Dietary and hypothyroid hypercholesterolemia induces hepatic apolipoprotein E expression in the rat: direct role of cholesterol

Ser-473 is solely phosphorylated in vivo in the tail region of neurofilament L (NF-L). With peptides including the native phosphorylation site, it was not possible to locate responsible kinases. We therefore adopted full-length dephosphorylated NF-L as the substrate, and employed MALDI/TOF (matrix-assisted laser desorption and ionization/time of flight) mass spectrometry and a site-specific phosphorylation-dependent antibody recognizing Ser-473 phosphorylation. The antibody showed that casein kinase I (CK I) as well as casein kinase II (CK II) phosphorylated Ser-473 in vitro, while neither GSK-3beta nor calcium/calmodulin-dependent protein kinase II did so. However, the mass spectra of the tail fragments of the phosphorylated NF-L indicated that CK II was the kinase mediating Ser-473 phosphorylation in vitro as opposed to CK I, because CK I phosphorylated another site as well as Ser-473 in vitro. The antibody also demonstrated that NF-L phosphorylated at Ser-473 was abundant in the neuronal perikarya of the rat cortex, indicating that phosphorylation of Ser-473 may take place there. This result may support the suggestion that CK II is the kinase responsible for Ser-473 phosphorylation. Despite many reports showing that CK I mediates phosphorylation of neurofilaments, CK II may phosphorylate NF-L in vivo.     

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.     

Identification of Ser-55 as a major protein kinase A phosphorylation site on the 70-kDa subunit of neurofilaments. Early turnover during axonal transport.

The 70-kDa neurofilament protein subunit (NF-L) is phosphorylated in vivo on at least three sites (L1 to L3) (Sihag, R. K. and Nixon, R. A. (1989) J. Biol. Chem. 264, 457-464). The turnover of phosphate groups on NF-L during axonal transport was determined after the neurofilaments in retinal ganglion cells were phosphorylated in vivo by injecting mice intravitreally with [32P]orthophosphate. Two-dimensional phosphopeptide maps of NF-L from optic axons of mice 10 to 90 h after injection showed that radiolabel decreased faster from peptides L2 and L3 than from L1 as neurofilaments were transported. To identify phosphorylation sites on peptide L2, axonal cytoskeletons were phosphorylated by protein kinase A in the presence of heparin. After the isolated NF-L subunits were digested with alpha-chymotrypsin, 32P-peptides were separated by high performance liquid chromatography on a reverse-phase C8 column. Two-dimensional peptide mapping showed that the alpha-chymotrypsin 32P-peptide accepting most of the phosphates from protein kinase A migrated identically with the in vivo-labeled phosphopeptide L2. The sequence of this peptide (S-V-R-R-S-Y) analyzed by automated Edman degradation corresponded to amino acid residues 51-56 of the NF-L sequence. A synthetic 13-mer (S-L-S-V-R-R-S-Y-S-S-S-S-G) corresponding to amino acid residues 49-61 of NF-L was also phosphorylated by protein kinase A. alpha-Chymotryptic digestion of the 13-mer generated a peptide which contained most of the phosphates and co-migrated with the phosphopeptide L2 on two-dimensional phosphopeptide maps. Edman degradation of the phosphorylated 13-mer identified serine residue 55 which is located within a consensus phosphorylation sequence for protein kinase A as the major site of phosphorylation. Since protein kinase A-mediated phosphorylation influences intermediate filament assembly/disassembly in vitro, we propose that the phosphopeptide L2 region is a neurofilament-assembly domain and that the cycle of phosphorylation and dephosphorylation of Ser-55 on NF-L, which occurs relatively early after subunit synthesis in vivo, regulaaes a step in neurofilament assembly or initial interactions during axonal transport.     

In vivo and in vitro phosphorylation at Ser-493 in the glutamate (E)-segment of neurofilament-H subunit by glycogen synthase kinase 3beta

Neurofilament (NF), a major neuronal intermediate filament, is composed of three subunits, NF-L, NF-M, and NF-H. All three subunits contain a well conserved glutamate (E)-rich region called "E-segment" in the N terminus of the tail region. Although the E-segments of NF-L and NF-M are phosphorylated by casein kinases, it has not been observed in NF-H. Using mass spectrometric analysis, we identified phosphorylation of the E-segment of NF-H, prepared from rat spinal cords, at Ser-493 and Ser-501 in the Ser-Pro sequences. The E-segment kinase was isolated from rat brain extract using column chromatography and identified as glycogen synthase kinase (GSK) 3beta. GSK3beta was shown to phosphorylate at Ser-493 in vitro by phosphopeptide mapping and site-directed mutagenesis, and in vivo in HEK293 cells using the phospho-Ser-493 antibody, but did not phosphorylate Ser-501. GSK3beta preferred Ser-493 to the KSP-repeated sequences for phosphorylation sites in the NF-H tail domain. Moreover, Ser-493 was a better phosphorylation site for GSK3beta than other proline-directed protein kinases, Cdk5/p35 and ERK. GSK3beta in the spinal cord extract was associated with NF cytoskeletons. Taken together, we concluded that Ser-493 in the E-segment of NF-H is phosphorylated by GSK3beta in rat spinal cords.     

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.     

Protein kinase C phosphorylation of desmin at four serine residues within the non-alpha-helical head domain

We reported that phosphorylation by either cAMP-dependent protein kinase or protein kinase C (Ca2+/phospholipid-dependent enzyme) in vitro induces disassembly of the desmin filaments (Inagaki, M., Gonda, Y., Matsuyama, M., Nishizawa, K., Nishi, Y., and Sato, C. (1988) J. Biol. Chem. 263, 5970-5978). For this subunit protein, Ser-29, Ser-35, and Ser-50 within the non-alpha-helical head domain were shown to be the sites of phosphorylation for cAMP-dependent protein kinase (Geisler, N., and Weber, K. (1988) EMBO J. 7, 15-20). In the present work, we identified the sites of desmin phosphorylated in vitro by other protein kinase which affects the filament structure. The protein kinase C-phosphorylated desmin was hydrolyzed with trypsin, and the phosphorylated peptides were isolated by reverse-phase chromatography. Sequential analysis of the purified phosphopeptides, together with the known primary sequence, revealed that Ser-12, Ser-29, Ser-38, and Ser-56 were phosphorylated by protein kinase C. All four sites are located within the non-alpha-helical head domain of desmin. Ser-12, Ser-38, and Ser-56, specifically phosphorylated by protein kinase C, have arginine residues at the carboxyl-terminal side (Arg-14, Arg-42, and Arg-59, respectively). Ser-29 phosphorylated by both protein kinase C and cAMP-dependent protein kinase has arginine residues at the amino and carboxyl termini (Arg-27 and Arg-33). These findings support the view that the head domain-specific phosphorylation strongly influences desmin filament structure; however, each protein kinase differed with regard to site recognition on this domain.     

Studies on the phosphorylation of myelin basic protein by protein kinase C and adenosine 3':5'-monophosphate-dependent protein kinase

The substrate specificity of protein kinase C was studied and compared with that of cyclic AMP-dependent protein kinase (protein kinase A) by using bovine brain myelin basic protein as a model substrate. This basic protein was phosphorylated at multiple sites by both of these protein kinases. In this analysis, the basic protein was thoroughly phosphorylated in vitro with [gamma-32P]ATP and each protein kinase, and then digested with trypsin. The resulting radioactive phosphopeptides were isolated by gel filtration followed by high performance liquid chromatography on a reverse-phase column. Subsequent amino acid analysis and/or sequential Edman degradation of the purified phosphopeptides, together with the known primary sequence of this protein, revealed that Ser-46 and Ser-151 were specifically phosphorylated by protein kinase C, whereas Thr-34 and Ser-115 were phosphorylated preferentially by protein kinase A. Both kinases reacted with Ser-8, Ser-11, Ser-55, Ser-110, Ser-132, and Ser-161 at various reaction velocities. Contrary to protein kinase A, protein kinase C appears to react preferentially with seryl residues that are located at the amino-terminal side close to lysine or arginine. The seryl residues that are phosphorylated commonly by these two protein kinases have basic amino acids at both the amino- and carboxyl-terminal sides. These results provide some clues to understanding the rationale that these kinases may show different but sometimes similar functions depending on the structure of target phosphate acceptor proteins.     

Determination of the phosphorylation sites of calmodulin catalyzed by casein kinase 2

Calmodulin is specifically phosphorylated by casein kinase 2 (CK 2), but not by casein kinase 1, A kinase, or C kinase. In the present report, the stoichiometry of the phosphorylation of calmodulin by CK 2 in the presence and absence of polylysine and its phosphorylation sites were examined. In the absence of polylysine, the radioactive phosphate incorporated into calmodulin by CK 2 was only 0.01 mol/mol and the phosphorylation occurred at Ser-101. In the presence of polylysine, 1.2 mol of radioactive phosphate was incorporated into 1 mol of calmodulin. In this case, Thr-79 in addition to Ser-101 was phosphorylated, but Ser-81 was not. The sequence around the phosphorylated Thr is Asp-Thr(P)-Asp-Ser-Glu-Glu-Glu-.     

Domain- and sequence-specific phosphorylation of vimentin induces disassembly of the filament structure

We reported that stoichiometric phosphorylation by either cAMP-dependent protein kinase or protein kinase C induces disassembly of vimentin filaments [Inagaki, M., Nishi, Y., Nishizawa, K., Matsuyama, M., & Sato, C. (1987) Nature 328, 649-652; Inagaki, M., Gonda, Y., Matsuyama, M., Nishizawa, K., Nishi, Y., & Sato, C. (1988) J. Biol. Chem. 263, 5970-5978]. In the present work, we attempted to identify the sites of vimentin phosphorylated by each protein kinase. Sequential analysis of the purified phosphopeptides, together with the known primary sequence, revealed that Ser-8, Ser-9, Ser-20, Ser-25, Ser-33, and Ser-41 were specifically phosphorylated by protein kinase C, whereas Ser-46 was phosphorylated preferentially by cAMP-dependent protein kinase. Both kinases reacted with Ser-6, Ser-24, Ser-38, Ser-50, and Ser-65. Specific phosphorylation sites for protein kinase C are mostly located close to the amino-terminal side of arginine while those for cAMP-dependent protein kinase are located close to the carboxyl-terminal side of arginine. The phosphorylation sites exclusively occur in the amino-terminal non-alpha-helical head domain, particularly at the beta-turn region. These results provide clues to the molecular mechanisms of phosphorylation-dependent disassembly of vimentin filaments.     

Phosphorylation of native and reassembled neurofilaments composed of NF-L, NF-M, and NF-H by the catalytic subunit of cAMP-dependent protein kinase.

Phosphorylation of neurofilament-L protein (NF-L) by the catalytic subunit of cAMP-dependent protein kinase (A-kinase) inhibits the reassembly of NF-L and disassembles filamentous NF-L. The effects of phosphorylation by A-kinase on native neurofilaments (NF) composed of three distinct subunits: NF-L, NF-M, and NF-H, however, have not yet been described. In this paper, we examined the effects of phosphorylation of NF proteins by A-kinase on both native and reassembled filaments containing all three NF subunits. In the native NF, A-kinase phosphorylated each NF subunit with stoichiometries of 4 mol/mol for NF-L, 6 mol/mol for NF-M, and 4 mol/mol for NF-H. The extent of NF-L phosphorylation in the native NF was nearly the same as that of purified NF-L. However, phosphorylation did not cause the native NFs to disassemble into oligomers, as was the case for purified NF-L. Instead, partial fragmentation was detected in sedimentation experiments and by electron microscopic observations. This is probably not due to the presence of the three NF subunits in NF or to differences in phosphorylation sites because reassembled NF containing all three NF subunits were disassembled into oligomeric forms by phosphorylation with A-kinase and the phosphorylation by A-kinase occurred at the head domain of NF-L whether NF were native or reassembled. Disassembling intermediates of reassembled NF containing all three NF subunits were somewhat different from disassembling intermediates of NF-L. Thinning and loosening of filaments was frequently observed preceding complete disassembly. From the fact that the thinning was also observed in the native filaments phosphorylated by A-kinase, it is reasonable to propose the native NF is fragmented through a process of thinning that is stimulated by phosphorylation in the head domain of the NF subunits.     

Autophosphorylation-dependent protein kinase phosphorylates Ser25, Ser38, Ser65, Ser71, and Ser411 in vimentin and thereby inhibits cytoskeletal intermediate filament assembly

The autophosphorylation-dependent protein kinase has been identified as a potent vimentin kinase that incorporates 2 mol of phosphates per mol of protein and generates five major phosphorylation sites in vimentin. Tryptic phosphopeptide mapping by high-performance liquid chromatography followed by sequential manual Edman degradation and direct peptide sequence analysis revealed that Ser-25, Ser-38, Ser-65, and Ser-71 in the amino-terminal domain and Ser-411 in the carboxyl-terminal domain are the phosphorylation sites in vimentin phosphorylated by this kinase, indicating that autophosphorylation-dependent protein kinase is a potent and unique vimentin kinase. Functional study further revealed that phosphorylation of vimentin by autophosphorylation-dependent protein kinase can completely inhibit polymerization and assembly of the cytoskeletal intermediate filament as demonstrated by electron microscopic analysis. Taken together, the results provide initial evidence that the autophosphorylation-dependent protein kinase may function as a vimentin kinase involved in the structure-function regulation of the cytoskeletal system. The results also support the notion that this cyclic nucleotide- and calcium-independent protein kinase may function as a multisubstrate/multifunctional protein kinase involved in the regulation of diverse cell functions.     

Autophosphorylation-dependent protein kinase phosphorylates Ser25, Ser38, Ser65, Ser71, and Ser411 in vimentin and thereby inhibits cytoskeletal intermediate filament assembly

The autophosphorylation-dependent protein kinase has been identified as a potent vimentin kinase that incorporates 2 mol of phosphates per mol of protein and generates five major phosphorylation sites in vimentin. Tryptic phosphopeptide mapping by high-performance liquid chromatography followed by sequential manual Edman degradation and direct peptide sequence analysis revealed that Ser-25, Ser-38, Ser-65, and Ser-71 in the amino-terminal domain and Ser-411 in the carboxyl-terminal domain are the phosphorylation sites in vimentin phosphorylated by this kinase, indicating that autophosphorylation-dependent protein kinase is a potent and unique vimentin kinase. Functional study further revealed that phosphorylation of vimentin by autophosphorylation-dependent protein kinase can completely inhibit polymerization and assembly of the cytoskeletal intermediate filament as demonstrated by electron microscopic analysis. Taken together, the results provide initial evidence that the autophosphorylation-dependent protein kinase may function as a vimentin kinase involved in the structure-function regulation of the cytoskeletal system. The results also support the notion that this cyclic nucleotide- and calcium-independent protein kinase may function as a multisubstrate/multifunctional protein kinase involved in the regulation of diverse cell functions.     

Further studies on the phosphorylation and kinetics of rat liver fructose-1,6-bisphosphatase: importance of the three-dimensional structure of a substrate to protein kinase A.

Rat liver fructose-1,6-bisphosphatase was phosphorylated by cAMP-dependent protein kinase to 2.6 mol phosphate/mol subunit but not by Ca2+/phospholipid-dependent and Ca2+/calmodulin-dependent protein kinases. It was demonstrated that phosphorylation of Ser-341 and Ser-356, and to a much lower extent, Ser-338, was dependent on the presence of intact arginine residues. This observation implicates that the intact three-dimensional structure of the substrate is necessary for phosphorylation of Ser-356 since the closest arginine is located at a six amino acid residue distance.     

Identification of the phosphorylation sites of the murine small heat shock protein hsp25.

Native phosphorylated mouse small heat shock protein hsp25 from Ehrlich ascites tumor cells was isolated and the in vivo phosphorylation sites of the protein were determined. Furthermore, native hsp25 was phosphorylated by the endogenous kinase(s) in a cell-free system as well as recombinant hsp25 was phosphorylated in vitro by protein kinase C and catalytic subunit of cAMP-dependent protein kinase. The two major phosphorylation sites of native and recombinant hsp25 were determined as Ser-15 and Ser-86. There are no differences in the hsp25 phosphorylation sites phosphorylated by the protein kinase C, the catalytic subunit of cAMP-dependent protein kinase and the unknown intracellular kinase(s). The serine residues identified exist in all known small mammalian stress proteins and are located in the conserved kinase recognition sequence Arg-X-X-Ser.     

Proline-Directed Protein Kinase (p34cdc2/p58cyclin A) Phosphorylates Bovine Neurofilaments

Proline-directed protein kinase (PDPK), a complex of p34cdc2 and p58cyclin A, phosphorylates bovine neurofilaments (NFs) in vitro. Incubation of intact filaments with PDPK led to strong labeling of the heavy (NF-H) and middle (NF-M) molecular weight NF proteins and weaker labeling of the low molecular weight protein (NF-L). All three proteins were phosphorylated in solution, with the best substrate being NF-H. Proteins that had been dephosphorylated by enzymatic treatment were better substrates than native proteins--as many as 6 mol of phosphate were incorporated per mole of NF-H. Partial proteolytic cleavage experiments combined with two-dimensional peptide mapping indicated that NF-H and NF-M were phosphorylated predominantly in the tail domains, with some phosphate also appearing in the heads. Soluble NF-L is phosphorylated on the head domain peptide L-3, whereas NF-L within intact filaments is phosphorylated only on the tail domain peptide L-1. Phosphorylation does not lead to filament disassembly. A possible role for PDPK in NF phosphorylation in vivo is discussed.     

Site-specific Phosphorylation of CXCR4 Is Dynamically Regulated by Multiple Kinases and Results in Differential Modulation of CXCR4 Signaling

The chemokine receptor CXCR4 is a widely expressed G protein-coupled receptor that has been implicated in a number of diseases including human immunodeficiency virus, cancer, and WHIM syndrome, with the latter two involving dysregulation of CXCR4 signaling. To better understand the role of phosphorylation in regulating CXCR4 signaling, tandem mass spectrometry and phospho-specific antibodies were used to identify sites of agonist-promoted phosphorylation. These studies demonstrated that Ser-321, Ser-324, Ser-325, Ser-330, Ser-339, and two sites between Ser-346 and Ser-352 were phosphorylated in HEK293 cells. We show that Ser-324/5 was rapidly phosphorylated by protein kinase C and G protein-coupled receptor kinase 6 (GRK6) upon CXCL12 treatment, whereas Ser-339 was specifically and rapidly phosphorylated by GRK6. Ser-330 was also phosphorylated by GRK6, albeit with slower kinetics. Similar results were observed in human astroglia cells, where endogenous CXCR4 was rapidly phosphorylated on Ser-324/5 by protein kinase C after CXCL12 treatment, whereas Ser-330 was slowly phosphorylated. Analysis of CXCR4 signaling in HEK293 cells revealed that calcium mobilization was primarily negatively regulated by GRK2, GRK6, and arrestin3, whereas GRK3, GRK6, and arrestin2 played a primary role in positively regulating ERK1/2 activation. In contrast, GRK2 appeared to play a negative role in ERK1/2 activation. Finally, we show that arrestin association with CXCR4 is primarily driven by the phosphorylation of far C-terminal residues on the receptor. These studies reveal that site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases resulting in both positive and negative modulation of CXCR4 signaling.     

Site-specific phosphorylation of neurofilament-L is mediated by calcium/calmodulin-dependent protein kinase II in the apical dendrites during long-term potentiation

Neurofilament-L (NF-L), one subunit of the neuronal intermediate filaments, is a major element of neuronal cytoskeletons. The dynamics of NF-L are regulated by phosphorylation of its head domain. The phosphorylation sites of the NF-L head domain by protein kinase A, protein kinase C, and Rho-associated kinase have been previously identified, and those by calcium/calmodulin-dependent protein kinase II (CaMKII) were identified in this study. A series of site- and phosphorylation state-specific antibodies against NF-L was prepared to investigate NF-L phosphorylation in neuronal systems. Long-term potentiation (LTP) is a cellular model of neuronal plasticity that is thought to involve the phosphorylation of various proteins. NF-L is considered a possible substrate for phosphorylation. During LTP stimulation of mouse hippocampal slices, the series of antibodies demonstrated the increase in the phosphorylation level of Ser(57) in NF-L and the visualization of the localized distribution of Ser(57) phosphorylation in a subpopulation of apical dendrites of the pyramidal neurons. Furthermore, Ser(57) phosphorylation during LTP is suggested to be mediated by CaMKII. Here we show that NF-L is phosphorylated by CaMKII in a subpopulation of apical dendrites during LTP, indicating that Ser(57) is a novel phosphorylation site of NF-L in vivo related to the neuronal signal transduction.     

Phosphorylation sites linked to glial filament disassembly in vitro locate in a non-alpha-helical head domain

Glial fibrillary acidic protein (GFAP), the intermediate filament component of astroglial cells, can serve as an excellent substrate for both cAMP-dependent protein kinase and protein kinase C, in vitro. GFAP phosphorylated by each protein kinase does not polymerize, and the filaments that do polymerize tend to depolymerize after phosphorylation. Dephosphorylation of phospho-GFAP by phosphatase led to a recovery of the polymerization competence of GFAP. Most of the phosphorylation sites for cAMP-dependent protein kinase and protein kinase C on GFAP are the same, Ser-8, Ser-13, and Ser-34. cAMP-dependent protein kinase has one additional phosphorylation site, Thr-7. All the sites are located within the amino-terminal non-alpha-helical head domain of GFAP. These observations pave the way for in vivo studies on organization of glial filaments.