Gudrun S. Bennett

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

Abstract: 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.

Lithium chloride inhibits the phosphorylation of newly synthesized neurofilament protein, NF-M, in cultured chick sensory neurons.

Abstract: The middle and high molecular weight members of the neurofilament triplet, NF‐M and NF‐H, undergo extensive posttranslational polyphosphorylation, a process requiring 24 h or more for completion. We have investigated ways of perturbing this process in intact cells and have found that phosphorylation of newly synthesized NF‐M in cultured chick sensory neurons is inhibited by Li+. [35S]Methionine pulse‐chase experiments were carried out with pure neuronal cultures, and the phosphorylation of newly synthesized NF‐M was monitored by following the accompanying change, with chase time, in apparent size and charge of the polypeptide. Addition of LiCl to the medium inhibited this mobility shift in a dose‐dependent manner over concentrations between 2 and 25 mM. Incorporation of 32P into NF‐M, as well as NF‐H, was also inhibited, whereas incorporation into the low molecular weight neurofilament protein, β‐tubulin, and total protein was unaffected. Protein synthesis was not altered.

Characterization of a neurofilament-associated kinase that phosphorylates the middle molecular mass component of chicken neurofilaments

We have examined the properties of a chicken neurofilament (NF) kinase partially purified from NF-enriched preparations. This kinase cosediments with NFs following extraction with Triton X-100 and can be separated in an active form from NFs by treatment with 0.8 M KCl. Sequential chromatography of the salt extract on DEAE-cellulose and phosphocellulose results in an approximately 500-fold increase in specific activity over endogenous NF preparations as measured by 32P-incorporation into the middle molecular mass component of NFs (NF-M). The kinase is Mg(2+)-dependent, second messenger-independent and inhibited by high concentrations of heparin. It shows selectivity for NF-M and evidence is presented that the kinase phosphorylates NF-M solely in the tail domain. The kinase can also phosphorylate the microtubule-associated proteins tau and MAP2 as well as mammalian NF-M, all of which share putative phosphorylation sequences with chicken NF-M.

Identification of Ser-Pro and Thr-Pro phosphorylation sites in chicken neurofilament-M tail domain

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 Glu‐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.

Relationship Between Casein Kinase I Isoforms and a Neurofilament-Associated Kinase

Abstract : Purified neurofilaments (NFs) contain an associated kinase (NFAK) activity that phosphorylates selectively a subset of sites in the tail of NF‐M and has properties consistent with casein kinase I (CKI). Because CKI consists of a family of as many as seven genes (α, β, γ1‐3, δ, and ε), we investigated the extent to which different CKI isoforms contribute to NFAK activity. Using an NF‐M‐derived substrate, we determined that NFAK activity copurified with casein kinase activity through two purification steps. In an in‐gel kinase assay, NFAK activity occurred at 36‐40 kDa, corresponding to the size of CKIα isoforms. Chicken neurons express transcripts encoding four alternatively spliced variants of CKIα (CKIα, CKIαS, CKIαL, and CKIαLS) differing in the presence or absence of two inserts, L and S. Using antibodies against different isoforms or with broad CKI specificity, we determined that all four CKIα variants, as well as other CKI family members, are present in chicken brain. However, only CKIα and CKIαS could be detected in purified NFAK. Also, immunoprecipitation studies showed that CKIα and CKIαS together account for NFAK activity. These findings raise the possibility that only a subset of CKI isoforms may be able to associate with and/or phosphorylate NFs.

A Neurofilament-Associated Kinase Phosphorylates Only a Subset of Sites in the Tail of Chicken Midsize Neurofilament Protein

Although neurofilaments are among the most highly phosphorylated proteins extant, relatively little is known about the kinases involved in their phosphorylation. The majority of the phosphates present on the two higher‐molecular‐mass neurofilament subunits are added to multiply repeated sequence motifs in the tail. We have examined the specificity of a neurofilament‐associated kinase (NFAK) partially purified from chicken spinal cord that selectively phosphorylates the middle‐molecular‐mass neurofilament subunit, NF‐M. Two‐dimensional phosphopeptide mapping of 32P‐labeled NF‐M shows that, in vitro, NFAK phosphorylates a subset of peptides phosphorylated in vivo in cultured neurons. The absence of a complete complement of labeled phosphopeptides following in vitro phosphorylation, compared with phosphorylation in vivo, is not due to a lack of availability of phosphorylation sites because the same maps are obtained when enzymatically dephosphorylated NF‐M is used as an in vitro substrate. Phosphopeptide maps from in vitro‐phosphorylated NF‐M and those from a recombinant fusion protein containing only a segment of the tail piece of chicken NF‐M reveal identical labeled peptides. The fusion protein lacks a segment containing 17 KXX(S/T)P putative phosphorylation sites contained in the tail of chicken NF‐M but contains a segment that includes four KSPs and a KSD site also present in the intact tail. These results suggest (a) that NFAK mediates the phosphorylation of some, but not all, potential phosphorylation sites within the tail of NF‐M and (b) that multiple kinases are necessary for complete phosphorylation of the NF‐M tail.

Rapid Degradation of Newly Synthesized Tubulin in Lithium-Treated Sensory Neurons

Abstract: When cultured chick sensory neurons were labeled with [35S]methionine for 1 h or longer in the presence of 5–25 mM LiCl, we found a dose‐dependent reduction in the level of radiolabeled tubulin, to one third of control levels, with no noticeable effect on other proteins. The magnitude of this response was identical after a 1‐h or 72‐h preincubation in 25 mM LiCl and returned to control values within 1 h after removal of LiCl. Short (5‐min) pulse‐chase experiments revealed that tubulin synthesis was not affected by Li+, but that newly synthesized tubulin was rapidly degraded, such that 50% of the labeled β‐tubulin was lost within 5 min. There was no enhanced degradation of tubulin present before exposure to Li+. Addition of LiCl at various times before and after a 10‐min pulse suggested that tubulin becomes completely refractory to Li+‐induced degradation within 10 min after translation. Although Li+ treatment resulted in a decrease in the fraction of extant tubulin present in the unassembled form, the Li+‐induced degradation of nascent tubulin is not a consequence of shifts in assembly state, because colcemid or taxol treatment did not lead to rapid degradation of newly synthesized tubulin, and neither drug altered the response to Li+. We suggest that Li+ interferes with the corre ct folding of tubulin polypeptides, exposing sites, normally hidden, to the action of a protease(s).

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

In order to characterize the phosphorylation of neurofilaments (NF) in intact neurons, we examined the ability of several protein kinase inhibitors to interfere with the incorporation 32P into individual NF polypeptides of sensory neurons in culture. We also examined their effect on the post-translational mobility shift on SDS-PAGE that accompanies phosphorylation of newly synthesized NF-M. Several agents known to inhibit cyclic nucleotide-, Ca2+/calmodulin-, and Ca2+/phospholipid-dependent protein kinases (H7, HA1004, trifluoperizine, sphingosine) had no effect on the phosphorylation of any NF polypeptide, in either assay. In contrast, two broadly active protein kinase inhibitors, staurosporine and K252a, inhibited the incorporation of 32P into NF-M by 60-70% and also blocked the post-translational mobility shift. They had no effect on NF-L. The action of staurosporine and K252a was identical to that of 25 mM LiCl. Proteolytic cleavage and phosphopeptide mapping of 32P-labeled NF-M from control and treated cultures revealed that the phosphorylation of only one subset of phosphopeptides was affected by staurosporine, K252a, and LiCl. These were contained within a single chymotryptic fragment of the NF-M tail segment, probably containing most of the 17 repeats of a KXXS/TP motif. The phosphorylation of another subset of phosphopeptides was insensitive to these inhibitors. They were contained within a different chymotryptic fragment of the tail segment which contains a KSD and four KSP potential phosphorylation sites. This differential sensitivity to protein kinase inhibitors distinguishes two different types of effector-independent kinases that phosphorylate, in vivo, different sites within the NF-M tail.