Reversible hyperphosphorylation and reorganization of vimentin intermediate filaments by okadaic acid in 9L rat brain tumor cells.

Okadaic acid (OA), a protein phosphatase inhibitor, was found to induce hyperphosphorylation and reorganization of vimentin intermediate filaments in 9L rat brain tumor cells. The process was dose dependent. Vimentin phosphorylation was initially enhanced by 400 nM OA in 30 min and reached maximal level (about 26-fold) when cells were treated with 400 nM OA for 90 min. Upon removal of OA, dephosphorylation of the hyperphosphorylated vimentin was observed and the levels of phosphorylation returned to that of the controls after the cells recovered under normal growing conditions for 11 h. The phosphorylation and dephosphorylation of vimentin induced by OA concomitantly resulted in reversible reorganization of vimentin filaments and alteration of cell morphology. Cells rounded up as they were entering mitosis in the presence of OA and returned to normal appearance after 11 h of recovery. Immuno-staining with anti-vimentin antibody revealed that vimentin filaments were disassembled and clustered around the nucleus when the cells were treated with OA but subsequently returned to the filamentous states when OA was removed. Two-dimensional electrophoresis analysis further revealed that hyperphosphorylation of vimentin generated at least seven isoforms having different isoelectric points. Furthermore, the enhanced vimentin phosphorylation was accompanied by changes in the detergent-solubility of the protein. In untreated cells, the detergent-soluble and -insoluble vimentins were of equal amounts but the solubility could be increased when vimentins were hyperphosphorylated in the presence of OA. Taken together, the results indicated that OA could be involved in reversible hyperphosphorylation and reorganization of vimentin intermediate filaments, which may play an important role in the structure-function regulation of cytoskeleton in the cell.     

An Eight Amino Acid Segment Controls Oligomerization and Preferred Conformation of the two Non-visual Arrestins

G protein coupled receptors signal through G proteins or arrestins. A long-standing mystery in the field is why vertebrates have two non-visual arrestins, arrestin-2 and arrestin-3. These isoforms are ~75% identical and 85% similar; each binds numerous receptors, and appear to have many redundant functions, as demonstrated by studies of knockout mice. We previously showed that arrestin-3 can be activated by inositol-hexakisphosphate (IP₆). IP₆ interacts with the receptor-binding surface of arrestin-3, induces arrestin-3 oligomerization, and this oligomer stabilizes the active conformation of arrestin-3. Here, we compared the impact of IP₆ on oligomerization and conformational equilibrium of the highly homologous arrestin-2 and arrestin-3 and found that these two isoforms are regulated differently. In the presence of IP₆, arrestin-2 forms “infinite” chains, where each promoter remains in the basal conformation. In contrast, full length and truncated arrestin-3 form trimers and higher-order oligomers in the presence of IP₆; we showed previously that trimeric state induces arrestin-3 activation (Chen et al., 2017). Thus, in response to IP₆, the two non-visual arrestins oligomerize in different ways in distinct conformations. We identified an insertion of eight residues that is conserved across arrestin-2 homologs, but absent in arrestin-3 that likely accounts for the differences in the IP₆ effect. Because IP₆ is ubiquitously present in cells, this suggests physiological consequences, including differences in arrestin-2/3 trafficking and JNK3 activation. The functional differences between two non-visual arrestins are in part determined by distinct modes of their oligomerization. The mode of oligomerization might regulate the function of other signaling proteins.     

Chromatographic and immunological evidence that chloroplastic and cytosolic pea (Pisum sativum L.) NADP-isocitrate dehydrogenases are distinct isoenzymes

Two NADP-isocitrate dehydrogenase isoenzymes designated as NADP-IDH₁ and NADP-IDH₂ (EC 1.1.1.42) were identified in pea (Pisum sativum) leaf extracts by diethylaminoethylcellulose chromatography. The predominant form was found to be NADP-IDH₁ while NADP-IDH₂ represented only about 4% of the total leaf enzyme activity. These enzymes share few common epitopes as NADP-IDH₂ was poorly recognized by the specific polyclonal antibodies raised against NADP-IDH₁, and as a consequence NADP-IDH₂ does not result from a post-translational modification of NADP-IDH₁. Subcellular fractionation and isolation of chloroplasts through a Percoll gradient, followed by the identification of the associated enzymes, showed that NADP-IDH₁ is restricted to the cytosol and NADP-IDH₂ to the chloroplasts. Compared with the cytosolic isoenzyme, NADP-IDH₂ was more thermolabile and exhibited a lower optimum pH. The data reported in this paper constitute the first report that the chloroplastic NADP-IDH and the cytosolic NADP-IDH are two distinct isoenzymes. The possible functions of the two isoenzymes are discussed.Abbreviations BSA bovine serum albumin - DEAE diethylaminoethyl - NADP-IDH NADP-isocitrate dehydrogenase - NADP-IDH₁ cytosolic NADP-IDH - NADP-IDH₂ chloroplastic NADP-IDH     

Tissue-specific analogues of erythrocyte protein 4.1 retain functional domains

Analogues of the human erythroid membrane skeletal component protein 4.1 have been identified in perfused rat tissues and human T and B lymphocyte cell lines. olyclonal antibodies were used which are specific for all domains of protein 4.1, the spectrin-actin-promoting 8-Kd peptide, the membrane-binding 30-Kd domain, and the 50-Kd domain. Antibody reactivity, by Western blotting of tissue homogenates, shows reactivity with proteins varying in molecular weight from 175 Kd to 30 Kd. Further, these protein 4.1 analogues appear to be expressed in a tissue-specific fashion. Of the analogues detected there appear to be at least three classes: analogues containing all erythroid protein 4.1 domains, analogues containing all domains but with modified antigenic epitopes, and analogues containing only some domains. Chemical cleavage at cysteine linkages indicates that in analogues containing the 30-Kd region the location of cysteine is highly conserved. This datum suggests that in nonerythroid 4.1 isoforms of higher molecular weight the additional protein mass is added to the amino terminal end (30 Kd end).     

The Effect of Light and Exogenously Supplied Ammonium Ions on Glutamate Dehydrogenase Activity and Isoforms in Young Mustard (Sinapis alba L.) Seedlings

Glutamate dehydrogenase (GDH, E.C. 1.4.1.3) of mustard cotyledons was investigated during the first 4 days of seedling development. The enzyme was found to be composed of seven catalytically active isoforms (each with a molecular mass of 270 kDa) which exhibited a charge heterogeneity when investigated by isoelectric focusing. Antibodies against the purified isoform 7, raised in rabbits, cross-reacted with each of the isoforms in Western blotting experiments. In addition, each of the isoforms was composed of four immunopositive reacting polypeptides with 19, 21, 23 and 25 kDa. During development of the seedlings, a shift in the isoform pattern towards the more acidic forms was found which was more pronounced when the seedlings were supplied with 15 mM NH₄Cl. The time course of changes in total GDH level can be correlated with the time course of disappearance of storage proteins. Both parameters are negatively regulated by light possibly via the photoreceptor, phytochrome. There are some indications that GDH in young mustard cotyledons mainly acts in the deaminating direction.     

Type I protein kinase C isozyme in the visual-information-processing pathway of monkey brain

Previously using PKC isozyme-specific antibodies for immunoblot analysis, we demonstrated the heterogeneous distribution of PKC isozymes in various regions of monkey and rat brains and that type I PKC was most abundant in cerebellum, hippocampus, amygdala, and cerebral cortex (Huang et al.: J Biol Chem 262:15714-15720, 1987). Using these antibodies, we have also demonstrated that type I, II, and III PKC are products of PKC genes gamma, beta, and alpha, respectively (Huang et al.: Biochem Biophys Res Commun 149:946-952, 1987). By immunocytochemical analysis, type I PKC-specific antibody showed strong reactivity in various types of neuron in hippocampal formation, amygdala, cerebellum, and neocortex. In hippocampal formation, granule cells of dentate gyrus and pyramidal cells of hippocampus were heavily stained. By immunoblot analysis, relative levels of PKC isozymes in several areas of monkey cerebral cortex involved in the visual information processing and storage were determined. Both type II and III PKCs appeared to be evenly distributed and at moderate levels, type I PKC formed a gradient of increasing concentration rostral along the cerebral cortex of occipital to temporal and then to the limbic areas. Neurobehavioral studies have demonstrated that the neocortical and limbic areas of the anterior and medial temporal regions participate more directly than the striate, prestriate, and posterior temporal regions in the storage of visual representations and that both hippocampus and amygdala are important in the memory formation. As type I PKC is present at high levels in hippocampus, amygdala, and anterior temporal lobe, we predict that the type I protein kinase C may participate in the plastic changes important for mnemonic function.     

Isoforms of Na,K-ATPase inArtemia salina: II. Tissue distribution and kinetic characterization

Summary To characterize the molecular properties conveyed by the isoforms of the subunit of Na,K-ATPase, the two major transepithelial transporting organs in the brine shrimp (Artemia salina), the salt glands and intestines, were isolated in pure form. The isoforms were quantified by ATP-sensitive fluorescein isothiocyanate (FITC) labeling. The salt gland enzyme exhibits only the 1 isoform, whereas the intestinal enzyme exhibits both the 1 and the 2 isoforms. After 32 hours of development, Na,K-ATPase activity [in mol P_i/mg protein/hr (1u)] in whole homogenates was 32±6 in the salt glands and 12±3 in the intestinal preparations (mean±sem). The apparent half-maximal activation constants (K _1/2) of the salt gland enzyme as compared to the intestinal enzyme were 3.7±0.6mm vs. 23.5±4mm (P<0.01) for Na⁺, 16.6±2.2mm vs. 8.29±1.5mm for K⁺ (P<0.01), and 0.87±0.8mm vs. 0.79±1.1mm for ATP (NS). The apparentK _i's for ouabain inhibition were 1.1×10^–4 m vs. 2×10^–5 m, respectively. Treatment of whole homogenates with deoxycholic acid (DOC) produced a maximal Na,K-ATPase activation of 46% in the salt gland as compared to 23% in the intestinal enzyme. Similar differences were found with sodium dodecyl sulfate (SDS). The two distinct forms of Na,K-ATPase isolated from the brine shrimp differed markedly in three kinetic parameters as well as in detergent sensitivity. The differences inK _1/2 for Na⁺ and K⁺ are more marked than those reported for the mammalian Na,K-ATPase isoforms. These differences may be attributed to the relative abundances of the subunit isoforms; other potential determinants (e.g. differences in membrane lipids), however, have not been investigated.During the tenure of an Educational Commission For Foreign Medical Graduates Visiting Associate Professorship.     

Rat RI beta isoform of type I regulatory subunit of cyclic adenosine monophosphate-dependent protein kinase: cDNA sequence analysis, mRNA tissue specificity, and rat/mouse difference in expression in testis

A rat complementary DNA (cDNA) for the RI beta isoform of type I cyclic adenosine monophosphate (cAMP)-dependent protein kinase regulatory subunit was cloned and sequenced and was found to contain the entire protein coding and 3'-untranslated regions, with a single (ATTAAA) poly-adenylation site. The largest open reading frame was preceded by a short out-of-phase open reading frame, which is not seen in the corresponding mouse RI beta cDNA due to a single base substitution. The rat RI beta cDNA clone was 2,374 bases long and detected a rat mRNA of approximately 2.8 kilobases. Rat RI beta mRNA was abundant in adult rat brain and testis but was undetectable in other rat tissues. The rat RI beta cDNA also detected RI beta mRNA in mouse brain, but not mouse testis, from 10-week-old BALB/c or 10- and 6-week-old Swiss Webster mice. Thus, despite a 96% nucleotide identity in the coding region of RI beta in rat vs. mouse, there are at least two differences in these closely related species. First, there is a short open reading frame, which precedes the coding region in the rat but not the mouse. Second, unlike the mouse testis, the rat testis contains abundant levels of RI beta mRNA.