 2003, Cell.

[29]  I. Meinertzhagen,et al.  Axonal Transport of Mitochondria to Synapses Depends on Milton, a Novel Drosophila Protein , 2002, Neuron.

[30]  I. Robinson,et al.  The C2B Ca2+-binding motif of synaptotagmin is required for synaptic transmission in vivo , 2002, Nature.

[31]  Nancy Ratner,et al.  Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin‐based motility , 2002, The EMBO journal.

[32]  R. Roncarati,et al.  Jun NH2-terminal Kinase (JNK) Interacting Protein 1 (JIP1) Binds the Cytoplasmic Domain of the Alzheimer's β-Amyloid Precursor Protein (APP)* , 2002, The Journal of Biological Chemistry.

[33]  Kei Ito,et al.  Embryonic and larval development of the Drosophila mushroom bodies: concentric layer subdivisions and the role of fasciclin II. , 2002, Development.

[34]  L. Goldstein,et al.  Kinesin-mediated axonal transport of a membrane compartment containing β-secretase and presenilin-1 requires APP , 2001, Nature.

[35]  Kunihiro Matsumoto,et al.  UNC-16, a JNK-Signaling Scaffold Protein, Regulates Vesicle Transport in C. elegans , 2001, Neuron.

[36]  J. Blenis,et al.  Cargo of Kinesin Identified as Jip Scaffolding Proteins and Associated Signaling Molecules , 2001, The Journal of cell biology.

[37]  L. Goldstein,et al.  Kinesin-Dependent Axonal Transport Is Mediated by the Sunday Driver (SYD) Protein , 2000, Cell.

[38]  L. Luo,et al.  Drosophila Lis1 is required for neuroblast proliferation, dendritic elaboration and axonal transport , 2000, Nature Cell Biology.

[39]  M. F. Stock,et al.  Kinesin’s IAK tail domain inhibits initial microtubule-stimulated ADP release , 2000, Nature Cell Biology.

[40]  G. Hauptmann,et al.  Multicolor whole-mount in situ hybridization. , 2000, Methods in molecular biology.

[41]  M. Hatten,et al.  A Mouse Serine/Threonine Kinase Homologous to C. elegans UNC51 Functions in Parallel Fiber Formation of Cerebellar Granule Neurons , 1999, Neuron.

[42]  W. Saxton,et al.  Cytoplasmic dynein, the dynactin complex, and kinesin are interdependent and essential for fast axonal transport. , 1999, Molecular biology of the cell.

[43]  Ronald D. Vale,et al.  Single-molecule analysis of kinesin motility reveals regulation by the cargo-binding tail domain , 1999, Nature Cell Biology.

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

[45]  K. White,et al.  Neuronal overexpression of APPL, the Drosophila homologue of the amyloid precursor protein (APP), disrupts axonal transport , 1999, Current Biology.

[46]  C. Tokunaga,et al.  Mammalian Homologue of the Caenorhabditis elegans UNC-76 Protein Involved in Axonal Outgrowth Is a Protein Kinase C ζ–interacting Protein , 1999, The Journal of cell biology.

[47]  D. Klionsky,et al.  Vacuolar import of proteins and organelles from the cytoplasm. , 1999, Annual review of cell and developmental biology.

[48]  L. Goldstein,et al.  Kinesin Light Chains Are Essential for Axonal Transport in Drosophila , 1998, The Journal of cell biology.

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

[50]  Y. Ohshima,et al.  The UNC-14 protein required for axonal elongation and guidance in Caenorhabditis elegans interacts with the serine/threonine kinase UNC-51. , 1997, Genes & development.

[51]  Lairdbloom Andh . Roberthorvitz.  The Caenorhabditis elegans gene unc-76 and its human homologs define a new gene family involved in axonal outgrowth and fasciculation , 1997 .

[52]  W. Saxton,et al.  Kinesin mutations cause motor neuron disease phenotypes by disrupting fast axonal transport in Drosophila. , 1996, Genetics.

[53]  R. Vallee,et al.  Targeting of Motor Proteins , 1996, Science.

[54]  N. Hirokawa,et al.  The neuron-specific kinesin superfamily protein KIF1A is a uniqye monomeric motor for anterograde axonal transport of synaptic vesicle precursors , 1995, Cell.

[55]  I. Mori,et al.  Caenorhabditis elegans unc-51 gene required for axonal elongation encodes a novel serine/threonine kinase. , 1994, Genes & development.

[56]  E. Suzuki,et al.  Immunolocalization of a Drosophila phosphatidylinositol transfer protein (rdgB) in normal and rdgA mutant photoreceptor cells with special reference to the subrhabdomeric cisternae. , 1994, Journal of electron microscopy.

[57]  S. Benzer,et al.  Paralysis and early death in cysteine string protein mutants of Drosophila. , 1994, Science.

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

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

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

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

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

[63]  E. Raff,et al.  Drosophila kinesin: characterization of microtubule motility and ATPase. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[64]  J. Culotti,et al.  Axonal guidance mutants of Caenorhabditis elegans identified by filling sensory neurons with fluorescein dyes. , 1985, Developmental biology.

[65]  P. Lawrence,et al.  Genes in development , 1977, Nature.