Phosphorylation Of Glial Fibrillary Acidic Protein Research Articles

Site-specific phosphorylation and caspase cleavage of GFAP are new markers of Alexander disease severity.

Alexander disease (AxD) is a fatal neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP), which supports the structural integrity of astrocytes. Over 70 GFAP missense mutations cause AxD, but the mechanism linking different mutations to disease-relevant phenotypes remains unknown. We used AxD patient brain tissue and induced pluripotent stem cell (iPSC)-derived astrocytes to investigate the hypothesis that AxD-causing mutations perturb key post-translational modifications (PTMs) on GFAP. Our findings reveal selective phosphorylation of GFAP-Ser13 in patients who died young, independently of the mutation they carried. AxD iPSC-astrocytes accumulated pSer13-GFAP in cytoplasmic aggregates within deep nuclear invaginations, resembling the hallmark Rosenthal fibers observed in vivo. Ser13 phosphorylation facilitated GFAP aggregation and was associated with increased GFAP proteolysis by caspase-6. Furthermore, caspase-6 was selectively expressed in young AxD patients, and correlated with the presence of cleaved GFAP. We reveal a novel PTM signature linking different GFAP mutations in infantile AxD.

Enteric GFAP expression and phosphorylation in Parkinson's disease.

Enteric glial cells (EGCs) are in many respects similar to astrocytes of the central nervous system and express similar proteins including glial fibrillary acidic protein (GFAP). Changes in GFAP expression and/or phosphorylation have been reported during brain damage or central nervous system degeneration. As in Parkinson's disease (PD) the enteric neurons accumulate α-synuclein, and thus are showing PD-specific pathological features, we undertook the present survey to study whether the enteric glia in PD become reactive by assessing the expression and phosphorylation levels of GFAP in colonic biopsies. Twenty-four PD, six progressive supranuclear palsy (PSP), six multiple system atrophy (MSA) patients, and 21 age-matched healthy controls were included. The expression levels and the phosphorylation state of GFAP were analyzed in colonic biopsies by western blot. Additional experiments were performed using real-time PCR for a more precise analysis of the GFAP isoforms expressed by EGCs. We showed that GFAPκ was the main isoform expressed in EGCs. As compared to control subjects, patients with PD, but not PSP and MSA, had significant higher GFAP expression levels in their colonic biopsies. The phosphorylation level of GFAP at serine 13 was significantly lower in PD patients compared to control subjects. By contrast, no change in GFAP phosphorylation was observed between PSP, MSA and controls. Our findings provide evidence that enteric glial reaction occurs in PD and further reinforce the role of the enteric nervous system in the initiation and/or the progression of the disease. We showed that GFAP is over-expressed and hypophosphorylated in the enteric glial cells (EGCs) of Parkinson's disease (PD) patients as compared to healthy subjects and patients with atypical parkinsonism (MSA, multiple system atrophy and PSP, progressive supranuclear palsy). Our findings provide evidence that enteric glial reaction occurs in PD but not in PSP and MSA and further reinforce the role of the enteric nervous system in the pathophysiology of PD.

Phosphorylation of GFAP is Associated with Injury in the Neonatal Pig Hypoxic-Ischemic Brain

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed in the astrocyte cytoskeleton that plays an important role in the structure and function of the cell. GFAP can be phosphorylated at six serine (Ser) or threonine (Thr) residues but little is known about the role of GFAP phosphorylation in physiological and pathophysiological states. We have generated antibodies against two phosphorylated GFAP (pGFAP) proteins: p8GFAP, where GFAP is phosphorylated at Ser-8 and p13GFAP, where GFAP is phosphorylated at Ser-13. We examined p8GFAP and p13GFAP expression in the control neonatal pig brain and at 24 and 72 h after an hypoxic-ischemic (HI) insult. Immunohistochemistry demonstrated pGFAP expression in astrocytes with an atypical cytoskeletal morphology, even in control brains. Semi-quantitative western blotting revealed that p8GFAP expression was significantly increased at 24 h post-insult in HI animals with seizures in frontal, parietal, temporal and occipital cortices. At 72 h post-insult, p8GFAP and p13GFAP expression were significantly increased in HI animals with seizures in brain regions that are vulnerable to cellular damage (cortex and basal ganglia), but no changes were observed in brain regions that are relatively spared following an HI insult (brain stem and cerebellum). Increased pGFAP expression was associated with poor neurological outcomes such as abnormal encephalography and neurobehaviour, and increased histological brain damage. Phosphorylation of GFAP may play an important role in astrocyte remodelling during development and disease and could potentially contribute to the plasticity of the central nervous system.

Phosphoproteomic Analysis Reveals Site-Specific Changes in GFAP and NDRG2 Phosphorylation in Frontotemporal Lobar Degeneration

Frontotemporal lobar degeneration (FTLD) is a progressive neurodegenerative disease characterized by behavioral abnormalities, personality changes, language dysfunction, and can co-occur with the development of motor neuron disease. One major pathological form of FTLD is characterized by intracellular deposition of ubiquitinated and phosphorylated TAR DNA binding protein-43 (TDP-43), suggesting that dysregulation in phosphorylation events may contribute to disease progression. However, to date systematic analysis of the phosphoproteome in FTLD brains has not been reported. In this study, we employed immobilized metal affinity chromatography (IMAC) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify phosphopeptides from FTLD and age-matched control post-mortem human brain tissue. Using this approach, we identified 786 phosphopeptides in frontal cortex (control and FTLD), in which the population of phosphopeptides represented approximately 50% of the total peptides analyzed. Label-free quantification using spectral counts revealed six proteins with significant changes in the FTLD phosphoproteome. N-myc-Downstream regulated gene 2 (NDRG2) and glial fibrillary acidic protein (GFAP) had an increased number of phosphospectra in FTLD, whereas microtubule associated protein 1A (MAP1A), reticulon 4 (RTN4; also referred to as neurite outgrowth inhibitor (Nogo)), protein kinase C gamma (PRKCG), and heat shock protein 90 kDa alpha, class A member 1(HSP90AA1) had significantly fewer phosphospectra compared to control brain. To validate these differences, we examined NDRG2 phosphorylation in FTLD brain by immunoblot analyses, and using a phosphoserine-13 (pSer13) GFAP monoclonal antibody we show an increase in pSer13 GFAP levels by immunoblot concomitant with increased overall GFAP levels in FTLD cases. These data highlight the utility of combining proteomic and phosphoproteomic strategies to characterize post-mortem human brain tissue.

Propionic Acid Induces Cytoskeletal Alterations in Cultured Astrocytes From Rat Cerebral Cortex

Severe neurological symptoms, cerebral edema, and atrophy are common features of the inherited metabolic disorder propionic acidemia. However, the pathomechanisms involved in the neuropathology of this disease are not well established. In this study, we investigate the effects of propionic acid (PA), a metabolite accumulating in this disorder, on cytoskeletal reorganization, on cell viability, and on the in vitro phosphorylation of glial fibrillary acidic protein (GFAP) and vimentin in cultured astrocytes from cerebral cortex of neonatal rats. We observed that the astrocytes changed their usual polygonal morphology when exposed to 5 mM PA for 72 h, leading to the appearance of fusiform or process-bearing cells, without elicit cell death. We also noticed that after 72 h treatment with 5 mM PA cells showed retracted cytoplasm with bipolar processes containing packed GFAP filaments and disorganized actin stress fibers, as revealed by immunocytochemistry. In addition, the morphological alterations were accompanied by increased in vitro 32P incorporation into GFAP and vimentin recovered into the high-salt Triton-insoluble cytoskeletal fraction. In conclusion, our results indicate that PA lead to cytoskeletal reorganization and to increased in vitro phosphorylation of Triton-insoluble GFAP and vimentin. On the basis of our results we could suppose that Triton-insoluble GFAP and vimentin hyperphosphorylation could be implicated in the reorganization of cellular structure and these findings could be involved in the brain damage characteristic of propionic acidemia patients.

Ontogenetic Changes in Glial Fibrillary Acid Protein Phosphorylation, Glutamate Uptake and Glutamine Synthetase Activity in Olfactory Bulb of Rats

Phosphorylation of the glial fibrillary acidic protein (GFAP) in hippocampal and cerebellar slices from immature rats is stimulated by glutamate. This effect occurs via a group II metabotropic glutamate receptor in the hippocampus and an NMDA ionotropic receptor in the cerebellum. We investigated the glutamate modulation of GFAP phosphorylation in the olfactory bulb slices of Wistar rats of different ages (post-natal day 15 = P15, post-natal day 21 = P21 and post-natal day 60 = P60). Our results showed that glutamate stimulates GFAP phosphorylation in young animals and this is mediated by NMDA receptors. We also observed a decrease in glutamate uptake at P60 compared to P15, a finding similar to that found in the hippocampus. The activity of glutamine synthetase was elevated after birth, but was found to decrease with development from P21 to P60. Together, these data confirm the importance of glutamatergic transmission in the olfactory bulb, its developmental regulation in this brain structure and extends the concept of glial involvement in glutamatergic neuron-glial communication.

Branched-Chain α-Keto Acids Accumulating in Maple Syrup Urine Disease Induce Reorganization of Phosphorylated GFAP in C6-Glioma Cells

In this study we investigate the effects of the branched-chain keto acids (BCKA) alpha-ketoisocaproic (KIC), alpha-ketoisovaleric (KIV), and alpha-keto-beta-methylvaleric (KMV) acids, metabolites accumulating in maple syrup urine disease (MSUD), on the in vitro phosphorylation of glial fibrillary acidic protein (GFAP) and cytoskeletal reorganization in C6-glioma cells. We observed that after 3 h treatment with KIC, KIV, or KMV cells showed retracted cytoplasm with bipolar processes containing packed GFAP filaments as revealed by immunocytochemistry. Western Blot analysis by anti-GFAP monoclonal antibody demonstrated that BCKA were not able to alter GFAP immunocontent in total cell homogenate, but the immunocontent as well as the in vitro (32)P incorporation into GFAP recovered into the high salt Triton-insoluble cytoskeletal fraction were significantly increased. Western Blot using monoclonal antiphosphoserine antibody showed that BCKA induced increased immunocontent of phosphoserine-containing amino acids in several proteins in total cell homogenate. In addition, the immunocontent of phosphoserine-containing amino acids was also greatly increased in GFAP recovered in the high-salt Triton insoluble cytoskeletal fraction, corresponding to the polymerized intermedite filament (IF) proteins present in the cell. In conclusion, our results indicate that KIC, KIV, or KMV increased the serine/threonine in vitro phosphorylation of GFAP leading to increased Triton-insoluble GFAP immunocontent and cytoskeletal reorganization. Considering IF networks can be regulated by phosphorylation of polypeptide subunits leading to reorganization of the IF filamentous structure, we could suppose that GFAP hyperphosphorylation and disorganization of cellular structure could be involved in the brain damage characteristic of MSUD patients.

Role of intracellular calcium stores on the effect of metabotropic glutamate receptors on phosphorylation of glial fibrillary acidic protein in hippocampal slices from immature rats.

Phosphorylation of glial fibrillary acidic protein (GFAP) in slices from immature rats is stimulated by glutamate via a group II metabotropic glutamate receptor (mGluR II) and by absence of external Ca2+ in reactions that are not additive (Wofchuk and Rodnight, Neurochem. Int. 24:517-523, 1994). These observations suggested that glutamate, via an mGluR, inhibits Ca(2+)-entry through L-type Ca2+ channels and down-regulates a Ca(2+)-dependent dephosphorylation event coupled to GFAP. Because ryanodine receptors are present on internal Ca2+ stores and are associated with L-type Ca(2+)-channels, we investigated the possibility that the glutamatergic modulation of GFAP phosphorylation involves internal Ca2+ stores regulated by ryanodine receptors and whether the Ca2+ originating from these stores acts in a similar manner to external Ca2+. The results showed that the ryanodine receptor-agonists, caffeine and ryanodine and thapsigargin, all of which in appropriate doses increase cytoplasmic Ca2+, reversed the stimulation of GFAP phosphorylation given by 1S,3R-ACPD, an mGluR II agonist.

S100B-mediated inhibition of the phosphorylation of GFAP is prevented by TRTK-12.

S100B belongs to a family of calcium-binding proteins involved in cell cycle and cytoskeleton regulation. We observed an inhibitory effect of S100B on glial fibrillary acidic protein (GFAP) phosphorylation, when stimulated by cAMP or Ca2+/calmodulin, in a cytoskeletal fraction from primary astrocyte cultures. We found that S100B has no direct effect on CaM KII activity, the major kinase in this cytoskeletal fraction able to phosphorylate GFAP. The inhibition of GFAP phosphorylation is most likely due to the binding of S100B to the phosphorylation sites on this protein and blocking the access of these sites to the protein kinases. This inhibition was dependent on Ca2+. However, Zn2+ could substitute for Ca2+. The inhibitory effect of S100B was prevented by TRTK-12, a peptide that blocks S100B interaction with several target proteins including glial fibrillary acidic protein. These data suggest a role for S100B in the assembly of intermediate filaments in astrocytes.

Phosphorylation of glial fibrillary acidic protein is stimulated by glutamate via NMDA receptors in cortical microslices and in mixed neuronal/glial cell cultures prepared from the cerebellum

In previous work we showed that phosphorylation of glial fibrillary acidic protein (GFAP), an astrocyte marker, is increased by glutamate in hippocampal slices from immature rats via a type II metabotropic receptor. In the present work we show that glutamate also stimulates GFAP phosphorylation in microslices prepared from immature cerebellar cortex, but by a different receptor mechanism from that observed in the hippocampus. Thus, in cerebellar microslices, NMDA consistently stimulated GFAP phosphorylation, whereas no effect of metabotropic or non-NMDA ionotropic agonists was observed. Glutamate and NMDA also stimulated GFAP phosphorylation in mixed neuronal/glial cell cultures from the cerebellum, although no effect of these agonists was observed in primary cultures of cerebellar astrocytes. In both models, the effects of glutamate and NMDA were dependent on external Ca 2+, were reversed by the NMDA receptor antagonist AP5 and were not blocked by tetrodotoxin. In the slice study the effect of NMDA was confined to a period starting with the first detectable expression of GFAP at 10 days and finishing at 16 days postnatal, as previously observed with metabotropic agonists in hippocampal slices. This period in the rat corresponds to the start of synaptogenesis when astrocyte hypertrophy is occurring. The results are discussed in the light of information in the literature on the occurrence of functional NMDA receptor subunits in glia.

Distribution of phosphorylated glial fibrillary acidic protein in the mouse central nervous system.

Glial fibrillary acidic protein (GFAP) is the principal component of intermediate filaments (IFs) in mature astrocytes in the central nervous system (CNS). Like other IF proteins, GFAP has multiple phosphorylation sites in the N-terminal head domain. The distribution of phospho-GFAP in vivo has not been elucidated. We generated Gfap(hwt) knock-in mice, in which the coding region for the head domain of GFAP is replaced with the corresponding human sequence. In combination with a series of monoclonal antibodies (mAbs) reactive to human phospho-GFAP, we visualized the distribution of phospho-GFAP in vivo in mice. GFAP phosphorylated at Thr7, Ser8 and/or Ser13 increased postnatally in the CNS of these mice. Limited populations of GFAP-positive astrocytes were labelled with anti-phospho-GFAP mAbs in most brain areas, whereas almost all the astrocytes in the optic nerve and spinal cord were labelled. Astrocytes in the subventricular zone and rostral migratory stream preferentially contained phospho-GFAP. In a cold injury model of the cerebral cortex, we detected phospho-GFAP in reactive astrocytes at 2-3 weeks after the injury. Phospho-GFAP provides a molecular marker indicating the heterogeneity of astrocytes, and Gfap(hwt) knock-in mice will aid in monitoring intracellular conditions of astrocytes, under various conditions. Our results suggest that the phosphorylation of GFAP plays a role in non-dividing astrocytes in vivo.

GFAP phosphorylation studied in digitonin-permeabilized astrocytes: standardization of conditions

Cycles of assembly/disassembly of the intermediate filaments of astrocytes are modulated by the phosphorylation of glial fibrillary acidic protein (GFAP). The sites on GFAP are localized at the N-terminal where they are phosphorylated by cAMP-dependent and Ca 2+-dependent protein kinases. Phosphorylation of GFAP has been investigated in brain slices, astrocyte cultures, cytoskeletal fractions and purified systems. Here we describe a different approach to study GFAP phosphorylation. We show that permeabilization of astrocytes in culture with digitonin allows direct access to the systems phosphorylating GFAP. Conditions for the permeabilization were established with an assay based on the exclusion of Trypan blue. Incubation of permeabilized cells with cAMP and Ca 2+ increased the phosphorylation state of GFAP. Immunocytochemistry with anti-GFAP showed that permeabilized astrocytes retained their typical flat, fibroblast morphology and exhibited well preserved glial filaments. On incubation with cAMP the filaments apparently condensed to form long processes. The results suggest the approach of studying structural changes in glial filaments in parallel to protein phosphorylation, in the presence of specific modulators of protein kinases and phosphatases has considerable potential.

Identification of a Novel Phosphorylation Site on Histone H3 Coupled with Mitotic Chromosome Condensation

Histone H3 (H3) phosphorylation at Ser(10) occurs during mitosis in eukaryotes and was recently shown to play an important role in chromosome condensation in Tetrahymena. When producing monoclonal antibodies that recognize glial fibrillary acidic protein phosphorylation at Thr(7), we obtained some monoclonal antibodies that cross-reacted with early mitotic chromosomes. They reacted with 15-kDa phosphoprotein specifically in mitotic cell lysate. With microsequencing, this phosphoprotein was proved to be H3. Mutational analysis revealed that they recognized H3 Ser(28) phosphorylation. Then we produced a monoclonal antibody, HTA28, using a phosphopeptide corresponding to phosphorylated H3 Ser(28). This antibody specifically recognized the phosphorylation of H3 Ser(28) but not that of glial fibrillary acidic protein Thr(7). Immunocytochemical studies with HTA28 revealed that Ser(28) phosphorylation occurred in chromosomes predominantly during early mitosis and coincided with the initiation of mitotic chromosome condensation. Biochemical analyses using (32)P-labeled mitotic cells also confirmed that H3 is phosphorylated at Ser(28) during early mitosis. In addition, we found that H3 is phosphorylated at Ser(28) as well as Ser(10) when premature chromosome condensation was induced in tsBN2 cells. These observations suggest that H3 phosphorylation at Ser(28), together with Ser(10), is a conserved event and is likely to be involved in mitotic chromosome condensation.

Effects of transient cerebral ischemia on glial fibrillary acidic protein phosphorylation and immunocontent in rat hippocampus

Transient global cerebral ischemia induced in rats by four-vessel occlusion for 20 min produced an increase in the immunocontent of glial fibrillary acidic protein and a protein phosphorylation response that was different in the CA1 and dentate gyrus areas of the hippocampus. We studied different times of reperfusion (one, four, seven, 14 and 30 days) and observed that the immunocontent and in vitro rate of phosphorylation of glial fibrillary acidic protein in the CA1 region was significantly increased at all intervals after the ischemic insult, indicating that the astrocytic response was maintained for at least 30 days. After reperfusion for 14 days a significant increase in the ratio “ in vitro phosphorylation rate/immunocontent” in the CA1 region was observed when compared to control values, to other intervals and to the dentate gyrus, suggesting a hyperphosphorylation of this intermediate filament protein at this interval. In the dentate gyrus, an area less vulnerable to the insult, labelling and immunocontent of glial fibrillary acidic protein were equally increased from four days of reperfusion and the increase remained significant until 30 days, confirming that neuronal death is not the only determining factor for gliosis to occur. In control sham-operated animals, neither the CA1 region nor the dentate gyrus showed significant increases in labelling or immunocontent. Changes in the phosphorylation of glial fibrillary acidic protein may be essential for the plastic response of astrocytes to neuronal damage, as neurons and astrocytes can act as functional units involved in homeostasis, plasticity and neurotransmission.

Regulation of the phosphorylation of glial fibrillary acidic protein (GFAP) by glutamate and calcium ions in slices of immature rat spinal cord: comparison with immature hippocampus

The effect of glutamate and lack of external Ca 2+ on the phosphorylation of the astrocyte cell marker glial fibrillary acidic protein (GFAP) was studied in slices of hippocampus and thoracic spinal cord from immature (P12–16) rats. Confirming previous work with immature hippocampal slices (Wofchuk, S.T. and Rodnight, R., Neurochem. Int., 24 (1994) 517–523; Wofchuk, S.T. and Rodnight, R., Dev. Brain Res., 85 (1995) 181–186), glutamate strongly stimulated GFAP phosphorylation in media with Ca 2+ and in media lacking Ca 2+ a quantitatively similar stimulation of basal GFAP phosphorylation was observed. By contrast in slices of immature thoracic spinal cord, glutamate had no effect on GFAP phosphorylation and in media lacking Ca 2+ phosphorylation of GFAP was inhibited. Since GFAP phosphorylation is Ca 2+-dependent and is not stimulated by glutamate in slices of adult hippocampus, the present results suggest that astrocytic functions in the rat spinal cord mature more rapidly than in the hippocampus. The possibility that the difference in the control of GFAP phosphorylation in the two structures is related to differences in the control of GFAP dephosphorylation was investigated by incubating spinal cord slices with the calcineurin inhibitor FK506 in the presence of Ca 2+. In contrast to results obtained with hippocampal slices FK506 had no effect on the phosphorylation state of GFAP in spinal cord slices.

The S100B protein inhibits phosphorylation of GFAP and vimentin in a cytoskeletal fraction from immature rat hippocampus.

The S100B protein belongs to a family of small Ca2+-binding proteins involved in several functions including cytoskeletal reorganization. The effect of S 100B on protein phosphorylation was investigated in a cytoskeletal fraction prepared from immature rat hippocampus. An inhibitory effect of 5 microM S100B on total protein phosphorylation, ranging from 25% to 40%, was observed in the presence of Ca2+ alone, Ca2+ plus calmodulin or Ca2+ plus cAMP. Analysis by two dimensional electrophoresis revealed a Ca2+/calmodulin-dependent and a Ca2+/cAMP-dependent inhibitory effect of S100B, ranging from 62% to 67% of control, on the phosphorylation of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. The fact that S100B binds to the N-terminal domain of GFAP and that the two proteins are co-localized in astrocytes suggests a potential in vivo role for S100B in modulating the phosphorylation of intermediate filament proteins in glia.

Evidence for a role for calcium ions in the dephosphorylation of glial fibrillary acidic protein (GFAP) in immature hippocampal slices and in astrocyte cultures from the rat

Evidence was sought for a role for Ca 2+ in the dephosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in immature hippocampal slices. Although previous work showed that the main phosphatase dephosphorylating GFAP in this preparation is a Ca 2+-independent type 1 enzyme, a role for Ca 2+ was suggested by the observation that the incorporation of [ 32P]phosphate into GFAP in immature slices is inhibited by external Ca 2+. This inhibition is strikingly different to the situation in mature slices where GFAP phosphorylation is completely dependent on Ca 2+. Pure astrocyte cultures were probed by immunoblotting for the presence of the Ca 2+-dependent phosphatase calcineurin. An enzyme content, amounting to about 2% of that found in fresh hippocampal tissue, was detected for both the catalytic (α) and regulatory (β) subunits. The direct or indirect association of calcineurin with GFAP was suggested by observations showing that FK506, a specific inhibitor of calcineurin, increased the phosphorylation state of GFAP in immature slices and of GFAP and vimentin in astrocyte cultures.

Calcium-dependent phosphorylation of glial fibrillary acidic protein (GFAP) in the rat hippocampus: a comparison of the kinase/phosphatase balance in immature and mature slices using tryptic phosphopeptide mapping

In previous work we showed that phosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in hippocampal slices from adult rats is dependent on external Ca 2+, whereas in slices from immature rats aged 12–16 days postnatal 32P incorporation into GFAP is inhibited by external Ca 2+. The nature of this late developmental change in Ca 2+ sensitivity for GFAP phosphorylation was investigated in the present work by comparing in immature and adult animals phosphorylation of GFAP by endogenous protein kinase activity in cytoskeletal fractions and tryptic phosphopeptide maps prepared from cytoskeletal fractions labelled with [γ- 32P]ATP and from slices labelled with [ 32P]phosphate. Cytoskeletal fractions prepared from immature and adult hippocampus both contained endogenous protein kinase activity towards GFAP and other proteins stimulated by Ca 2+/calmodulin and by cyclic AMP. The maps of GFAP isolated from the cytoskeletal fractions labelled in the presence of Ca 2+/calmodulin were very similar and exhibited two major and several minor phosphopeptides. Comparison with maps derived from these fractions labelled in the presence of cyclic AMP showed that one of the major phosphopeptides was either directly or indirectly phosphorylated by Ca 2+/calmodulin-stimulated kinase activity. Maps derived from GFAP isolated from adult slices labelled in the presence of Ca 2+ and immature slices labelled in the absence of Ca 2+ were qualitatively identical, with minor differences from the cytoskeletal maps. At both ages the slice maps displayed the phosphopeptide phosphorylated through the activity of a Ca 2+/calmodulin kinase in the cytoskeletal fractions. By its migration properties this peptide appears to correspond to a sequence containing a site shown by other workers to be phosphorylated in vitro by CaM kinase II, suggesting that even in the absence of external Ca 2+, kinase activity directly or indirectly dependent on Ca 2+ was occurring in the immature slices. The near identity of the phosphorylation sites at the two ages suggest that the change in Ca 2+ sensitivity of GFAP phosphorylation during development is not due to a change in the balance of kinase and phosphatase activities, but rather to a change in the mechanism(s) whereby Ca 2+ controls the relative activity of these enzymes.

Phosphorylation of Glial Fibrillary Acidic Protein at the Same Sites by Cleavage Furrow Kinase and Rho-associated Kinase

Site- and phosphorylation state-specific antibodies are useful to analyze spatiotemporal distribution of site-specific phosphorylation of target proteins in vivo. Using several polyclonal and monoclonal antibodies that can specifically recognize four phosphorylated sites on glial fibrillary acidic protein (GFAP), we have previously reported that Thr-7, Ser-13, and Ser-34 on this intermediate filament protein are phosphorylated at the cleavage furrow during cytokinesis. This observation suggests that there exists a protein kinase named cleavage furrow kinase specifically activated at metaphase-anaphase transition (Matsuoka, Y., Nishizawa, K., Yano, T., Shibata, M., Ando, S., Takahashi, T., and Inagaki, M. (1992) EMBO J. 11, 2895-2902; Sekimata, M., Tsujimura, K., Tanaka, J., Takeuchi, Y., Inagaki, N., and Inagaki, M. (1996) J. Cell Biol. 132, 635-641). Here we report that GFAP is phosphorylated specifically at Thr-7, Ser-13, and Ser-34 by Rho-associated kinase (Rho-kinase), which binds to the small GTPase Rho in its GTP-bound active form. The kinase activity of Rho-kinase toward GFAP is dramatically stimulated by guanosine 5'-(3-O-thio)-triphosphate-bound RhoA. Furthermore, the phosphorylation of GFAP by Rho-kinase results in a nearly complete inhibition of its filament formation in vitro. The possibility that Rho-kinase is a candidate for cleavage furrow kinase is discussed.

Control of the phosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in the immature rat hippocampus by glutamate and calcium ions: possible key factor in astrocytic plasticity.

The present review describes recent research on the regulation by glutamate and Ca2+ of the phosphorylation state of the intermediate filament protein of the astrocytic cytoskeleton, glial fibrillary acidic protein (GFAP), in immature hippocampal slices. The results of this research are discussed against a background of modern knowledge of the functional importance of astrocytes in the brain and of the structure and dynamic properties of intermediate filament proteins. Astrocytes are now recognized as partners with neurons in many aspects of brain function with important roles in neural plasticity. Site-specific phosphorylation of intermediate filament proteins, including GFAP, has been shown to regulate the dynamic equilibrium between the polymerized and depolymerized state of the filaments and to play a fundamental role in mitosis. Glutamate was found to increase the phosphorylation state of GFAP in hippocampal slices from rats in the post-natal age range of 12-16 days in a reaction that was dependent on external Ca2+. The lack of external Ca2+ in the absence of glutamate also increased GFAP phosphorylation to the same extent. These effects of glutamate and Ca2+ were absent in adult hippocampal slices, where the phosphorylation of GFAP was completely Ca(2+)-dependent. Studies using specific agonists of glutamate receptors showed that the glutamate response was mediated by a G protein-linked group II metabotropic glutamate receptor (mGluR). Since group II mGluRs do not act by liberating Ca2+ from internal stores, it is proposed that activation of the receptor by glutamate inhibits Ca2+ entry into the astrocytes and consequently down-regulates a Ca(2+)-dependent dephosphorylation cascade regulating the phosphorylation state of GFAP. The functional significance of these results may be related to the narrow developmental window when the glutamate response is present. In the rat brain this window corresponds to the period of massive synaptogenesis during which astrocytes are known to proliferate. Possibly, glutamate liberated from developing synapses during this period may signal an increase in the phosphorylation state of GFAP and a consequent increase in the number of mitotic astrocytes.