Ikuko Iwamoto

Phosphorylation of αB-crystallin in Mitotic Cells and Identification of Enzymatic Activities Responsible for Phosphorylation

The immunofluorescence localization of αB-crystallin in U373 MG human glioma cells with an antibody specific for αB-crystallin that had been phosphorylated at Ser-45 revealed an intense staining of cells in the mitotic phase of the cell cycle. Phosphorylated forms of αB-crystallin in mitotic cells were detected in all cell lines examined and in tissue sections of mouse embryos. Increases in the levels of αB-crystallin that had been phosphorylated at Ser-45 and Ser-19, but not at Ser-59, were detected biochemically by isoelectric focusing or SDS-polyacrylamide gel electrophoresis and a subsequent Western blot analysis of extracts of cells collected at the mitotic phase. When we estimated the phosphorylation activity specific for αB-crystallin in extracts of mitotic U373 MG cells, using the amino-terminal 72-amino acid peptide derived from unphosphorylated αB2-crystallin as the substrate, we found that the activities responsible for the phosphorylation of Ser-45 and Ser-19 were markedly enhanced but that the activity responsible for the phosphorylation of Ser-59 was suppressed. The protein kinases responsible for the phosphorylation of Ser-45 and Ser-59 in the amino-terminal 72-amino acid peptide were partially purified from extracts of cells that had been stimulated by exposure to H2O2 in the presence of calyculin A. The activities responsible for the phosphorylation of Ser-45 and Ser-59 were eluted separately from a column of Superdex 200 at fractions corresponding to about 40 and 60 kDa, respectively, while the kinase for Ser-19 was unstable. p44/42 mitogen-activated protein (MAP) kinase and MAP kinase-activated protein (MAPKAP) kinase-2 were concentrated in the Ser-45 kinase fraction and Ser-59 kinase fraction, respectively. Recombinant human p44 MAP kinase and MAPKAP kinase-2 purified from rabbit muscle selectively phosphorylated Ser-45 and -59, respectively. The Ser-45 kinase fraction and Ser-59 kinase fraction phosphorylated myelin basic protein and hsp27, respectively. These results suggest that the phosphorylations of Ser-45 and Ser-59 in αB-crystallin are catalyzed by p44/42 MAP kinase and MAPKAP kinase-2, respectively, in cells and that the phosphorylation of Ser-45 by p44/42 MAP kinase is enhanced while the phosphorylation of Ser-59 by MAPKAP kinase-2 is suppressed during cell division.

Stimulation of the stress-induced expression of stress proteins by curcumin in cultured cells and in rat tissues in vivo.

Curcumin, a major component of turmeric, a seasoning commonly used in Indian food, and a known antioxidant, anti-inflammatory and anti-carcinogenic agent, is a potent stimulator of the stress-induced expression of Hsp27, alphaB crystallin and Hsp70. When C6 rat glioma cells were exposed to arsenite (100 microM for 1 h), CdCl2 (100 microM for 1 h) or heat (42 degrees C for 30 min) in the presence of 3-10 microM curcumin, induction of the synthesis of all three proteins was markedly stimulated, as detected by specific immunoassays, Western blot analysis and Northern blot analysis. A gel mobility shift assay revealed that curcumin prolonged the stress-induced activation of the heat shock element-binding (HSE-binding) activity of heat shock transcription factor (Hsf) in the cultured cells. The stimulatory effect of curcumin on the responses to stress was also observed in BRL-3A rat liver cells and Swiss 3T3 mouse fibroblasts. Induction of Hsp27, alphaB crystallin and Hsp70 in the liver and adrenal glands of heat-stressed (42 degrees C for 20 min) rats was also enhanced by prior injection of curcumin (20 mg/kg body weight). As curcumin is a potent inhibitor of arachidonic acid metabolism, it is suggested that the mechanism of the stimulation by curcumin of the stress responses might be similar to that of salicylate, indomethacin and nordihydroguaiaretic acid.

Dysbindin-1, WAVE2 and Abi-1 form a complex that regulates dendritic spine formation.

Genetic variations in dysbindin-1 (dystrobrevin-binding protein-1) are one of the most commonly reported variations associated with schizophrenia. As schizophrenia could be regarded as a neurodevelopmental disorder resulting from abnormalities of synaptic connectivity, we attempted to clarify the function of dysbindin-1 in neuronal development. We examined the developmental change of dysbindin-1 in rat brain by western blotting and found that a 50 kDa isoform is highly expressed during the embryonic stage, whereas a 40 kDa one is detected at postnatal day 11 and increased thereafter. Immunofluorescent analyses revealed that dysbindin-1 is enriched at the spine-like structure of primary cultured rat hippocampal neurons. We identified WAVE2, but not N-WASP, as a binding partner for dysbindin-1. We also found that Abi-1, a binding molecule for WAVE2 involved in spine morphogenesis, interacts with dysbindin-1. Although dysbindin-1, WAVE2 and Abi-1 form a ternary complex, dysbindin-1 promoted the binding of WAVE2 to Abi-1. RNA interference-mediated knockdown of dysbindin-1 led to the generation of abnormally elongated immature dendritic protrusions. The present results indicate possible functions of dysbindin-1 at the postsynapse in the regulation of dendritic spine morphogenesis through the interaction with WAVE2 and Abi-1.

Possible role of Rho/Rhotekin signaling in mammalian septin organization

There is growing evidence for crosstalk between septin filaments and actin cytoskeleton which is regulated by Rho family of GTPases. Here we show that active Rho disrupts septin filament structures in rat embryonic fibroblast REF52 cells. Among Rho effector molecules tested, Rhotekin induced morphological changes of septin filaments similar to those by activated Rho. The center region of Rhotekin was sufficient for the septin reorganization in the cells, and likely to interact indirectly with the C-terminal half of a septin Sept9b, where a GTPase domain is located. Rhotekin and Sept9b are colocalized mainly in perinuclear regions in serum-starved REF52 cells. Upon stimulation with lysophosphatidic acid, they translocated to actin stress fibers in 10 min and then redistributed again to cytoplasm after 90 min treatment. In neuroblastoma Neuro2a cells, Rhotekin and Sept9b were enriched in the tip of neurites, a location where cortical actin reorganization is induced upon stimulation with lysophosphatidic acid. Taken together, we propose that Rhotekin is a novel regulator organizing mammalian septin structures and provide a new link between the septin and Rho-signaling.

Protein kinase inhibitors can suppress stress-induced dissociation of Hsp27

Abstract We previously showed that the aggregated form of Hsp27 in cultured cells becomes dissociated as a result of phosphorylation with various types of stress. In order to clarify the signal transduction cascade involved, the effects of various inhibitors of protein kinases and dithiothreitol on the dissociation of Hsp27 were here examined by means of an immunoassay after fractionation of cell extracts by sucrose density gradient centrifugation. The dissociation of Hsp27 induced by exposure of U251 MG human glioma cells to metals (NaAsO2 and CdCl2), hypertonic stress (sorbitol and NaCl), or anisomycin, an activator of p38 mitogen–activated protein (MAP) kinase, was completely suppressed by the presence of SB 203580 or PD 169316, inhibitors of p38 MAP kinase, but not by PD 98059 and Uo 126, inhibitors of MAP kinase kinase (MEK), nor by staurosporine, Go 6983, and bisindolylmaleimide I, inhibitors of protein kinase C. Phorbol ester (PMA)–induced dissociation of Hsp27 was completely suppressed by staurosporine, Go 6983, or bisindolylmaleimide I and partially suppressed by SB 203580, or PD 169316 but not by PD 98059 or Uo 126, indicating mediation by 2 cascades. The presence of 1 mM dithiothreitol in the culture medium during exposure to chemicals suppressed the dissociation of Hsp27 induced by arsenite and CdCl2 but not by other chemicals. These results suggest that the phosphorylation of Hsp27 is catalyzed by 2 protein kinases, p38 MAP kinase–activated protein (MAPKAP) kinase- 2/3 and protein kinase C. In addition, metal-induced signals are sensitive to reducing power.

Septin 14 Is Involved in Cortical Neuronal Migration via Interaction with Septin 4

Septins are a family of conserved GTP/GDP-binding proteins implicated in a variety of cellular functions. We found that knockdown of Septin 14 or Septin 4 resulted in inhibition of cortical neuronal migration and defective leading process formation. These results suggest a novel function of septin in cortical development.

Ser-59 is the major phosphorylation site in alphaB-crystallin accumulated in the brains of patients with Alexander's disease.

The phosphorylation state of αB‐crystallin accumulated in the brains of two patients with Alexanders disease (one infantile and one juvenile type) was determined by means of SDS‐PAGE or isoelectric focusing of soluble and insoluble fractions of brain extracts and subsequent western blot analysis with specific antibodies against αB‐crystallin and each of three phosphorylated serine residues. The level of mammalian small heat shock protein of 25–28 kDa (Hsp27) in the same fraction was also estimated by western blot analysis. The majority of αB‐crystallin was detected in the insoluble fraction of brain homogenates and phosphorylation was preferentially observed at Ser‐59 in both cases. A significant level of phosphorylation at Ser‐45 but not Ser‐19 was also detected. Hsp27 was found at considerable levels in the insoluble fractions. αB‐crystallin and phosphorylated forms were detected in the cerebrospinal fluid of patient with the juvenile type. αB‐crystallin and phosphorylated forms were also detectable at considerable levels in the insoluble fraction of brain homogenates from patients with Alzheimers disease and aged controls. The phosphorylation site was mostly at Ser‐59 in all cases. Immunohistochemically, αB‐crystallin was stained in Rosenthal fibers in brains of patients with Alexanders disease and their peripheral portions were immunostained with antibodies recognizing phosphorylated Ser‐59. These results indicate that the major phosphorylation site in αB‐crystallin in brains of patients with Alexanders disease or Alzheimers disease as well as in aged controls is Ser‐59.

Innervation-dependent phosphorylation and accumulation of αB-crystallin and Hsp27 as insoluble complexes in disused muscle 1

Levels and phosphorylation states of the two small molecular chaperones, αB‐crystallin and Hsp27, in disused rat soleus muscles were determined by Western blot analysis of extracts with antibodies recognizing each of the two proteins and their phosphorylated serine residues. Increased phosphorylation and relocalization to insoluble fractions were found within a few days of hind‐limb suspension. High phosphorylation of αB‐crystallin at Ser‐59 (and to a certain extent, at Ser‐45) and of Hsp27 at Ser‐15 and Ser‐85, along with phosphorylated, active states of p38 and p44/42 mitogen‐activated protein kinases were maintained during hind‐limb suspension but promptly returned to control levels within a 5‐day recovery period. These results are similar to those observed with U373 MG glioma cells exposed to proteasome inhibitors (16). However, the responses of αB‐crystallin and Hsp27 in suspended soleus muscles did not appear with ipsilateral transection of the sciatic nerve trunk, indicating mediation by nerve activity. The fact that ubiquitinated proteins accumulated in the insoluble fractions of suspended soleus muscle further suggests participation of αB‐crystallin and Hsp27 in quality control of proteins in disused soleus muscle, with involvement of nerve activity‐dependent processes.

Sept8 controls the binding of vesicle-associated membrane protein 2 to synaptophysin.

Septins, a conserved family of GTP/GDP‐binding proteins, are present in organisms as diverse as yeast and mammals. We analyzed the distribution of five septins, Sept6, Sept7, Sept8, Sept9 and Sept11, in various rat tissues by western blot analyses and found all septins to be expressed in brain. We also examined the developmental changes of expression of these septins in the rat brain and found that the level of Sept8 increased during post‐natal development. Morphological analyses revealed that Sept8 is enriched at pre‐synapses. Using yeast two‐hybrid screening, we identified vesicle‐associated membrane protein 2 (VAMP2), a soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE), as an interacting protein for Sept8. Synaptophysin is reported to associate with and recruit VAMP2 to synaptic vesicles and dissociate prior to forming the SNARE complex consisting of VAMP2, syntaxin and synaptosome‐associated protein of 25 kDa. We showed that Sept8 suppresses the interaction between VAMP2 and synaptophysin through binding to VAMP2. In addition, we found that Sept8 forms a complex with syntaxin1A, and the Sept8‐VAMP2 interaction is disrupted by synaptosome‐associated protein of 25 kDa. These results suggest that Sept8 may participate in the process of the SNARE complex formation and subsequent neurotransmitter release.

Endoplasmic reticulum stress induces the phosphorylation of small heat shock protein, Hsp27

There are several reports describing participation of small heat shock proteins (sHsps) in cellular protein quality control. In this study, we estimated the endoplasmic reticulum (ER) stress‐induced response of Hsp27 and αB‐crystallin in mammalian cells. Treatment targeting the ER with tunicamycin or thapsigargin induced the phosphorylation of Hsp27 but not of αB‐crystallin in U373 MG cells, increase being observed after 2–10 h and decline at 24 h. Similar phosphorylation of Hsp27 by ER stress was also observed with U251 MG and HeLa but not in COS cells and could be blocked using SB203580, an inhibitor of p38 MAP kinase. Other protein kinase inhibitors, like Gö6983, PD98059, and SP600125, inhibitors of protein kinase C (PKC), p44/42 MAP kinase, and JNK, respectively, were without major influence. Prolonged treatment with tunicamycin but not thapsigargin for 48 h caused the second induction of the phosphorylation of Hsp27 in U251 MG cells. Under these conditions, the intense perinuclear staining of Hsp27, with some features of aggresomes, was observed in 10%–20% of the cells.