Mitogen-Activated Protein Kinases (Erk1,2) Phosphorylate Lys-Ser-Pro (KSP) Repeats in Neurofilament Proteins NF-H and NF-M

Mammalian neurofilament proteins, particularly midsized (NF-M) and heavy (NF-H) molecular weight neurofilament proteins, are highly phosphorylated in axons. Neurofilament function depends on the state of phosphorylation of the numerous serine/threonine residues in these proteins. Most phosphorylation occurs in the lys-ser-pro (KSP) repeats in the C-terminal tail domains of NF-H and NF-M. In our previous study, cyclin-dependent kinase 5 (cdk5) was shown to phosphorylate specifically the KSPXK repeats in rat NF-H. Because 80% of the repeats are of the KSPXXXK type, it was of interest to determine which kinase phosphorylates these motifs. Using a synthetic KSPXXXK peptide to screen for a specific kinase, we fractionated rat brain extracts by column chromatography and identified extracellular signal-regulated kinase (Erk2) activated by an upstream activator, the mitogen-activated protein kinase kinase MAPKK (MEK), by Western blot analysis, sequence identification, and inhibition by a specific MEK inhibitor (PD 98059). The fraction containing Erk2, as well as bacterially expressed Erk1 and Erk2, phosphorylated all types of KSP motifs in peptides (KSPXK, KSPXXK, KSPXXXK, and KSPXXXXK) derived from NF-M and NF-H. They also phosphorylated an expressed 24 KSPXXXK repeat NF-H polypeptide, an expressed NF-H as well as dephosphorylated native rat NF-H, and NF-M proteins with accompanying decreases in their respective electrophoretic mobilities. A comparative kinetic study of Erk2 and cdk5 phosphorylation of KSPXK and KSPXXXK peptides revealed that, in contrast to cdk5, which phosphorylated only the KSPXK peptide, Erk2 could phosphorylate both. The preferred substrate for Erk2 was KSPXXXK peptide. The MEK inhibitor PD 98059 also inhibited phosphorylation of NF-H, NF-M, and microtubule-associated protein (MAP) in primary rat hippocampal cells and caused a decrease in neurite outgrowth, suggesting that Erk1,2 may play an important role in neurite growth and branching. These data suggest that neuronal Erk1 and Erk2 are capable of phosphorylating serine residues in diverse KSP repeat motifs in NF-M and NF-H.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  B. Giasson,et al.  Study of Proline-Directed Protein Kinases Involved in Phosphorylation of the Heavy Neurofilament Subunit , 1997, The Journal of Neuroscience.

[3]  Veeranna,et al.  Phosphorylation of human high molecular weight neurofilament protein (hNF-H) by neuronal cyclin-dependent kinase 5 (cdk5) , 1997, Brain Research.

[4]  J. Julien,et al.  Neurofilaments and motor neuron disease. , 1997, Trends in cell biology.

[5]  G. Bennett,et al.  Identification of Ser‐Pro and Thr‐Pro Phosphorylation Sites in Chicken Neurofilament‐M Tail Domain , 1997, Journal of neurochemistry.

[6]  N. Ahn,et al.  Interdependent domains controlling the enzymatic activity of mitogen-activated protein kinase kinase 1. , 1996, Biochemistry.

[7]  B. Giasson,et al.  Aberrant Stress-induced Phosphorylation of Perikaryal Neurofilaments* , 1996, The Journal of Biological Chemistry.

[8]  Christopher C. J. Miller,et al.  Cellular phosphorylation of neurofilament heavy-chain by cyclin-dependent kinase-5 masks the epitope for monoclonal antibody N52 , 1996, Neuroscience Letters.

[9]  R. Liem,et al.  Phosphorylation of the High Molecular Weight Neurofilament Protein (NF-H) by Cdk5 and p35* , 1996, The Journal of Biological Chemistry.

[10]  L. Tsai,et al.  Differential Cellular Phosphorylation of Neurofilament Heavy Side‐Arms by Glycogen Synthase Kinase‐3 and Cyclin‐Dependent Kinase‐5 , 1996, Journal of neurochemistry.

[11]  M. Greenberg,et al.  Ca2+-Dependent Routes to Ras: Mechanisms for Neuronal Survival, Differentiation, and Plasticity? , 1996, Neuron.

[12]  Jerry H. Wang,et al.  Interaction of Cyclin-dependent Kinase 5 (Cdk5) and Neuronal Cdk5 Activator in Bovine Brain (*) , 1996, The Journal of Biological Chemistry.

[13]  G. Shaw,et al.  Localization of Sites in the Tail Domain of the Middle Molecular Mass Neurofilament Subunit Phosphorylated by a Neurofilament‐Associated Kinase and by Casein Kinase I , 1996, Journal of neurochemistry.

[14]  Philip R. Cohen,et al.  PD 098059 Is a Specific Inhibitor of the Activation of Mitogen-activated Protein Kinase Kinase in Vitro and in Vivo(*) , 1995, The Journal of Biological Chemistry.

[15]  E. Krebs,et al.  Association of mitogen-activated protein kinase with the microtubule cytoskeleton. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[16]  A. Bridges,et al.  A synthetic inhibitor of the mitogen-activated protein kinase cascade. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[17]  A. Saltiel,et al.  Inhibition of MAP Kinase Kinase Blocks the Differentiation of PC-12 Cells Induced by Nerve Growth Factor(*) , 1995, The Journal of Biological Chemistry.

[18]  Veeranna,et al.  Neuronal Cyclin‐Dependent Kinase‐5 Phosphorylation Sites in Neurofilament Protein (NF‐H) Are Dephosphorylated by Protein Phosphatase 2A , 1995, Journal of neurochemistry.

[19]  Paul N. Hoffman,et al.  Review : The Synthesis, Axonal Transport, and Phosphorylation of Neurofilaments Determine Axonal Caliber in Myelinated Nerve Fibers , 1995 .

[20]  H. Gainer,et al.  Identification of Endogenously Phosphorylated KSP Sites in the High‐Molecular‐Weight Rat Neurofilament Protein , 1994, Journal of neurochemistry.

[21]  A. Murray,et al.  A MAP kinase-dependent spindle assembly checkpoint in Xenopus egg extracts , 1994, Cell.

[22]  J. Trojanowski,et al.  Modulation of axon diameter and neurofilaments by hypomyelinating Schwann cells in transgenic mice , 1994, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[23]  R. Nixon,et al.  Phosphorylation on carboxyl terminus domains of neurofilament proteins in retinal ganglion cell neurons in vivo: influences on regional neurofilament accumulation, interneurofilament spacing, and axon caliber , 1994, The Journal of cell biology.

[24]  J. W. Gloor,et al.  Mitogen-activated protein (MAP) kinase phosphorylation of MAP kinase kinase: determination of phosphorylation sites by mass spectrometry and site-directed mutagenesis. , 1994, Journal of biochemistry.

[25]  J. H. Wang,et al.  Substrate specificity characterization of a cdc2-like protein kinase purified from bovine brain. , 1993, The Journal of biological chemistry.

[26]  M. Cobb,et al.  MAP kinase becomes stably activated at metaphase and is associated with microtubule-organizing centers during meiotic maturation of mouse oocytes. , 1993, Developmental biology.

[27]  H. Pant,et al.  cdc2-like kinase from rat spinal cord specifically phosphorylates KSPXK motifs in neurofilament proteins: isolation and characterization. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[28]  K. Ishiguro,et al.  Tau protein kinase II has a similar characteristic to cdc2 kinase for phosphorylating neurofilament proteins. , 1993, Journal of Biological Chemistry.

[29]  Jonathan A. Cooper,et al.  p42 mitogen-activated protein kinase in brain: Prominent localization in neuronal cell bodies and dendrites , 1993, Neuroscience.

[30]  J. Wood,et al.  p44mpk MAP kinase induces aizheimer type alterations in tau function and in primary hippocampal neurons , 1993 .

[31]  V. Ingram,et al.  Brain protein kinase PK40erk converts TAU into a PHF-like form as found in Alzheimer's disease. , 1993, Biochemical and biophysical research communications.

[32]  T. Sakaguchi,et al.  Reduced diameter and conduction velocity of myelinated fibers in the sciatic nerve of a neurofilament-deficient mutant quail , 1993, Neuroscience Letters.

[33]  H. Pant,et al.  Bovine neurofilament-enriched preparations contain kinase activity similar to casein kinase I — Neurofilament phosphorylation by casein kinase I (CKI) , 1993, Neuroscience Letters.

[34]  M. Cobb,et al.  Regulation and properties of extracellular signal-regulated protein kinases 1 and 2 in vitro. , 1993, The Journal of biological chemistry.

[35]  R. Liem,et al.  Molecular biology of neuronal intermediate filaments. , 1993, Current opinion in cell biology.

[36]  J. H. Wang,et al.  Brain proline-directed protein kinase is a neurofilament kinase which displays high sequence homology to p34cdc2. , 1992, The Journal of biological chemistry.

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

[38]  P. Cohen,et al.  p42 map kinase phosphorylation sites in microtubule‐associated protein tau are dephosphorylated by protein phosphatase 2A1 Implications for Alzheimer's disease , 1992, FEBS letters.

[39]  L. Tsai,et al.  A family of human cdc2‐related protein kinases. , 1992, The EMBO journal.

[40]  J. H. Wang,et al.  Purification and characterization of a novel proline-directed protein kinase from bovine brain. , 1992, The Journal of biological chemistry.

[41]  G. Drewes,et al.  Mitogen activated protein (MAP) kinase transforms tau protein into an Alzheimer‐like state. , 1992, The EMBO journal.

[42]  Philip R. Cohen,et al.  MAP kinase activator from insulin‐stimulated skeletal muscle is a protein threonine/tyrosine kinase. , 1992, The EMBO journal.

[43]  Z. S. Xu,et al.  Identification of six phosphorylation sites in the COOH-terminal tail region of the rat neurofilament protein M. , 1992, The Journal of biological chemistry.

[44]  D. Fink,et al.  Phosphorylation-dependent neurofilament epitopes are reduced at the node of Ranvier , 1992, Journal of neurocytology.

[45]  M. DeMott,et al.  Internal protein sequence analysis: enzymatic digestion for less than 10 micrograms of protein bound to polyvinylidene difluoride or nitrocellulose membranes. , 1992, Analytical biochemistry.

[46]  H. Pant,et al.  Association of cyclic-AMP-dependent protein kinase with neurofilaments. , 1992, The Biochemical journal.

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

[48]  T. Kishimoto,et al.  Phosphorylation of neurofilament H subunit at the tail domain by CDC2 kinase dissociates the association to microtubules. , 1991, The Journal of biological chemistry.

[49]  HM Roder,et al.  Two novel kinases phosphorylate tau and the KSP site of heavy neurofilament subunits in high stoichiometric ratios , 1991, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[50]  R. Nixon,et al.  Neurofilament phosphorylation: a new look at regulation and function , 1991, Trends in Neurosciences.

[51]  C. Crews,et al.  Mouse Erk-1 gene product is a serine/threonine protein kinase that has the potential to phosphorylate tyrosine. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[52]  B. Khatra,et al.  Phosphorylation of bovine neurofilament proteins by protein kinase FA (glycogen synthase kinase 3). , 1991, The Journal of biological chemistry.

[53]  E. Krebs,et al.  Multiple components in an epidermal growth factor-stimulated protein kinase cascade. In vitro activation of a myelin basic protein/microtubule-associated protein 2 kinase. , 1991, The Journal of biological chemistry.

[54]  E. Nishida,et al.  Activation of microtubule-associated protein kinase by microtubule disruption in quiescent rat 3Y1 cells. , 1991, Experimental cell research.

[55]  N. Hirokawa,et al.  Dephosphorylation-induced interactions of neurofilaments with microtubules. , 1990, The Journal of biological chemistry.

[56]  K. Angelides,et al.  Effect of phosphorylation on 68 KDa neurofilament subunit protein assembly by the cyclic AMP dependent protein kinase in vitro. , 1990, Biochemical and biophysical research communications.

[57]  M. Inagaki,et al.  Involvement of protein kinase C in the regulation of assembly-disassembly of neurofilaments in vitro. , 1990, Biochemical and biophysical research communications.

[58]  Ram K. Sihag,et al.  Phosphorylation of the amino-terminal head domain of the middle molecular mass 145-kDa subunit of neurofilaments. Evidence for regulation by second messenger-dependent protein kinases. , 1990, The Journal of biological chemistry.

[59]  M. Inagaki,et al.  Domain- and sequence-specific phosphorylation of vimentin induces disassembly of the filament structure. , 1989, Biochemistry.

[60]  R. Nixon,et al.  In vivo phosphorylation of distinct domains of the 70-kilodalton neurofilament subunit involves different protein kinases. , 1989, The Journal of biological chemistry.

[61]  H. Pant Dephosphorylation of neurofilament proteins enhances their susceptibility to degradation by calpain. , 1988, The Biochemical journal.

[62]  T. Sturgill,et al.  Characterization of insulin-stimulated microtubule-associated protein kinase. Rapid isolation and stabilization of a novel serine/threonine kinase from 3T3-L1 cells. , 1988, The Journal of biological chemistry.

[63]  J. Eyer,et al.  Influence of the phosphorylation state of neurofilament proteins on the interactions between purified filaments in vitro. , 1988, The Biochemical journal.

[64]  L. Otvos,et al.  Identification of the major multiphosphorylation site in mammalian neurofilaments. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[65]  J. Trojanowski,et al.  Two-stage expression of neurofilament polypeptides during rat neurogenesis with early establishment of adult phosphorylation patterns , 1987, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[66]  M. Inagaki,et al.  Site-specific phosphorylation induces disassembly of vimentin filaments in vitro , 1987, Nature.

[67]  H. Gainer,et al.  Biochemical and immunocytochemical characterization and distribution of phosphorylated and nonphosphorylated subunits of neurofilaments in squid giant axon and stellate ganglion , 1987, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[68]  C. Marotta,et al.  Posttranslational modification of neurofilament proteins by phosphate during axoplasmic transport in retinal ganglion cell neurons , 1987, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[69]  L. Sternberger,et al.  Phosphorylation protects neurofilaments against proteolysis , 1987, Journal of Neuroimmunology.

[70]  M. Glicksman,et al.  Posttranslational modification of neurofilament polypeptides in rabbit retina. , 1987, Journal of neurobiology.

[71]  Y. Miyata,et al.  Binding of microtubule-associated protein 2 and tau to the intermediate filament reassembled from neurofilament 70-kDa subunit protein. Its regulation by calmodulin. , 1986, The Journal of biological chemistry.

[72]  W. Schlaepfer,et al.  The structure, biochemical properties, and immunogenicity of neurofilament peripheral regions are determined by phosphorylation state. , 1985, The Journal of biological chemistry.

[73]  K H Jones,et al.  An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide. , 1985, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[74]  J. Julien,et al.  The distribution of phosphorylation sites among identified proteolytic fragments of mammalian neurofilaments. , 1983, The Journal of biological chemistry.

[75]  J. Julien,et al.  Multiple phosphorylation sites in mammalian neurofilament polypeptides. , 1982, The Journal of biological chemistry.

[76]  N. Hirokawa,et al.  Cross-linker system between neurofilaments, microtubules and membranous organelles in frog axons revealed by the quick-freeze, deep-etching method , 1982, The Journal of cell biology.

[77]  P G Nelson,et al.  Mouse spinal cord in cell culture. I. Morphology and intrinsic neuronal electrophysiologic properties. , 1977, Journal of neurophysiology.

[78]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[79]  J. Mandell,et al.  Compartmentation of alpha-internexin and neurofilament triplet proteins in cultured hippocampal neurons , 1996, Journal of neurocytology.

[80]  R. Moritz,et al.  Rapid separation of proteins and peptides using conventional silica-based supports: Identification of 2-D gel proteins following in-gel proteolysis , 1995 .

[81]  Veeranna,et al.  Neurofilament phosphorylation. , 1995, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[82]  R. Nixon,et al.  Dynamics of neuronal intermediate filaments: a developmental perspective. , 1992, Cell motility and the cytoskeleton.

[83]  N. Hirokawa,et al.  Effects of phosphorylation of the neurofilament L protein on filamentous structures. , 1990, Cell regulation.

[84]  H. Sambrook Molecular cloning : a laboratory manual. Cold Spring Harbor, NY , 1989 .

[85]  E. Lazarides Intermediate filaments: a chemically heterogeneous, developmentally regulated class of proteins. , 1982, Annual review of biochemistry.