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  • 1
    Electronic Resource
    Electronic Resource
    Dynamics of Phosphorylation and Assembly of the High Molecular Weight Neurofilament Subunit in NB2a/d1 Neuroblastoma (1990)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 55 (1990), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: In neuronal systems thus far studied, newly synthesized neurofilament subunits rapidly associate with the Triton-insoluble cytoskeleton and subsequently undergo extensive phosphorylation. However, in the present study we demonstrate by biochemical and immunological criteria that NB2a/dl neuroblastoma cells also contain Triton-soluble, extensively phosphorylated 200-kDa high molecular weight neurofilament subunits (NF-H). High-speed centrifugation (100,000 g) of the Triton-soluble fraction for 1 h sedimented some, but not all, soluble NF-H subunits; immunoelectron microscopic analyses of the resulting pellet indicated that a portion of the NF-H subunits in this fraction are assembled into (Triton-soluble) neurofilaments. When cells were pulse labeled for 15 min with [35S]methionine, radiolabel was first associated with the Triton-soluble 200-kDa NF-H variants. Because only extensively phosphorylated NF-H subunits migrate at 200 kDa, whereas hypophosphorylated subunits migrate instead at 160 kDa, these findings suggest that some newly synthesized subunits were phosphorylated before they polymerized. In pulse-chase analyses, radiolabeled 200-kDa NF-H migrated into the 100,000 g particulate fraction of Triton-soluble extracts before its arrival in the Triton-insoluble cytoskeleton. Undifferentiated cells, which do not possess axonal neurites and lack a significant amount of Triton-insoluble, extensively phosphorylated NF-H, contain a sizeable pool of Triton-soluble extensively phosphorylated NF-H subunits and polymers. We interpret these data to indicate that the integration of newly synthesized NF-H into the cytoskeleton occurs in a progression of distinct stages, and that assembly of NF-H into neurofilaments and integration into the Triton-insoluble cytoskeleton are not prerequisites for the incorporation of certain phosphate groups on these polypeptides. Because NF-H can be extensively phosphorylated in perikarya, additional mechanisms besides differential localization of the responsible kinase systems must account for the segregation of Triton-insoluble NF-H in NB2a/d1 neurites.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1990.tb04969.x
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  • 2
    Electronic Resource
    Electronic Resource
    Brain β-Spectrin Phosphorylation: Phosphate Analysis and Identification of Threonine-347 as a Heparin-Sensitive Protein Kinase Phosphorylation Site (1998)
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 71 (1998), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Phosphorylation of brain spectrin was studied by a combination of in vivo and in vitro approaches. Chemical analysis of phosphate groups on electrophoretically purified mouse brain β-spectrin yielded a stoichiometry of 3.2 ± 0.18 mol of PO4/mol of β-spectrin. The spectrin isolated by chromatographic methods from mouse brain, pig brain, and human erythrocytes yielded 4.1, 5.6, and 3.2 mol of PO4/mol of spectrin heterodimer, respectively. The 32P labeling of spectrin in retinal ganglion cell neurons or NB 2a/d1 neuroblastoma cells with [32P]orthophosphate showed phosphorylation of only β-spectrin in vivo. Two-dimensional phosphopeptide map analyses showed that most of the in vivo sites on β-spectrin were phosphorylated by either a heparin-sensitive endogenous cytoskeleton-associated protein kinase or protein kinase A. Phosphoamino acid analysis of in vivo and in vitro phosphorylated β-spectrin showed that [32P]phosphate groups were incorporated into both serine (〉90%) and threonine residues. In vitro, phosphate groups were incorporated into threonine residues by the heparin-sensitive endogenous protein kinase. The amino acid sequence VQQQLQAFNTY of an α-chymotryptic 32P-labeled peptide phosphorylated by the heparin-sensitive cytoskeleton-associated endogenous protein kinase corresponded to amino acid residues 338–348 on the β1 repeat of β-spectrinG (βSPIIa) gene. These data suggest that phosphorylation of Thr347, which is localized on the presumptive synapsin I binding domain of β-spectrinG, may play a role in synaptic function by regulating the binding of spectrin to synaptic vesicles.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1471-4159.1998.71052220.x
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  • 3
    Electronic Resource
    Electronic Resource
    Serine-23 Is a Major Protein Kinase A Phosphorylation Site on the Amino-Terminal Head Domain of the Middle Molecular Mass Subunit of Neurofilament Proteins (1999)
    Oxford UK : Blackwell Science Ltd.
    Journal of neurochemistry 72 (1999), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract : We have shown previously that phosphate groups on theamino-terminal head domain region of the middle molecular mass subunit ofneurofilament proteins (NF-M) are added by second messenger-dependent proteinkinases. Here, we have identified Ser23 as a specific proteinkinase A phosphorylation site on the native NF-M subunit and on two syntheticpeptides, S1 (14RRVPTETRSSF24) andS2 (21RSSFSRVSGSPSSGFRSQSWS41), localizedwithin the amino-terminal head domain region. Ser23 was identifiedas a phosphorylation site on the 32P-labeled α-chymotrypticpeptide that carried 〉80% of the 32P-phosphates incorporatedinto the NF-M subunit by protein kinase A. The synthetic peptidesS1 and S2 were phosphorylated 18 and two times moreefficiently by protein kinase A than protein kinase C, respectively. Neitherof the peptides was phosphorylated by casein kinase II. The sequence analysesof the chemically modified phosphorylated serine residues showed thatSer23 was the major site of phosphorylation for protein kinase A onboth S1 and S2 peptides. Low levels of incorporation of32P-phosphates into Ser22, Ser28, andSer32 by protein kinase A were also observed. Protein kinase Cincorporated 32P-phosphates into Ser22,Ser23, Ser25, Ser28, Ser32, and athreonine residue, but none of these sites could be assigned as a major siteof phosphorylation. Analyses of the phosphorylated synthetic peptides byliquid chromatography-tandem mass spectrometry also showed that protein kinaseA phosphorylated only one site on peptide S1 and that ions with upto four phosphates were detected on peptide S2. Analysis of thedata from the tandem ion trap mass spectrometry by using the computer programPEPSEARCH did not unequivocally identify the specific sites of phosphorylationon these serine-rich peptides. Our data suggest that Ser23 is amajor protein kinase A-specific phosphorylation site on the amino-terminalhead region of the NF-M subunit. Phosphorylation of Ser23 on the NF-M subunit by protein kinase A may play a regulatory role in neurofilament assembly and/or the organization of neurofilaments in the axon.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1471-4159.1999.0720491.x
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  • 4
    Electronic Resource
    Electronic Resource
    [32P]orthophosphate and [35S]methionine label separate pools of neurofilaments with markedly different axonal transport kinetics in mouse retinal ganglion cells in vivo (1994)
    Springer
    Neurochemical research 19 (1994), S. 1445-1453 
    Details
    ISSN: 1573-6903
    Keywords: Neurofilaments ; axonal transport ; phosphorylation ; retinal ganglion cells ; optic axons
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Newly synthesized neurofilament proteins become highly phosphorylated within axons. Within 2 days after intravitreously injecting normal adult mice with [32P]orthophosphate, we observed that neurofilaments along the entire length of optic axons were radiolabeled by a soluble32P-carrier that was axonally transported faster than neurofilaments.32P-incorporation into neurofilament proteins synthesized at the time of injection was comparatively low and minimally influenced the labeling pattern along axons.32P-incorporation into axonal neurofilaments was considerably higher in the middle region of the optic axons. This characteristic non-uniform distribution of radiolabel remained nearly unchanged for at least 22 days. During this interval, less than 10% of the total32P-labeled neurofilaments redistributed from the optic nerve to the optic tract. By contrast, newly synthesized neurofilaments were selectively pulse-labeled in ganglion cell bodies by intravitreous injection of [35S]methionine and about 60% of this pool translocated by slow axoplasmic transport to the optic tract during the same time interval. These findings indicate that the steady-state or resident pool of neurofilaments in axons is not identical to the newly synthesized neurofilament pool, the major portion of which moves at the slowest rate of axoplasmic transport. Taken together with earlier studies, these results support the idea that, depending in part on their phosphorylation state, transported neurofilaments can interact for short or very long periods with a stationary but dynamic neurofilament lattice in axons.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00972474
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