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.

Ultrastructure of feeding in the karyorelictean ciliate Tracheloraphis examined by scanning electron microscopy.

The karyorelictean ciliate Tracheloraphis is considered to be among the most primitive of the extant ciliates based on both nuclear and somatic characters. These organisms lack the elaborate oral ciliation present in most ciliates. Their mode of ingestion is a type of phagocytosis through a non-ciliated region, the glabrous stripe, which runs the length of the cell. This type of ingestion is reminiscent of feeding in amoebae and some flagellate groups. It is possible that ciliate oral structures evolved within the karyorelictean ciliates from an ancestor resembling Tracheloraphis. We studied the ingestion process in a Tracheloraphis species from the Chesapeake Bay using scanning electron microscopy. The results indicate that the anterior terminus of the organism is not involved in the actual ingestion process, only the glabrous stripe. There is some interaction between the food particle and the surface of the stripe, possibly mediated by a substance secreted by the underlying extrusomes. The somatic cilia do not appear to be involved. The stripe invaginates at the ingestion site engulfing the particle. The cell becomes greatly distended at this site, but neither the anterior nor posterior terminus is affected.

Lithium chloride alters cytoskeletal organization in growing, but not mature, cultured chick sensory neurons.

Biochemical analysis indicates that lithium ion (Li+) has deleterious effects on the metabolism of at least two elements of the cytoskeleton in cultured chick dorsal root ganglia (DRG) neurons. Phosphorylation of newly synthesized middle molecular mass neurofilament polypeptide (NF-M) is inhibited by 10-25 mM LiCl, and tubulin (Tb) synthesized in the presence of Li+ is subject to rapid degradation. These Li(+)-induced metabolic abnormalities are accompanied by alterations in cellular and cytoskeletal morphology. Treatment of cultures having vigorously growing neurites with 25 mM LiCl results in the cessation of net neurite growth, without causing neurite retraction. Indirect immunofluorescence reveals that in these cultures Li+ provokes an aggregation of NF protein into a dense knot in the cell body/proximal neurite region. The knots contain accumulations of all three NF polypeptides and electron microscopic observation demonstrates that the knots contain intact, but disorganized, filaments. Both the inhibition of neurite outgrowth and NF collapse are reversible. Tubulin and intact microtubules are redistributed in immature cultures treated with Li+ insofar as they are excluded from the NF knots. Neurons in established cultures (e.g., 7 days and beyond) fail to show any difference between Li+ treatment and control conditions in the morphology of the cytoskeletal elements examined.

Expression and phosphorylation of the mid-sized neurofilament protein NF-M during chick spinal cord neurogenesis.

The middle molecular weight polypeptide of neurofilaments (NF-M) is modified posttranslationally by extensive phosphorylation. This modification is slow in mature neurons, requiring approximately 24-48 hr for completion and probably occurs outside of the cell soma (Bennett and DiLullo: J Cell Biol 100:1799, 1985c). Thus, NF-M synthesis and phosphorylation are separate events both temporally and spatially. Although it is known that NF-M is among the earliest neuron-specific gene products to be expressed during nervous system development, it is not known what the temporal relationship is between the initiation of NF-M translation and its phosphorylation. To address this question, we have produced an antiserum against the dephosphorylated form of NF-M (NF-M130) and have used this antiserum, together with a previously characterized antiserum against completely phosphorylated NF-M (NF-M160), in an immunohistochemical examination of neurogenesis and the initial period of neuronal differentiation in chick spinal cord. We found that 1) nonphosphorylated and partially phosphorylated NF-M cannot be detected prior to the completion of the terminal mitosis; 2) most postmitotic neuroblasts begin expressing NF-M as they commence migration, but do not contain the completely phosphorylated polypeptide until some time after completion of migration; and 3) those precursor cells of a subpopulation of neuroblasts that begin expressing completely phosphorylated NF-M during their terminal cell cycle (Bennett and DiLullo: Dev Biol 107:94, 1985a) contain no detectable nonphosphorylated or partially phosphorylated NF-M. These cells probably complete the phosphorylation step more rapidly than do mature neurons.

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.

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 (CKl) family based on the following criteria: (1) inhibition of NFAK phosphorylation by the selective CKl inhibitor CKl-7, (2) the similarity in substrate specificity of NFAK and authentic CKl, (3) the correspondence of two-dimensional phosphopeptide maps of NF-M phosphorylated in vitro by NFAK with those generated by CKl under similar conditions, and (4) immunological cross-reactivity of NFAK with an antibody raised against CKl. We have also identified Ser502, SER528, and Ser536 as phosphorylation sites by NFAK/CKl in vitro, each of which is also phosphorylated in vivo. All three serines are found in peptides with CKl 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.

Linkage of a nucleolin-related protein and casein kinase II with the detergent-stable photoreceptor cytoskeleton.

Vertebrate photoreceptors are highly polarized sensory neurons with a complex microtubule and actin-based cytoskeletal organization. In the present study, we have used a detergent-extracted cytokeleton preparation from bovine photoreceptors to test the hypothesis that protein kinases and their substrates co-purify with the photoreceptor cytoskeleton. We incubated the cytoskeletal preparation in the presence of [gamma-32P]ATP. Following SDS-PAGE and autoradiography, we found two principal phosphoproteins with apparent molecular weights of 55 kDa (pp55) and 112 kDa (pp112). We have additionally identified the kinase responsible for phosphorylation of pp112 (and possibly pp55) as a casein kinase II-like enzyme. pp55 was identified as beta-tubulin based on Western blotting and its position on two-dimensional gels. Microsequencing revealed that 16 of the first 17 amino acids of pp112 were identical to human nucleolin, a nuclear protein. Western blotting, mobility in SDS PAGE and in two-dimensional gels, predominant localization within the nucleus, and phosphorylation by a casein kinase II all support the conclusion that pp112 is a nucleolin-related protein. Immunocytochemistry revealed a significant extranuclear pool of nucleolin-immunoreactivity within the cell bodies of photoreceptors. These findings suggest an important extranuclear role for nucleolin or a related protein in photoreceptors.

Rapid degradation of newly synthesized tubulin in lithium-treated sensory neurons.

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 beta-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 correct 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.

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.

Evidence for kinesin-related proteins associated with the axoneme of retinal photoreceptors.

Situated at the junction between inner and outer segment, the connecting cilium of retinal photoreceptors supports regulated transport of molecules that function distally, while restricting diffusion of membrane proteins from one plasmalemmal domain to the other. Both functions are thought to be performed by a group of proteins stably or transiently associated with the axoneme. We have identified two types of unique polypeptides which associated with the axoneme in a nucleotide-dependent manner: they bind to the axonemes in the presence of adenosine monophosphate (AMP)-PNP, and are solubilized in the presence of adenosine triphosphate (ATP). The first group contained glyconjugates, previously shown to be part of the axoneme-plasmalemma cross-linkers at the connecting cilium. The second group cross-reacted with antibodies to two different conserved peptide sequences (called LAGSE and HIPYR) of kinesin-related proteins, and included polypeptides of approximately 85-97 kDa. Immunofluorescence microscopy of whole-mounted axonemes with the two anti-kinesin antibodies showed labeling throughout the axoneme, including the connecting cilium-basal body region. These results suggest that the identified proteins may serve as motor molecules for transport of material to the outer segment via the connecting cilium.

Selective intra-arterial embolization of bone tumors. A useful adjunct in the management of selected lesions.

Our initial experience with therapeutic selective arterial embolization of 3 highly vascular bone tumors is discussed. In 2 benign lesions embolization of Gelfoam particles through a large bore selective catheter facilitated complete resection by limiting hemorrhage at the time of surgery. Embolization of a vascular malignant tumor provided temporary palliation.

Characterization of additional casein kinase I sites in the C-terminal "tail" region of chicken and rat neurofilament-M.

In previous studies we have identified Ser502, Ser528, and Ser534 as target sites in chicken neurofilament middle molecular mass protein (NF-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 (VEEIIEET-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 alpha-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.