Phosphorylation of αB-crystallin, a member of the hsp27 family, in human glioma (U373 MG) cells was stimulated by exposure of the cells to various stimuli, which included heat, arsenite, phorbol 12-myristate 13-acetate (PMA), okadaic acid, H₂O₂, anisomycin, and high concentrations of NaCl or sorbitol, but not in response to agents that elevated intracellular levels of cyclic AMP. Cells exposed to PMA together with okadaic acid yielded three bands of ³²P-labeled αB-crystallin when immunoprecipitated samples were subjected to electrophoresis on an isoelectric focusing gel. All of the phosphorylated residues were identified as serine, an indication that three different serine residues can act as sites of phosphorylation in αB-crystallin. Structural analysis by mass spectrometry revealed that phosphorylation of αB-crystallin occurred at serines 19, 45, and 59. Dithiothreitol and staurosporine selectively inhibited the phosphorylation induced by arsenite and the phorbol ester, respectively. SB202190, an inhibitor of p38 mitogen-activated protein (MAP) kinase, suppressed the phosphorylation induced by arsenite, anisomycin, H₂O₂, sorbitol, NaCl, and heat shock, but not that induced by PMA and okadaic acid. The PMA-induced phosphorylation was selectively suppressed by an inhibitor of p44 MAP kinase kinase, PD98059. Although PMA and arsenite preferentially stimulated the phosphorylation of Ser-45 and Ser-59, respectively, as determined with antibodies that recognized the respective phosphorylated forms of αB-crystallin, all three sites were phosphorylated in response to each stimulus. These results suggest that p38 MAP kinase or p44 MAP kinase might be involved in the signal transduction cascade that leads to the phosphorylation of αB-crystallin. The phosphorylation of αB-crystallin was also enhanced in the heart and diaphragm when rats were exposed to heat stress (42 °C for 20 min).

AlphaB-crystallin in cells can be phosphorylated at three serine residues in response to stress or during mitosis (Ito, H., Okamoto, K., Nakayama, H., Isobe, T., and Kato, K. (1997) J. Biol. Chem. 272, 29934-29941 and Kato, K., Ito, H., Kamei, K., Inaguma, Y., Iwamoto, I., and Saga, S. (1998) J. Biol. Chem. 273, 28346-28354). In the present study, we determined effects of phosphorylation of alphaB-crystallin on its oligomerization state, mainly by using site-directed mutagenesis, in which all three phosphorylation sites were substituted with aspartate to mimic the phosphorylation state (3D-alphaB). From results of sucrose density gradient centrifugation, we found that wild type alphaB-crystallin (wt-alphaB) and 3D-alphaB sedimented in fractions corresponding to apparent molecular masses of about 500 and 300 kDa, respectively. Chaperone-like activity of 3D-alphaB was significantly weaker than that of wt-alphaB. When wt-alphaB and 3D-alphaB were expressed in COS-m6 cells, they sedimented at positions corresponding to apparent molecular masses of about 500 and 100 kDa, respectively. In U373 MG human glioma cells, alphaB-crystallin was observed as large oligomers with apparent molecular masses about 500 kDa and the oligomerization size was reduced after phosphorylation induced by phorbol 12-myristate 13-acetate and okadaic acid. Coexpression of luciferase and wt-alphaB or 3D-alphaB in Chinese hamster ovary cells caused protection of the enzyme from heat inactivation although the degree of protection with 3D-alphaB was less than that with wt-alphaB. From these observations, it is suggested that phosphorylation of alphaB-crystallin causes dissociation of large oligomers to smaller sizes molecules and reduction of chaperone-like activity, like in the case of HSP27.

The immunofluorescence localization of alphaB-crystallin in U373 MG human glioma cells with an antibody specific for alphaB-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 alphaB-crystallin in mitotic cells were detected in all cell lines examined and in tissue sections of mouse embryos. Increases in the levels of alphaB-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 alphaB-crystallin in extracts of mitotic U373 MG cells, using the amino-terminal 72-amino acid peptide derived from unphosphorylated alphaB2-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 alphaB-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.

Septins are a family of conserved guanosine triphosphate/guanosine diphosphate-binding proteins implicated in a variety of cellular functions such as cell cycle control and cytokinesis. Although several members of septin family, including Septin 14 (Sept14), are abundantly expressed in nervous tissues, little is known about their physiological functions, especially in neuronal development. Here, we report that Sept14 is strongly expressed in the cortical plate of developing cerebral cortex. Knockdown experiments by using the method of in utero electroporation showed that reduction of Sept14 caused inhibition of cortical neuronal migration. Whereas cDNA encoding RNA interference-resistant Sept14 rescued the migration defect, the C-terminal deletion mutant of Sept14 did not. Biochemical analyses revealed that C-terminal coiled-coil region of Sept14 interacts with Septin 4 (Sept4). Knockdown experiments showed that Sept4 is also involved in cortical neuronal migration in vivo. In addition, knockdown of Sept14 or Sept4 inhibited leading process formation in migrating cortical neurons. These results suggest that Sept14 is involved in neuronal migration in cerebral cortex via interaction with Sept4.

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.