Differential Sensitivity to Inhibitors Discriminates between Two Types of Kinases Responsible for in Vivo Phosphorylation of Different Sites in the Carboxy-Terminal Tail of Chicken Neurofilament-M

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Abstract:In previous studies we have identified Ser 502, Ser528, and Ser534 as target sites in chicken neurofilament middle molecular mass protein (N F-M) for casein kinase I (CKI) in vitro and have shown that these sites are also phosphorylated in vivo. We now make use of a combination of molecular biological and protein chemical techniques to show that two additional in vivo phosphorylation sites in chicken NF-M, Ser464 and Ser471, can also be phosphorylated by CKI in vitro. These two sites are conserved in higher vertebrate NF-M molecules, and recombinant protein constructs containing the homologous rat NF-M peptides can be phosphorylated by CKI in vitro, suggesting that phosphorylation of these sites is conserved at least in higher vertebrates. The two new sites are adjacent to a conserved peptide sequence (VEE-IIEET-V) found once in higher vertebrate NF-M molecules and twice in lamprey NF-180. Variants of this sequence are also found in neurofilament low and high molecular mass proteins (NF-L and NF-H) and a-internexin, and in mammalian NF-L are known to be associated with in vivo phosphorylation sites. We speculate that CKI phosphorylation in general, and these sites in particular, may be important in neurofilament function.

Section: Discussionsupporting

confidence: 53%

Section: Two-dimensional Peptide Mappingmentioning

confidence: 99%

mentioning

confidence: 99%

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Characterization of Additional Casein Kinase I Sites in the C‐Terminal “Tail” Region of Chicken and Rat Neurofilament‐M

Shaw¹,

Miller²,

Wang³

et al. 1997

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“…peptide maps to sort phosphopeptides into different groups based on differences in properties, such as sensitivity to selective protein kinase inhibitors (Bennett et a!., 1994) and ability to be phosphory!ated in vitro by endogenous protein kinase activity (Hollander et a!., 1993). As an extension of these investigations, we have carried out experiments to identify the actua!…”

Section: Proline-dependent Nf-m Phosphorylation Sites 535mentioning

confidence: 99%

“…The procedures used were as previously described (Hol-!ander et al, !993, !996;Bennett et al, 1994). Chicken brain NF proteins were mixed with the cytoskeleta!…”

Section: Proteolytic Fragmentation Of Nf-mmentioning

confidence: 99%

Identification of Ser‐Pro and Thr‐Pro Phosphorylation Sites in Chicken Neurofilament‐M Tail Domain

Bennett

Quintana²

1997

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Abstract:The tail domain of the midsize chicken neurofilament polypeptide (NF-M) contains several different types of Ser-Pro and Thr-Pro putative phosphorylation sites. We determined which of these sites are actually phosphorylated in vivo. Chick sensory neuron cultures were incubated in [ 32P]phosphate, and the cytoskeletal fraction was mixed with a neurofilament fraction prepared from adult chicken brain. NF-M was purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and digested with chymotrypsin, and two large fragments were isolated. These were individually cleaved with trypsin, endoprotease Lys-C, or endoprotease GIu-C, and peptides separated by two-dimensional high-voltage electrophoresis and thin-layer chromatography. 32P-labeled phosphopeptides were eluted from the cellulose plates and subjected to microsequencing and mass spectometry. We found that of 21 potential Ser-Pro and Thr-Pro phosphoacceptor sites, at least 20 are phosphorylated in vivo: all four Lys-Ser-Pro sites and at least 16 of the 17 Lys-Xaa-Xaa-Ser/Thr-Pro repeats. In addition, a novel Ser-Pro site in the extreme carboxy terminus is phosphorylated. This site, which has no proximal Lys residue, is also found in mammalian NF-M, but has not been reported to be phosphorylated. Together with three casein kinase I sites we have found recently in the acidic amino-terminal segment of the tail, a total of 24 or 25 Ser and Thr phosphoacceptor sites have now been located in the chicken NF-M tail.

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

Hollander¹,

Bennett

Shaw³

1996

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We have shown previously that a neurofilament (NF)‐associated kinase (NFAK) extracted from chicken NF preparations phosphorylates selectively the middle molecular mass NF subunit (NF‐M). Here we show that the major kinase activity in NFAK is indistinguishable from enzymes of the casein kinase I (CKI) family based on the following criteria: (1) inhibition of NFAK phosphorylation by the selective CKI inhibitor CKI‐7, (2) the similarity in substrate specificity of NFAK and authentic CKI, (3) the correspondence of two‐dimensional phosphopeptide maps of NF‐M phosphorylated in vitro by NFAK with those generated by CKI under similar conditions, and (4) immunological cross‐reactivity of NFAK with an antibody raised against CKI. We have also identified Ser502, Ser528, and Ser536 as phosphorylation sites by NFAK/CKI in vitro, each of which is also phosphorylated in vivo. All three serines are found in peptides with CKI phosphorylation consensus sequences, and Ser528 and Ser536 and flanking amino acids are highly conserved in higher vertebrate NF‐M sequences. Neither Ser502 nor Ser536 has been identified previously as NF‐M phosphorylation sites.