Expression and activation of Akt/protein kinase B in sexually immature and mature rat uterus

This study investigated the expression and activation of Akt/PKB in developing and adult rat uterus. Expression of Akt was observed in uteri from adult ovariectomized and 7–35-day-old rats and no changes were observed in response to in vivo estradiol treatment (1–100 μg/100 g b.w.). To examine the mechanisms of PKB/Akt activation, phosphorylation at Thr³⁰⁸ and Ser⁴⁷³ regulatory sites were studied in uteri. Akt was constitutively phosphorylated on Ser⁴⁷³ residue in the untreated, control uteri, while phosphorylation of Thr³⁰⁸ was observed only after estradiol 17β (E2) treatment. The effects of E2 treatment were age dependent, no response was induced in 11-day-old uteri, while in 28 days and older rats the activation of Akt at both regulatory sites, Ser⁴⁷³ and Thr³⁰⁸, increased, the first response was detected 2 h after treatment, reaching the highest rate at 6 h. The rate of phosphorylation was stronger at Ser⁴⁷³ residue. The results suggest that the regulation of Akt activation at two regulatory sites in rat uteri are different, phosphorylation of Thr³⁰⁸ seems to be entirely estrogen dependent, while the phosphorylation of Ser⁴⁷³ is regulated by other factors as well as estrogen.     

Calcium/calmodulin-dependent protein kinase I inhibits neuronal nitric-oxide synthase activity through serine 741 phosphorylation

We demonstrate here that neuronal nitric-oxide synthase (nNOS) is phosphorylated and inhibited by a constitutively active form of Ca²⁺/calmodulin (CaM)-dependent protein kinase I (CaM-K I1-293). Substitution of Ser⁷⁴¹ to Ala in nNOS blocked the phosphorylation and the inhibitory effect. Mimicking phosphorylation at Ser⁷⁴¹ by Ser to Asp mutation resulted in decreased binding of and activation by CaM, since the mutation was within the CaM-binding domain. CaM-K I1-293 gave phosphorylation of nNOS at Ser⁷⁴¹ in transfected cells, resulting in 60–70% inhibition of nNOS activity. Wild-type CaM-K I also did phosphorylate nNOS at Ser⁷⁴¹ in transfected cells, but either CaM-K II or CaM-K IV did not. These results raise the possibility of a novel cross-talk between nNOS and CaM-K I through the phosphorylation of Ser⁷⁴¹ on nNOS.     

Protein phosphatase type 2C dephosphorylates BAD

Reversible phosphorylation modulates a cells’ susceptibility to apoptosis. The phosphorylation status of BAD, a member of the Bcl-2 protein family, is an important checkpoint governing life-or-death decisions: Phosphorylation of serine residues 112, 136 and 155 on BAD prevents apoptosis. Here we report that BAD is a substrate for PP2C. Ser¹⁵⁵ is involved in heterodimerization with Bcl-X_L. We could demonstrate that PP1, PP2A and PP2C act on this site in vitro. However, only PP2C gives priority to P-Ser¹⁵⁵ compared to P-Ser¹¹² and P-Ser¹³⁶ on BAD. The results indicate that PP2C is an additional factor triggering the pro-apoptotic function of BAD.     

Distinct importin recognition properties of histones and chromatin assembly factors

The adhesive protein vitronectin (75 kDa) occurs in human blood fluid in a one-chain (Vn₇₅) or a two-chain form (Vn₆₅₊₁₀), and is produced by a specific cleavage (at Arg³⁷⁹–Ala³⁸⁰), by a proteinase not identified hitherto. These two forms were shown to be functionally different and therefore, this cleavage may have a regulatory significance in vivo. Here, we report the use of a tailored one-chain recombinant Vn, a specific protein kinase A phosphorylation at Ser³⁷⁸, and sequence analysis to show: (1) that none of the proteinases originating from blood, previously thought to be the endogenous proteinase (plasmin, thrombin, tPA, and uPA), is indeed the in vivo convertase; and (2) that furin, a serine endoproteinase residing in the secretory pathway of hepatocytes, where Vn is synthesized, specifically cleaves Vn at the endogenous cleavage site. Consequently, we propose that the Vn₇₅ to Vn₆₅₊₁₀ conversion takes place in the liver (not in blood) and is carried out by furin.     

Role of Serine-19 Phosphorylation in Regulating Tyrosine Hydroxylase Studied with Site- and Phosphospecific Antibodies and Site-Directed Mutagenesis

Abstract: The effects of depolarization by elevated potassium concentrations were studied in PC12 cells and in stably transfected AtT-20 cells expressing wild-type or [Leu¹⁹]-recombinant tyrosine hydroxylase (rTH). Changes in the phosphorylation states of Ser¹⁹ and Ser⁴⁰ in tyrosine hydroxylase (TH) were determined immunochemically using antibodies specific for the phosphorylated state of each site and compared with changes in TH activity in PC12 cell lysates and with changes in l -DOPA biosynthesis rates in intact AtT-20 cells. Treatment of either PC12 cells or AtT-20 cells expressing wild-type rTH with elevated potassium produced a transient increase in the phosphorylation state of Ser¹⁹ (up to 0.7 mol of phosphate/mol of subunit) in concert with a more gradual and sustained increase in Ser⁴⁰ phosphorylation. Elevated potassium treatment also increased TH activity in PC12 cell lysates, but these increases paralleled the temporal course of Ser⁴⁰, as opposed to Ser¹⁹, phosphorylation. Similarly, increases in DOPA accumulation produced by elevated potassium in AtT-20 cells expressing wild-type rTH paralleled the increases in the phosphorylation state of Ser⁴⁰ but not Ser¹⁹. Moreover, elevated potassium produced comparable increases in DOPA accumulation in AtT-20 cells expressing rTH in which Ser¹⁹ phosphorylation had been eliminated (by substitution of Leu for Ser¹⁹). Thus, depolarization-induced increases in the stoichiometry of Ser¹⁹ phosphorylation do not appear to influence directly the activity of TH in situ.     

Phosphorylation of Delta Sleep-Inducing Peptide (DSIP) by casein kinase II in vitro

A phosphorylated analogue of DSIP at Ser⁷ has been shown to exist endogenously by immunochemical studies. An enzyme which could phosphorylate DSIP has not yet been identified. In the present study, we examined DSIP as a substrate for in vitro phosphorylation by casein kinase II. DSIP was phosphorylated by the enzyme with apparent K_m and V_max values of 20 mM and 90.9 nmol/min/mg protein, respectively. Both ATP and GTP were utilized as phosphoryl donors. Phosphorylation of DSIP was inhibited by heparin and enhanced by spermine. These results demonstrate that DSIP can serve as a possible substrate for casein kinase II in vitro.     

Different kinases desensitize the human delta-opioid receptor (hDOP-R) in the neuroblastoma cell line SK-N-BE upon peptidic and alkaloid agonists

In a previous work, we described a differential desensitization of the human δ-opioid receptor (hDOP-R) by etorphine (a non-selective and alkaloid agonist) and δ-selective and peptidic agonists (DPDPE ([d-Pen^2,5]enkephalin) and deltorphin I (Tyr-d-Ala-Phe-Asp-Val-Val-Gly-NH₂)) in the neuroblastoma cell line SK-N-BE (Allouche et al., Eur. J. Pharmacol., 371, 235, 1999). In the present study, we explored the putative role of different kinases in this differential regulation.

First, selective chemical inhibitors of PKA, PKC and tyrosine kinases were used and we showed a significant reduction of etorphine-induced opioid receptor desensitization by the bisindolylmaleimide I (PKC inhibitor) while genistein (tyrosine kinase inhibitor) was potent to impair desensitization induced by the different agonists. When the PKA was inhibited by H89 pretreatment, no modification of opioid receptor desensitization was observed whatever the agonist used.

Second, we further studied the role of G protein-coupled receptor kinases (GRKs) and by using western-blot experiments we observed that only the GRK2 isoform was expressed in the SK-N-BE cells. Next, the neuroblastoma cells were transfected with the wild type GRK2 or its dominant negative mutant GRK2-K220R and the inhibition on cAMP level was determined in naïve and agonist-pretreated cells. We showed that over-expression of GRK2-K220R totally abolished etorphine-induced receptor desensitization while no effect was observed with peptidic agonists and over-expression of GRK2 selectively impaired cAMP inhibition promoted by etorphine suggesting that this kinase was involved in the regulation of hDOP-R activated only by etorphine.

Third, correlation between functional experiments and phosphorylation of the hDOP-R after agonist activation was assessed by western-blot using the specific anti-phospho-DOP-R Ser³⁶³ antibody. While all agonists were potent to increase phosphorylation of opioid receptor, we showed no impairment of receptor phosphorylation level after PKC inhibitor pretreatment. Upon agonist activation, no enhancement of receptor phosphorylation was observed when the GRK2 was over-expressed while the GRK2-K220R partially reduced the hDOP-R Ser³⁶³ phosphorylation only after peptidic agonists pretreatment.

In conclusion, hDOP-R desensitization upon etorphine exposure relies on the GRK2, PKC and tyrosine kinases while DPDPE and deltorphin I mediate desensitization at least via tyrosine kinases. Although the Ser³⁶³ was described as the primary phosphorylation site of the mouse DOP-R, we observed no correlation between desensitization and phosphorylation of this amino acid.     

Characterization of Additional Casein Kinase I Sites in the C-Terminal "Tail" Region of Chicken and Rat Neurofilament-M

Abstract: 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 (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 α-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.     

Manganese induces sustained Ser40 phosphorylation and activation of tyrosine hydroxylase in PC12 cells

Manganese (Mn²⁺) is an essential metal involved in normal functioning of a range of physiological processes. However, occupational overexposure to Mn²⁺ causes neurotoxicity. The dopaminergic system is a particular target for Mn²⁺ neurotoxicity. Tyrosine hydroxylase (TH) is the rate limiting enzyme for dopamine synthesis and is regulated acutely by phosphorylation at Ser40 and chronically by protein synthesis. In this study we used pheochromocytoma 12 cells to investigate the effects of Mn²⁺ exposure on the phosphorylation and activity of TH. Mn²⁺ treatment for 24 h caused a sustained increase in Ser40 phosphorylation and TH activity at a concentration of 100 μM, without altering the level of TH protein or PC12 cell viability. Inhibition of protein kinase A and protein kinase C and protein kinases known to be involved in sustained phosphorylation of TH in response to other stimuli did not block the effects of Mn²⁺ on Ser40 phosphorylation. A substantial increase in H₂O₂ production occurred in response to 100 μM Mn²⁺. The antioxidant Trolox^TM completely inhibited H₂O₂ production but did not block TH phosphorylation at Ser40, indicating that oxidative stress was not involved. Sustained TH phosphorylation at Ser40 and the consequent activation of TH both occurred at low concentrations of Mn²⁺ and this provides a potential new mechanism for Mn²⁺-induced neuronal action that does not involve H₂O₂-mediated cell death.     

Identification and characterization of SEB, a novel protein that binds to the acute undifferentiated leukemia-associated protein SET.

SET, the translocation breakpoint-encoded protein in acute undifferentiated leukemia (AUL), is a 39-kDa nuclear phosphoprotein and has an inhibitory activity for protein phosphatase 2A (PP2A). SET is fused to a putative oncoprotein, CAN/NUP214, in AUL and is thought to play a key role in leukemogenesis by its nuclear localization, protein-protein interactions and PP2A inhibitory activity. Here, we describe the isolation and characterization of a novel cDNA encoding a protein with 1542 amino-acid residues that specifically interacts in a yeast two-hybrid system as well as in human cells with SET. This new protein, which we name SEB (SET-binding protein), is identified as a 170-kDa protein by immunoprecipitation with a specific antibody and is localized predominantly in the nucleus. SEB1238--1434 is determined as a SET-binding region that specifically binds to SET182--223. SEB also has an oncoprotein Ski homologous region (amino acids 654--858), six PEST sequences and three sequential PPLPPPPP repeats at the C-terminus. SEB mRNA is expressed ubiquitously in all human adult tissues and cells examined. The SEB gene locus is assigned to the chromosome 18q21.1 that contains candidate tumor suppressor genes associated with deletions in cancer and leukemia. Although the function of SEB is not known, we propose that SEB plays a key role in the mechanism of SET-related leukemogenesis and tumorigenesis, perhaps by suppressing SET function or by regulating the transforming activity of Ski in the nucleus.     

Fibroblast growth factor 9 prevents MPP+-induced death of dopaminergic neurons and is involved in melatonin neuroprotection in vivo and in vitro

Oxidative stress and down-regulated trophic factors are involved in the pathogenesis of nigrostriatal dopamine(DA)rgic neurodegeneration in Parkinson's disease. Fibroblast growth factor 9 (FGF9) is a survival factor for various cell types; however, the effect of FGF9 on DA neurons has not been studied. The antioxidant melatonin protects DA neurons against neurotoxicity. We used MPP⁺ to induce neuron death in vivo and in vitro and investigated the involvement of FGF9 in MPP⁺ intoxication and melatonin protection. We found that MPP⁺ in a dose- and time-dependent manner inhibited FGF9 mRNA and protein expression, and caused death in primary cortical neurons. Treating neurons in the substantia nigra and mesencephalic cell cultures with FGF9 protein inhibited the MPP⁺-induced cell death of DA neurons. Melatonin co-treatment attenuated MPP⁺-induced FGF9 down-regulation and DA neuronal apoptosis in vivo and in vitro . Co-treating DA neurons with melatonin and FGF9-neutralizing antibody prevented the protective effect of melatonin. In the absence of MPP⁺, the treatment of FGF9-neutralizing antibody-induced DA neuronal apoptosis whereas FGF9 protein reduced it indicating that endogenous FGF9 is a survival factor for DA neurons. We conclude that MPP⁺ down-regulates FGF9 expression to cause DA neuron death and that the prevention of FGF9 down-regulation is involved in melatonin-provided neuroprotection.     

Requirements for the phosphorylation of the Escherichia coli EIIANtr protein in vivo

The nitrogen-related phosphotransferase system (Ntr-PTS) is a paralogous system working in parallel to the well-known carbohydrate:PTS. In a chain of phosphotransfer reactions, EI^Ntr and NPr (PtsO) deliver phosphoryl groups to the EIIA^Ntr (PtsN) protein. EIIA^Ntr is implicated in important regulatory processes such as the σ^E-dependent cell envelope stress response and regulation of K⁺ uptake. Phosphorylation is believed to trigger the output of EIIA^Ntr in these regulations. EIIA^Ntr is encoded within the gene cluster ptsN–yhbJ–ptsO , which is highly conserved in Proteobacteria . In this study, we investigated the phosphorylation of the Escherichia coli EIIA^Ntr protein in vivo by ³²P-labeling. We show that EIIA^Ntr is readily phosphorylated in wild-type cells. This phosphorylation occurs at a single site, the histidine 73 in EIIA^Ntr. YhbJ and NPr are dispensable for this phosphorylation. A detailed analysis revealed that both the energy coupling phosphotransferases of the Ntr-PTS as well as the 'sugar'-PTS contribute to the phosphorylation of EIIA^Ntr, suggesting cross talk between both systems.     

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.     

Protein Kinase FA/Glycogen Synthase Kinase 3α Predominantly Phosphorylates the In Vivo Sites of Ser502, Ser506, Ser603, and Ser666 in Neurofilament

Abstract: In this report, the phosphorylation sites of neurofilament protein of medium molecular mass (NF-M) by protein kinase F_A/glycogen synthase kinase 3α (kinase F_A/GSK-3α) were determined by two-dimensional electrophoresis/TLC, phosphoamino acid analysis, HPLC, Edman degradation, and peptide sequencing. Kinase F_A/GSK-3α phosphorylates NF-M predominantly on serine, residue. Three major tryptic phosphopeptide peaks were resolved by C₁₈ reverse-phase HPLC. Edman degradation and peptide sequence analysis revealed that AKS(p)PVSK is the phosphorylation site sequence for the first major peak. When mapping with the amino acid sequence of neurofilament, we finally demonstrate Ser⁶⁰³-Pro, one of the in vivo sites in NF-M, as the major site phosphorylated by kinase F_A/GSK-3α. By using the same approach, we also identified the in vivo sites of Ser⁵⁰²-Pro, Ser⁵⁰⁶-Pro, and Ser⁶⁶⁶-Pro as the other three major sites in NF-M phosphorylated by kinase F_A/GSK-3α. Taken together, the results provide initial evidence that kinase F_A/GSK-3α may represent a physiologically relevant protein kinase involved in the in vivo phosphorylation of NF-M. Because Ser⁵⁰², Ser⁵⁰⁶, Ser⁶⁰³, and Ser⁶⁶⁶ are all flanked by a carboxyl-terminal proline residue, the results provide further evidence that F_A/GSK-3α may represent a proline-directed protein kinase involved in the structure-function regulation of the neuronal cytoskeletal system.     

τ Protein Kinase II Is Involved in the Regulation of the Normal Phosphorylation State of τ Protein

Abstract: To study the phosphorylation state of τ in vivo, we have prepared antisera by immunizing rabbits with synthetic phosphopeptides containing phosphoamino acids at specific sites that are potential targets for τ protein kinase II. Immunoblot experiments using these antisera demonstrated that τ in microtubule-associated proteins is phosphorylated at Ser¹⁴⁴ and at Ser³¹⁵. Almost all τ variants separated on two-dimensional gel electrophoresis were phosphorylated at Ser¹⁴⁴ and nearly one-half of them at Ser³¹⁵. Phosphorylation at Ser¹⁴⁴ and at Thr¹⁴⁷ of τ isolated from heat-stable brain extracts was shown to be developmentally regulated, with the highest level of phosphorylation found at postnatal week 1. In vitro phosphorylation of τ by τ protein kinase I, a kinase responsible for abnormal phosphorylation of τ found in paired helical filaments of patients with Alzheimer's disease, was enhanced by prior phosphorylation of τ by τ protein kinase II. Thus, we suggest that τ protein kinase II is indirectly involved, at least in part, in the regulation of the phosphorylation state of τ in neuronal cells.     

Signaling Pathway Downstream of GABAA Receptor in the Growth Cone

Abstract: The growth cone is responsible for axonal elongation and pathfinding by responding to various modulators for neurite growth, including neurotransmitters, although the sensor mechanisms are not fully understood. Among neurotransmitters, GABA is most likely to demonstrate activity in vivo because GABA and the GABA_A receptor appear even in early stages of CNS development. We investigated the GABA_A receptor-mediated signaling pathway in the growth cone using isolated growth cones (IGCs). Both the GABA_A binding site and the benzodiazepine modulatory site were enriched in the growth cone membrane. In the intact IGC, GABA induced picrotoxin-sensitive Cl⁻ flux (not influx but efflux) and increased the intracellular Ca²⁺ concentration in a picrotoxin- and verapamil-sensitive manner. Protein kinase C (PKC)-dependent phosphorylation of two proteins identified as GAP-43 and MARCKS protein was enhanced in the intact IGC stimulated by GABA, resulting in the release of MARCKS protein and GAP-43 from the membrane. Collectively, our results suggest the following scheme: activation of the functional GABA_A receptor localized in the growth cone membrane → Cl⁻ efflux induction through the GABA_A-associated Cl⁻ channel → Ca²⁺ influx through an L-type voltage-sensitive Ca²⁺ channel → Ca²⁺-dependent phosphorylation of GAP-43 and MARCKS protein by PKC.     

Tyrosine Hydroxylase Phosphorylation in Digitonin-Permeabilized Bovine Adrenal Chromaffin Cells: The Effect of Protein Kinase and Phosphatase Inhibitors on Ser19 and Ser40 Phosphorylation

Abstract: The protein kinases and protein phosphatases that act on tyrosine hydroxylase in vivo have not been established. Bovine adrenal chromaffin cells were permeabilized with digitonin and incubated with [γ-³²P]ATP, in the presence or absence of 10 µ M Ca²⁺, 1 µ M cyclic AMP, 1 µ M phorbol dibutyrate, or various kinase or phosphatase inhibitors. Ca²⁺ increased the phosphorylation of Ser¹⁹ and Ser⁴⁰. Cyclic AMP, and phorbol dibutyrate in the presence of Ca²⁺, increased the phosphorylation of only Ser⁴⁰. Ser³¹ and Ser⁸ were not phosphorylated. The Ca²⁺-stimulated phosphorylation of Ser¹⁹ was incompletely reduced by inhibitors of calcium/calmodulin-stimulated protein kinase II (46% with KN93 and 68% with CaM-PKII 273–302), suggesting that another protein kinase(s) was contributing to the phosphorylation of this site. The Ca²⁺-stimulated phosphorylation of Ser⁴⁰ was reduced by specific inhibitors of protein kinase A (56% with H89 and 38% with PKAi 5–22 amide) and protein kinase C (70% with Ro 31-8220 and 54% with PKCi 19–31), suggesting that protein kinases A and C contributed to most of the phosphorylation of this site. Results with okadaic acid and microcystin suggested that Ser¹⁹ and Ser⁴⁰ were dephosphorylated by PP2A.     

Functional expression of the plant K channel KAT1 in insect cells

Following the biophysical analysis of plant K⁺ channels in their natural environment, three members from the green branch of the evolutionary tree of life KAT1, AKT1 and KST1 have recently been identified on the molecular level. Among them, we focused on the expression and characterization of the Arabidopsis thaliana K⁺ channel KAT1 in the insect cell line Sf9. The infection of Sf9 cells with KAT1-recombinant baculovirus resulted in functional expression of KAT1 channels, which was monitored by inward-rectifying, K⁺-selective (impermeable to Na⁺ and even NH₄⁺) ionic conductance in whole-cell patch-clamp recordings. A voltage threshold as low as −60 to −80 mV for voltage activation compared to other plant inward rectifiers in vivo, and to in vitro expression of KAT1 in Xenopus oocytes or yeast, may be indicative for channel modulation by the expression system. A rise in cytoplasmic Ca²⁺ concentration (up to 1 mM), a regulator of the inward rectifier in Vicia faba guard cells, did not modify the voltage dependence of KAT1 in Sf9 cells. The access to channel function on one side and channel protein on the other make Sf9 cells a suitable heterologous system for studies on the biophysical properties, post-translational modification and assembly of a green inward rectifier.     

Neuropathological Abnormalities in Transgenic Mice Harbouring a Phosphorylation Mutant Neurofilament Transgene

Abstract: Ser⁵⁵ within the head domain of neurofilament light chain (NF-L) is a target for phosphorylation by protein kinase A. To understand further the physiological role(s) of NF-L Ser⁵⁵ phosphorylation, we generated transgenic mice with a mutant NF-L transgene in which Ser⁵⁵ was mutated to Asp so as to mimic permanent phosphorylation. Two lines of NF-L(Asp) mice were created and these animals express the transgene in many neurones of the central and peripheral nervous systems. Both transgenic lines display identical, early onset, and robust pathological changes in the brain. These involve the formation of NF-L(Asp)-containing perikaryal neurofilament inclusion bodies and the development of swollen Purkinje cell axons. Development of these pathologies was rapid and fully established in mice as young as 4 weeks of age. The two transgenic lines show no elevation of NF-L, neurofilament middle chain (NF-M), or neurofilament heavy chain (NF-H), and transgenic NF-L(Asp) represents only a minor proportion of total NF-L protein. Because other published transgenic lines expressing higher levels of wild-type NF-L do not exhibit phenotypic changes that in any way resemble those in the NF-L(Asp) mice and because the two different NF-L(Asp) transgenic lines display identical neuropathological changes, it is likely that the pathological alterations observed in the NF-L(Asp) mice are the result of properties of the mutant NF-L. These results support the notion that phosphorylation of Ser⁵⁵ is a mechanism for regulating neurofilament organisation in vivo.