Phosphorylation of neurofilament proteins by protein kinase C

The low molecular mass (70 kDa) subunit of neurofilaments (NF-L) contains at least three phosphorylation sites in vivo and is phosphorylated by multiple kinases in a site-specific manner [(1987) J. Neurochem. 48, S101; Sihag, R.K. and Nixon, R.A. submitted]. In this study, we observed that the three subunits of neurofilament proteins from retinal ganglion cell neurons are substrates for purified mouse brain protein kinase C. Two-dimensional alpha-chymotryptic phosphopeptide map analyses of the NF-L subunit demonstrated that protein kinase C phosphorylates four polypeptide sites, two of which incorporate phosphate when retinal ganglion cells are pulse-radiolabeled with [32P]orthophosphate in vivo.     

Phosphorylation of neurofilament proteins by endogenous calcium/calmodulin-dependent protein kinase

A protein fraction containing neurofilaments was prepared from rat brain cytosol by differential centrifugation and gel filtration chromatography. These preparations were enriched for a calcium/calmodulin-dependent kinase activity that phosphorylated endogenous neurofilament proteins. The enzyme incorporated approximately 1 mol PO4/mol of each neurofilament triplet polypeptide. These data suggest that a calmodulin-dependent kinase may mediate some of the effects of calcium on cytoskeletal function by phosphorylation of neurofilament proteins.     

Characteristics of the protein kinase activity associated with rat neurofilament preparations

Some properties of the protein kinase activity associated with neurofilaments isolated from the brain stem and spinal cord of rats have been investigated. The activity had an apparent Km for ATP of 20 microM, a pH optimum of 8.0 and phosphorylated both serine and threonine residues in neurofilament proteins. Cyclic AMP had no effect on the in vitro reaction and casein was a preferred exogenous substrate in comparison to histone. Phosphopeptide mapping of the 145 kDa subunit from neurofilaments phosphorylated in the presence and absence of microtubule proteins indicated that the neurofilament-associated activity was distinct from the microtubule-associated protein kinase. Limited proteolysis of neurofilaments with chymotrypsin indicated that the enzyme activity was not associated with a domain of the 200 kDa subunit which may form the side-arm projections on neurofilaments.     

Cyclin-dependent protein kinase 5 (Cdk5) and the regulation of neurofilament metabolism.

Cyclin-dependent kinase 5 (Cdk5), a complex of Cdk5 and its activator p35 (Cdk5/p35), phosphorylates diverse substrates which have multifunctional roles in the nervous system. During development, it participates in neuronal differentiation, migration, axon outgrowth and synaptogenesis. Cdk5, acting together with other kinases, phosphorylates numerous KSPXK consensus motifs in diverse cytoskeletal protein target molecules, including neurofilaments, and microtubule associated proteins, tau and MAPs. Phosphorylation regulates the dynamic interactions of cytoskeletal proteins with one another during all aspects of neurogenesis and axon radial growth. In this review we shall focus on Cdk5 and its regulation as it modulates neurofilament metabolism in axon outgrowth, cytoskeletal stabilization and radial growth. We suggest that Cdk5/p35 forms compartmentalized macromolecular complexes of cytoskeletal substrates, other neuronal kinases, phosphatases and activators ('phosphorylation machines') which facilitate the dynamic molecular interactions that underlie these processes.     

Phosphorylation of bovine neurofilament proteins by protein kinase FA (glycogen synthase kinase 3).

A highly purified preparation of protein kinase FA (where FA is the activating factor for phosphatase 1)/glycogen synthase kinase 3 from rabbit muscle readily phosphorylated bovine neurofilaments. All three neurofilament proteins, the high, middle, and low molecular proteins (NF-H, NF-M, and NF-L), were phosphorylated when intact filaments were incubated with the kinase. Experiments with individual proteins showed that NF-M was the best substrate. At protein concentrations of 0.13 mg/ml, the initial rate of NF-M phosphorylation was 30% of that observed for glycogen synthase. Km values were 0.24 mg/ml (7 x 10(-7) M tetramer) for glycogen synthase and 0.10 mg/ml (5 x 10(-7) M dimer) for NF-M. Vmax values were 0.36 mumol/min/mg for glycogen synthase and 0.035 mumol/min/mg for NF-M. Dephosphorylated NF-M was phosphorylated only half as much as native NF-M; this is consistent with the known substrate specificity of the kinase. The possible involvement of FA/GSK-3 in the phosphorylation of neurofilaments in vivo is discussed.     

Brain proline-directed protein kinase is a neurofilament kinase which displays high sequence homology to p34cdc2.

The carboxyl-terminal regions of neurofilament high (NF-H) and middle (NF-M) molecular weight proteins have been suggested to be phosphorylated in vivo by a p34cdc2-like protein kinase, on the basis of the in vivo phosphorylation site motif and in vitro phosphorylation of the proteins by p34cdc2 kinase (Hisanaga, S.I., Kusubata, M., Okumura, E. and Kishimoto, T. (1991) J. Biol. Chem. 266, 21798-21803). A novel proline-directed protein kinase previously identified and purified from bovine brain has been found in this study to phosphorylate NF-H and NF-M at sites identical to those phosphorylated by HeLa cell p34cdc2 kinase. The proline-directed kinase is composed of a 33-kDa and a 25-kDa subunit. The 33-kDa kinase subunit was partially sequenced, and degenerate oligonucleotide primers corresponding to the amino acid sequence information were used to clone the subunit by polymerase chain reaction (PCR). Two overlapping PCR products comprised a complete open reading frame of 292 amino acids. The sequence contains all features of a protein kinase, suggesting that the 33-kDa peptide represents the catalytic subunit of the kinase. The 33-kDa subunit shows high and approximately equal homology to human p34cdc2 and human cdk2, with about 58 and 59% amino acid identity, respectively. These results suggest that the brain kinase represents a new category of the cdc2 family, and that some members of the cdc2 kinase family may have major functions unrelated to cell cycle control.     

Molecular architecture of the neurofilament. II. Reassembly process of neurofilament L protein in vitro

Reassembly of the neurofilament (NF) in vitro was studied by low-angle rotary shadowing electron microscopy. Various intermediate stages of the reassembly were reconstructed from the smallest molecular mass subunit (NF-L) under controlled reassembly conditions. NF-L in 6 M-urea took the form of spherical particles with a diameter of about 12 nm. NF-L aggregated into rodlets of 70 to 80 nm long in a low-salt solution at alkaline pH. By reducing the pH of the dialyzing solution to 6.6, a pair of rods was formed by association side-by-side. Increasing the temperature of low-salt solutions from 4 degrees C to 35 degrees C did not produce intermediate-sized filaments. The addition of Mg2+ to the dialyzing solution resulted in the formation of short intermediate-sized filaments even at 4 degrees C. Further dialysis of the short intermediate-sized filaments against reassembly solution containing both NaCl and MgCl2 at 37 degrees C failed to elongate them into longer filaments, suggesting that annealing does not contribute to the elongation of neurofilaments. Different roles for Mg+ and NaCl in neurofilament reassembly were indicated. While Mg2+ strengthened the lateral association between 70 to 80 nm rods, NaCl appeared to promote the end-to-end association of filaments preferentially. Longer filaments were formed by increasing the NaCl concentration. By dialyzing NF-L against a buffer containing 50 mM-NaCl in the absence of Mg2+, unraveled filaments were formed. The many unraveled filaments were composed of four 8 nm wide filaments, which have been called the subfilament or the protofibril. Time-course experiments of the reassembly were performed in the absence of Mg2+, in which condition the rate of neurofilament reassembly appeared to be reduced. Star-like clusters, about four protofibrils joined together at one end, were suggested to be the initial stage of the intermediate-sized filament formation. The following two-step elongation mechanism of neurofilaments was deduced from these results. The pairs of rods were added to the ends of the protofibrils of neurofilaments, and after all four protofibrils were elongated they were then packed into neurofilaments. Distribution of larger molecular mass subunits, NF-M and NF-H, was studied. Addition of NF-M or NF-H to NF-L did not change the assembly properties of neurofilaments. Unraveled filaments reconstituted from NF-L plus either NF-M or NF-H indicated that NF-M and NF-H are incorporated evenly into each protofibril.     

Properties of membrane-inserted protein kinase C

Protein kinase C (PKC) interacted with phospholipid vesicles in a calcium-dependent manner and produced two forms of membrane-associated PKC: a reversibly bound form and a membrane-inserted form. The two forms of PKC were isolated and compared with respect to enzyme stability, cofactor requirements, and phorbol ester binding ability. Membrane-inserted PKC was stable for several weeks in the presence of calcium chelators and could be rechromatographed on gel filtration columns in the presence of EGTA without dissociation of the enzyme from the membrane. The activity of membrane-inserted PKC was not significantly influenced by Ca2+, phospholipids, and/or PDBu. Partial dissociation of this PKC from phospholipid was achieved with Triton X-100, followed by dialysis to remove the detergent. The resulting free PKC appeared indistinguishable from original free PKC with respect to its cofactor requirements for activation (Ca2+, phospholipid, and phorbol esters), molecular weight, and phorbol 12,13-dibutyrate (PDBu) binding. The binding of PDBu to free and membrane-inserted PKC was measured under equilibrium conditions using gel filtration techniques. At 2.0 nM PDBu, free PKC bound PDBu with nearly 1:1 stoichiometry in the presence of Ca2+ and phospholipid. No PDBu binding to the free enzyme was observed in the absence of Ca2+. In contrast, membrane-inserted PKC bound PDBu in the presence or the absence of Ca2+; calcium did enhance the affinity of this interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular architecture of the neurofilament. I. Subunit arrangement of neurofilament L protein in the intermediate-sized filament

Using the smallest subunit (NF-L) of a neurofilament and a glial fibrillary acidic protein, the subunit arrangement in intermediate filaments was studied by low-angle rotary shadowing. NF-L formed a pair of 70 to 80 nm rods in a low ionic strength solution at pH 6.8. Two 70 to 80 nm rods appeared to associate in an antiparallel manner with an overlap of about 55 nm, almost the same length as the alpha-helix-rich central rod domain of intermediate filament proteins. The overlap extended for three-beaded segments, present at 22 nm intervals along the pairs of rods. The observations that (1) 70 to 80 nm rods were a predominant structure in a low ionic strength solution at pH 8.5, (2) the molecular weights of the rod and the pair were measured by sedimentation equilibrium as 190,000 and 37,000 respectively, and (3) the rods formed from the trypsin-digested NF-L had a length of about 47 nm, indicated that the 70 to 80 nm rod is the four-chain complex and the pair of rods is the eight-chain complex. Similar structures were observed with glial fibrillary acidic protein, indicating that these oligomeric structures are common to other intermediate filament proteins. NF-L assembled into short intermediate-sized filaments upon dialysis against a low-salt solution containing 1 to 2 mM-MgCl2 at 4 degrees C. The majority of these short filaments possessed four or five-beaded segments, suggesting that the pair of rods were arranged in a half-staggered fashion in neurofilaments. On the basis of these observations, we propose the following model for the intermediate filament subunit arrangement. (1) The four-chain complex is the 70 to 80 nm rod, in which two coiled-coil molecules align in parallel and in register. (2) Two four-chain complexes form the eight-chain complex by associating in an antiparallel fashion with the overlap of the entire central rod domain. (3) The eight-chain complex is the building block of the intermediate filament. The eight-chain complexes are arranged in a half-staggered fashion within the intermediate filament.     

Effect of phosphorylation on 68 KDa neurofilament subunit protein assembly by the cyclic AMP dependent protein kinase in vitro

The effect of phosphorylation by cyclic AMP dependent protein kinase on the assembly of the core-forming 68 KDa neurofilament subunit protein (NF-L) was studied in vitro by fluorescence energy transfer and electron microscopy. Phosphorylation of unassembled NF-L in a low ionic strength buffer by cyclic AMP dependent protein kinase led to the incorporation of 1-2 phosphate groups/mole protein. Assembly of this phosphorylated NF-L was inhibited significantly; compared to non-phosphorylated NF-L, the critical concentration of phosphorylated NF-L was raised by greater than 30-fold. Assembled NF-L filaments could also be phosphorylated by cyclic AMP dependent protein kinase indicating that the sites were accessible. Phosphorylation of NF-L in the filamentous state induced their disassembly. The results suggest that phosphorylation by cyclic AMP dependent protein kinase is a possible means to modulate the assembly state of NF-L.     

Cyclin-dependent kinase 5 in neurofilament function and regulation

Neurofilaments are neuron-specific intermediate filaments. They are classed into three groups according to their molecular masses: neurofilament heavy, middle and light chains (NF-H, NF-M and NF-L). Neurofilaments assemble and form through the association of their central alpha-helical coiled-coil rod domains. NF-H and NF-M are distinct from NF-L as they contain a carboxyl-terminal tail domain, which appears to form connections with adjacent structures and other neurofilaments. Together with other axonal components such as microtubules, they form the dynamic axonal cytoskeleton. They maintain and regulate neuronal cytoskeletal plasticity through the regulation of neurite outgrowth, axonal caliber and axonal transport. Neurofilaments contain KSP repeats that are consensus motifs for the proline-directed kinases and are extensively phosphorylated in vivo, and their functions are thought to be regulated through their phosphorylation. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed kinase, whose activity is restricted to the neuron through the neuronal-specific distribution of its activators p35 and p39. Cdk5 is the only kinase that affects the electrophoretic mobility of human NF-H and is thought to be the major neurofilament kinase. Cdk5 is involved in crosstalk with other signal transduction pathways such as the mitogen-activated protein kinase and myelin-associated glycoprotein pathways to influence the phosphorylation of neurofilaments and other cytoskeletal proteins. Both the hyperactivation of Cdk5 activity and subsequent hyperphosphorylation of neurofilaments and the microtubule-associated protein tau have been implicated in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease and amyotrophic lateral sclerosis. Here we review the functions of neurofilaments and the significance of Cdk5 phosphorylation of neurofilaments.     

Properties of the protein kinase C-phorbol ester interaction

The properties of the protein kinase C (PKC)-phorbol ester interaction were highly dependent on assay methods and conditions. Binding to cation-exchange materials or adsorption to gel matrices resulted in PKC that was capable of binding phorbol 12,13-dibutyrate (PDBu). The extraneous interactions were eliminated by measuring phorbol ester binding with a gel filtration chromatography assay in the presence of bovine serum albumin (BSA). In the absence of calcium, free PKC did not bind PDBu or phospholipids. Calcium caused structural changes in PKC which enhanced its interaction with surfaces such as the gel chromatography matrix. While BSA prevented this interaction, it did not interfere with PKC association with acidic phospholipids. Interaction of PKC with phospholipid resulted in two forms of membrane-associated PKC. The initial calcium-dependent and reversible form of membrane-associated PKC was capable of binding PDBu. Both PKC and PDBu were released from this complex by calcium chelation. Sustained interaction with phospholipid vesicles resulted in a PKC-membrane complex that could not be dissociated by calcium chelation and appeared to result from insertion of PKC into the hydrocarbon portion of the phospholipid bilayer. Membrane insertion was observed at calcium concentrations of 2-500 microM and with membrane compositions of 10-50% acidic phospholipid. However, the extent of insertion was dependent on the binding conditions and was promoted by high phospholipid to PKC ratios, high calcium, the presence of phorbol esters, high membrane charge, and long incubations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties and distribution of the protein inhibitor (Mr 17,000) of protein kinase C.

Ca2+-dependent hydrophobic-interaction chromatography is a powerful tool for the identification and isolation of a variety of Ca2+-binding proteins which expose a hydrophobic site(s) in the presence of Ca2+ [Gopalakrishna & Anderson (1982) Biochem. Biophys. Res. Commun. 104, 830-836; Walsh, Valentine, Ngai, Carruthers & Hollenberg (1984) Biochem. J. 224, 117-127; McDonald & Walsh (1985) Biochem. J. 232, 559-567]. Using this approach, we isolated two potent and specific protein inhibitors of protein kinase C, of 17 kDa [McDonald & Walsh (1985) Biochem. J. 232, 559-567] and 12 kDa [McDonald & Walsh (1986) Biochem. Soc. Trans. 14, 585-586]. Although these inhibitors were purified by Ca2+-dependent hydrophobic-interaction chromatography and exhibit properties similar to those of calmodulin and related Ca2+-binding proteins, we were unable to demonstrate high-affinity Ca2+ binding to these inhibitors, using equilibrium dialysis. Protein kinase C exhibited half-maximal activity at 0.6 microM-Ca2+ in the presence of phospholipid and diacylglycerol, and complete inhibition by both inhibitors was observed over the range of Ca2+ concentrations examined (10 nM-10 microM). These observations suggest that the inhibitory action of these proteins does not require Ca2+. The inclusion of proteinase inhibitors during isolation of the kinase C inhibitors, as well as two-dimensional peptide mapping and amino acid analysis of the isolated proteins, suggested that the 12 kDa inhibitor is a proteolytic fragment of the 17 kDa protein which is generated during purification. Antibodies raised in rabbits against the bovine brain 17 kDa inhibitor were shown to be specific by Western immunoblotting and the competitive enzyme-linked immunosorbent assay method and were used to study the tissue and species distribution of this protein. The inhibitor was found to be present in several bovine, murine, avian and human tissues, consistent with a role in the regulation of a variety of physiological functions involving the widely distributed protein kinase C.     

Slow posttranslational modification of a neurofilament protein

The synthesis and subsequent modification of neurofilament (NF) polypeptides has been examined in pulse-chase experiments, using cultured chick spinal cord neurons. Fluorography of the [35S]methionine-labeled cytoskeletal proteins, after separation by two-dimensional gel electrophoresis, revealed that (a) the mid-size chicken NF protein, NF-M160, is synthesized as a smaller and more basic precursor, NF-M130; (b) beginning approximately 8 h after translation, NF-M130 slowly and continuously becomes larger and more acidic, attaining the size and charge of NF-M160 16 or more h later, and undergoing no further change in mobility for many days thereafter; and (c) in contrast, the low molecular weight NF protein, NF-L, is synthesized as such, and undergoes no subsequent change in apparent size or charge. Additional experiments provided evidence that the conversion of NF-M130 to NF-M160 is due, at least in part, to phosphorylation: (a) Incubation of similar cultures in 32PO4 resulted in incorporation into NF-M160 and transitional forms, but not into NF-M130. (b) An antiserum to NF-M160 was found by immunoblot analysis to bind strongly to untreated NF-M160, but poorly to phosphatase-treated NF-M160, and not at all to NF-M130. It has already been demonstrated (Bennett, G. S., S. J. Tapscott, C. DiLullo, and H. Holtzer, 1984, Brain Res., 304:291-302) that this anti-NF-M160 fails to stain the soma of motor neurons in sections of chick spinal cord, but detects an increasing gradient of immunoreactivity in the proximal axons. These results, together with the known kinetics of axoplasmic transport of NF, suggest that the mid-size chicken NF protein is synthesized as NF-M130 and is extensively modified, at least in part by phosphorylation, to become NF-M160 during transport along proximal neurites. Once maximally modified, NF-M160 undergoes no further net change during transport along distal neurites.     

Properties of tyrosine-specific protein kinase from rat sarcoma cells

A procedure for measuring the activity of tyrosine-specific protein kinases was developed. The method is based on the fact that the phosphoryl groups of phosphotyrosine residues of phosphorylated protein substrates are not hydrolyzed in alkaline solutions, whereas the phosphoserine and phosphothreonine residues lose their phosphoryl groups upon heating in 2 N KOH. It was demonstrated that rat sarcoma XC cells in culture and in solid tumours contain tyrosine-specific protein kinase (TPK). The TPK is localized in the membrane fraction of the cells and is solubilized by 1% Triton X-100. Mn2+-ATP is a nucleotide substrate for TPK. Among other protein substrates, TPK strongly phosphorylates histones H5 and H2a, weakly phosphorylates histones H2b, H4 and H1 but does not phosphorylate protamine, casein, vinculin or angiotensin II. The optimal concentration of Mn2+ for the reaction is 2 mM; the Km values for ATP and histone H5 are 2-3 microM and 2.5 mg/ml, respectively. Tyrosine-specific protein kinases phosphorylating histone H5 were detected in the membrane fraction isolated from different mammalian tissues, e. g., spleen, heart, liver, brain and lungs, as well as from human intestinal mucosa and mucosal adenocarcinoma. These results suggest that tyrosine-specific protein kinases phosphorylating histone H5 are present in a vast majority of mammalian tissues.     

Properties of protein kinase C subspecies in human platelets

Protein kinase C (PKC) from human platelets was resolved into two fractions by hydroxyapatite column chromatography. One of the enzymes was indistinguishable from the brain type III PKC having alpha-sequence in its kinetic and immunological properties. The other enzyme was kinetically different from any of the brain PKC subspecies so far isolated, although it resembled the brain type II PKC having beta-sequence. With H1 histone as substrate, this platelet enzyme was not very sensitive to Ca2+, and activated partly by phosphatidylserine plus diacylglycerol or by free arachidonic acid. Both platelet enzymes could phosphorylate the P47 protein in vitro, but the enzyme physiologically responsible for the P47 protein phosphorylation in the activated platelets remains to be identified.     

Effects of phosphorylation of the neurofilament L protein on filamentous structures.

Effects of phosphorylation of the neurofilament L protein (NF-L) on the reassembly system were studied by both sedimentation experiments and low-angle rotary shadowing. Bovine spinal cord NF-L was phosphorylated with 3-4 mol/mol protein by either the catalytic subunit of cAMP-dependent protein kinase or protein kinase C. Phosphorylated NF-L could not assemble into filaments. Phosphorylation by either cAMP-dependent protein kinase or protein kinase C inhibited the same step of the reassembly process. Phosphorylated NF-L remained as an 8-chain complex even in favorable conditions for reassembly. The extent of the effect of phosphorylation on the filamentous structure of NF-L was also investigated by using the catalytic subunit of cAMP-dependent protein kinase. The amount of unassembled NF-L increased linearly with increased phosphorylation in the sedimentation experiments. Structural observations indicated that 1 or 2 mol of phosphorylation is enough to inhibit reassembly and to induce disassembly, and the disassembly process was also observed. The filaments were shown to unravel with disassembly. Star-like clusters, which we reported as being the initial stage of reassembly, were also identified.