Two-dimensional polyacrylamide gel electrophoresis analysis of phosphorylated, membrane-localized ras p21 proteins

The ras p21 oncogene product migrates as a heterogeneous series of polypeptides as resolved by both one- and two-dimensional polyacrylamide gel electrophoresis (PAGE). We have prepared polyclonal rat serum antibody to ras p21 and used this as well as monoclonal antibodies to immunoprecipitate forms of p21 synthesized in vivo in transformed NIH3T3 cells. Two-dimensional PAGE of p21 resolved two distinct groups of polypeptides, one acidic (pI 4.8-5.3) which we call the "A" forms, and one less acidic or more basic (pI 6.5-7.0) which we call the "B" forms. It is the membrane-localized, B forms of v-ras-Ha p21 that are predominantly phosphorylated in vivo.     

Growth-rate-related and hydroxysterol-induced changes in membrane fluidity of cultured hepatoma cells: correlation with 3-hydroxy-3-methyl glutaryl CoA reductase activity

Creatine kinase (CK) activity in plasma obtained non-invasively from adult healthy, Sprague-Dawley, male rats was found to be 528 +/- 270 U/L (N = 17), a value which was 7 times that obtained in human specimens. Agarose gel electrophoresis revealed that the only detectable CK isoenzyme present was CK-BB, in contrast to the human serum isoenzyme which was CK-MM. Furthermore, it was found that the rat CK-BB could be detected using an RIA technique designed to quantitate human CK-BB occasionally present in blood after brain injury (rat CK-BB = 84.5 +/- 55.2 micrograms/L, N = 17, human CK-BB: Not detectable). It was thus possible to calculate the CK-BB specific activity (SA) in rat plasma using total CK assay and RIA (rat CK-BB SA = 6.25 +/- 3.87 U/micrograms, N = 17). When six rats (156 +/- 23 g) were treated with lead acetate in the drinking water (26 mM) for 3 weeks, the CK-BB SA rose to 18 +/- 5.8 U/micrograms (P less than .02). At this point the electrophoresis pattern of the CK-BB showed a transient change from a single band to a doublet. The dose was then increased to 52 mM for 6 weeks, during which time the CK-BB SA declined steadily to 1.6 +/- 0.6, a level significantly less than that of the untreated animals (p less than .02). The results suggest that chronic lead treatment evokes a biphasic response in CK-BB SA with the initial release of enzyme of high SA from tissues. Further treatment apparently results in an inactivation of the enzyme within lead sensitive tissues.     

Biosynthesis of the D2-cell adhesion molecule: post-translational modifications, intracellular transport, and developmental changes

Posttranslational modifications and intracellular transport of the D2- cell adhesion molecule (D2-CAM) were examined in cultured fetal rat neuronal cells. Developmental changes in biosynthesis were studied in rat forebrain explant cultures. Two D2-CAM polypeptides with Mr of 187,000-210,000 (A) and 131,000-158,000 (B) were synthesized using radiolabeled precursors in cultured neurons. A and B were found to contain only N-linked complex oligosaccharides, and both polypeptides appeared to be polysialated as determined by [14C]mannosamine incorporation and precipitation with anti-polysialic acid antibody. The two polypeptides were sulfated in the trans-Golgi compartment and phosphorylated at the plasma membrane. D2-CAM underwent rapid intracellular transport, appearing at the cell surface within 35 min of synthesis. A and B were shown to be integral membrane proteins as seen by radioiodination by photoactivation employing a hydrophobic labeling reagent. In rat forebrain explant cultures, D2-CAM was synthesized as four polypeptides: A (195,000 Mr), B (137,000 Mr), C (115,000 Mr), and a group of polypeptides in the high molecular weight region (HMr) between 250,000 and 350,000. Peptide maps of the four polypeptides yielded similar patterns. Biosynthesis of C and HMr increased with age, relative to A and B. A and B were sulfated in embryonic brain, however, sulfation was not noticeable at postnatal ages. Phosphorylation, on the other hand, of A and B was observed at all ages examined. We suggest that D2-CAM function may be modified during development by changes in the relative synthesis of the different polypeptides, as well as by changes in their glycosylation and sulfation.     

Identification of three coated vesicle components as alpha- and beta- tubulin linked to a phosphorylated 50,000-dalton polypeptide

Coated vesicles are involved in the intracellular transport of membrane proteins between a variety of membrane compartments. The coats of bovine brain coated vesicles contain at least six polypeptides in addition to an 180,000-dalton polypeptide called clathrin. In this report we show that the 54,000- and 56,000-dalton coated vesicle polypeptides are alpha- and beta-tubulin, determined by immunoblotting and two-dimensional gel electrophoresis. An affinity-purified tubulin antiserum can precipitate coated vesicles. The tubulin polypeptides are tightly associated with a 50,000-dalton coated vesicle polypeptide, which is phosphorylated. The phosphorylated 50,000-dalton polypeptide appears to be related to brain microtubule-associated tau proteins since it can be specifically immunoprecipitated by an affinity-purified antiserum directed against these proteins. In addition, gel filtration experiments indicate that at least a fraction of the 50,000-dalton polypeptide may associate with the 100,000-dalton coated vesicle polypeptide. Since brain is a tissue rich in tubulins, liver coated vesicles were analyzed for the presence of alpha- and beta-tubulin. Like brain coated vesicles, liver coated vesicles also contain an endogenous kinase activity, which phosphorylates polypeptides of the same molecular weights and isoelectric points as the brain coated vesicle 50,000-dalton, tau-like polypeptide, and alpha- and beta-tubulin. The phosphorylated 50,000-dalton polypeptide may link the membrane and contents of coated vesicles with components of the cytoskeleton.     

τ Phosphorylation in Human, Primate, and Rat Brain: Evidence that a Pool of τ Is Highly Phosphorylated In Vivo and Is Rapidly Dephosphorylated In Vitro

Abstract: The extent of τ phosphorylation is thought to regulate the binding of τ to microtubules: Highly phosphorylated τ does not bind to tubules, whereas dephosphorylated τ can bind to microtubules. It is interesting that the extent of τ phosphorylation in vivo has not been accurately determined. τ was rapidly isolated from human temporal neocortex and hippocampus, rhesus monkey temporal neocortex, and rat temporal neocortex and hippocampus under conditions that minimized dephosphorylation. In brain slices, we observed that τ isolated under such conditions largely existed in several phosphorylated states, including a pool that was highly phosphorylated; this was determined using epitope-specific monoclonal and polyclonal antibodies. This highly phosphorylated τ was dephosphorylated during a 120-min time course in vitro, presumably as a result of neuronal phosphatase activity. The slow-mobility forms of τ were shifted to faster-mobility forms following in vitro incubation with alkaline phosphatase. Laser densitometry was used to estimate the percent of τ in slow-mobility, highly phosphorylated forms. Approximately 25% of immunoreactive τ was present as slow-mobility (66- and 68-kDa) forms of τ. The percentage of immunoreactive τ in faster-mobility pools (42–54 kDa) increased in proportion to the decrease in content of 66–68-kDa τ as a function of neuronal phosphatases or alkaline phosphatase treatment. These data suggest that the turnover of phosphorylated sites on τ is rapid and depends on neuronal phosphatases. Furthermore, τ is highly phosphorylated in normal-appearing human, primate, and rodent brain. The presence of a highly phosphorylated pool of τ in adult brain may modify the present hypotheses on how paired helical filaments of Alzheimer's disease are formed.     

Differential phosphorylation in vivo of cytoplasmic dynein associated with anterogradely moving organelles

Two microtubule-stimulated ATPases, cytoplasmic dynein, and kinesin, are believed to be responsible for the intracellular movement of membrane-bound organelles in opposite directions along microtubules. An unresolved component of this model is the mechanism by which cells regulate these two motors to direct various membrane-bound organelles to their proper locations. To determine if phosphorylation may play a role in the regulation of cytoplasmic dynein, the in vivo phosphorylation state of cytoplasmic dynein from two cellular pools was examined. The entire cellular pool of brain cytoplasmic dynein was metabolically labeled by the infusion of [32P]orthophosphate into the cerebrospinal fluid of rat brain ventricles. To characterize the phosphorylation of dynein associated with anterograde membrane-bound organelles, the optic nerve fast axonal transport system was used. Using a monoclonal antibody to the 74-kD polypeptide of brain cytoplasmic dynein, the native dynein complex was immunoprecipitated from the radiolabled tissue extracts. Autoradiographs of one and two dimensional gels showed labeling of nearly all of the polypeptide isoforms of cytoplasmic dynein from rat brain. These polypeptides are phosphorylated on serine residues. Comparison of the amount of 32P incorporated into the dynein polypeptides revealed differences in the phosphorylation of dynein polypeptides from the anterograde and the cellular pools. Most interestingly, the 530-kD heavy chain of dynein appears to be phosphorylated to a lesser extent in the anterograde pool than in the cellular pool. Since the anterograde pool contains inactive dynein, while the entire cellular pool contains both inactive and active dynein, these results are consistent with the hypothesis that phosphorylation regulates the functional activity of cytoplasmic dynein.     

Subcellular compartmentalization of phosphorylated neurofilament polypeptides in neurons

Immunocytochemistry and polyacrylamide gel electrophoresis have been used to study the distribution of phosphorylated forms of neurofilament antigens in rat brain. Immunostaining of tissue with an antisera produced against phosphatase-sensitive domains of the 200-kilodalton (kd) neurofilament polypeptide showed that phosphorylated forms of this polypeptide were present in virtually all axons and certain somata and dendrites of neurons in different brain regions. Immunoblots of whole brain homogenate or a neurofilament preparation from rat revealed that the affinity-purified anti-200-kd sera used to immunostain tissue labeled the neurofilament-associated 200-kd band in a phosphatase-sensitive manner. Fine structural analysis of this immunoreactivity in tissue showed that whenever the labeled organelle could be identified, it was a microtubule. In contrast, immunoblot analysis of twice-cycled microtubules from porcine brain revealed that microtubules in vitro did not possess the 200-kd antigen that was observed in situ. The results suggest that our antibody recognizes a phosphorylated domain on the neurofilament involved in cross-linking neurofilaments and microtubules, and that in vivo, phosphorylated epitopes of the 200-kd neurofilament polypeptide are capable of associating with microtubules.     

Purification and characterization of calmodulin-dependent protein kinase II from rat spleen: a new type of calmodulin-dependent protein kinase II

A calmodulin-dependent protein kinase has been purified from rat spleen. The enzyme showed a remarkably similar substrate specificity and kinetic parameters to those of rat brain calmodulin-dependent protein kinase II, and exhibited cross-reactivity to a monoclonal antibody against rat brain calmodulin-dependent protein kinase II, indicating that the enzyme might be a calmodulin-dependent protein kinase II isozyme. The sedimentation coefficient was 13.9S, the Stokes radius was 67 A, and the molecular weight was calculated to be 380,000. The purified enzyme gave five polypeptides bands, corresponding to molecular weights of 51,000, 50,000, 21,000, 20,000, and 18,000, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation of the purified enzyme with Ca2+, calmodulin, and ATP under phosphorylating conditions induced the phosphorylation of all five polypeptides. When the logarithm of the velocity of the phosphorylation was plotted against the logarithm of the enzyme concentration (van't Hoff plot), slopes of 0.89, 0.94, and 1.1 were obtained for the phosphorylation of the 50/51-kDa doublet, 20/21-kDa doublet, and 18-kDa polypeptide, respectively. These results indicate that the phosphorylation of the five polypeptides is an intramolecular process, and further indicate that all five polypeptides are subunits of this enzyme. Of the five polypeptides, only the 50- and 51-kDa polypeptides bound to [125I]calmodulin, the other polypeptides not binding to it. A number of isozymic forms of calmodulin-dependent protein kinase II so far demonstrated in various tissues are known to be composed of subunits with molecular weights of 50,000 to 60,000 which can bind to calmodulin. Thus a new type of calmodulin-dependent protein kinase II was demonstrated in the present study.     

GAP-43, a protein associated with axon growth, is phosphorylated at three sites in cultured neurons and rat brain.

GAP-43 is a neuronal calmodulin-binding phosphoprotein that is concentrated in growth cones and presynaptic terminals. By sequencing tryptic and endoproteinase Asp-N phosphopeptides and directly determining the release of radioactive phosphate, we have identified three sites (serines 41 and 96 and threonine 172) that are phosphorylated, both in cultured neurons and in neonatal rat brain. These three sites account for most of the 32PO4 that was incorporated into GAP-43 in cultured neurons; 8-15% of each site was occupied with phosphate in GAP-43 isolated from neonatal rat brain. Phosphorylation of serine 41 in cultured neurons was stimulated by phorbol ester, indicating that it is the only site phosphorylated by protein kinase C. The resemblance of the sequence surrounding the other two sites suggests that they may be substrates for the same protein kinase. None of the sites phosphorylated by casein kinase II in vitro was phosphorylated in living cells or in neonatal rat brain. These results show that GAP-43 is a substrate for at least one protein kinase in addition to protein kinase C in living cells and brain.     

Characterization of Soluble Neural Cell Adhesion Molecule in Rat Brain, CSF, and Plasma

The polypeptide composition and glycosylation of soluble isoforms of neural cell adhesion molecule (NCAM) in developing rat brain, CSF, and plasma were characterized. Soluble NCAM in rat brain consisted of several glycosylated isoforms. The degree of glycosylation was developmentally regulated. After desialylation, four polypeptides of M(r) values of approximately 190,000 (s1), 135,000 (s2), 115,000 (s3), and 110,000 (s4) were observed. Polypeptides s1, s2, and s3 were also present in CSF, whereas only s3 and s4 were observed in plasma. Treatment of soluble brain NCAM with N-glycosidase F, which removes N-linked carbohydrates, produced polypeptides of M(r) values of approximately 190,000, 125,000, and 108,000-97,000. The monoclonal antibody OB11, which recognizes an epitope on the cytoplasmic part of transmembrane forms of NCAM, did not react with any of the soluble isoforms. Purified soluble NCAM, consisting mainly of s3, contained an N-terminal sequence identical to that of membrane-associated NCAM. Gel filtration of s3 indicated that it was present as a dimer under the chosen conditions. NCAM-expressing glioma cells adhered specifically to immobilized soluble NCAM. This implies that functionally significant soluble forms of NCAM are present in the extracellular fluid.     

The apparent molecular size of native alpha-crystallin B in non-lenticular tissues

The apparent molecular size of the native alpha-crystallin B in cytosol preparations from rat heart, brain and retina was determined by gel permeation chromatography, detecting the protein by immunochemical assay (ELISA), using an alpha-crystallin specific antiserum. Native alpha-crystallin from cytosol preparations of rat lens cortex was used as a reference. alpha-Crystallin B present in all three cytosol preparations from non-lenticular tissues eluted in a single symmetrical peak, with the same elution volume as alpha-crystallin from lens cortex cytosol preparations, corresponding to an apparent average molecular size of 0.8 x 10(6) Da. No other species could be detected. The results indicate that the alpha-crystallin aggregates characterized by an apparent average molecular mass of 0.8 x 10(6) Da, and considered to be the native, physiological form of the protein in the lens, are indeed not specific to lens tissue. Furthermore, the size of these alpha-crystallin aggregates is independent of their polypeptide composition. Aggregates found in the lens, composed of alpha A and alpha B polypeptides and their respective phosphorylated forms alpha Ap and alpha Bp, are similar in size to those found in heart, brain and retina, containing the alpha B but not the alpha A polypeptide.     

Effect upon mitogenic stimulation of calcium-dependent phosphorylation of cytoskeleton-associated 350,000- and 80,000-mol-wt polypeptides in quiescent 3Y1 cells

Rabbit antiserum raised against highest molecular weight microtubule-associated protein (MAP-1) of brain immunoprecipitated 350,000-, 300,000-, and 80,000-mol-wt phosphoproteins of rat embryo fibroblasts (3Y1-B). The 350,000-mol-wt protein was sensitive to heat as was brain MAP-1, but the 300,000- and 80,000-mol-wt proteins were not. These polypeptides were hardly phosphorylated in cells in the quiescent G0 phase but were rapidly phosphorylated after addition of serum, epidermal growth factor, phorbol ester, insulin, or transferrin in the presence of calcium ions. All these agents also induced incorporation of [3H]-thymidine into DNA. These polypeptides were detected in isolated microtubules and cold-resistant filaments by immunoblotting. Since the 350,000-mol-wt polypeptide was detected in the membrane, the cytoskeletons, and the nucleus, and has been suggested to function as a linker, its rapid phosphorylation might represent an early process in transduction of the signal of mitogenic stimulation to the nucleus.     

Autophosphorylation of chick brain synaptic polypeptides. Phosphorylation of proteins during incubation of intact synaptosomes with 32PO4

Chick brain synaptosomes incorporated phosphate into proteins when incubated in physiological buffer containing energy sources. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that three synaptosomal polypeptides were significantly phosphorylated after 15 sec incubation while at least fifteen polypeptides were active kinase substrates after 15 min incubation. Labeled synaptosomes were hypotonically lysed and separated by centrifugation into soluble, membrane, and mitochondrial fractions. Every fraction exhibited significant phosphate incorporation. Electrophoresis revealed that each fraction had several unique phosphorylated polypeptides and a distinctive phosphorylation pattern. The same polypeptides appear to be labeled whether MgATP was added to synaptic plasma membranes or synaptic plasma membranes were isolated after synaptosomal autophosphorylation.     

Calmodulin-dependent multifunctional protein kinase. Evidence for isoenzyme forms in mammalian tissues

Calcium/calmodulin-dependent multifunctional protein kinases, extensively purified from rat brain (with apparent molecular mass 640 kDa), rabbit liver (300 kDa) and rabbit skeletal muscle (700 kDa), were analysed for their structural, immunological, and enzymatic properties. The immunological cross-reactivity with affinity-purified polyclonal antibodies to the 50-kDa catalytic subunit of the brain calmodulin-dependent protein kinase confirmed the presence of common antigenic determinants in all subunits of the protein kinases. One-dimensional phosphopeptide patterns, obtained by digestion of the autophosphorylated protein kinases with S. aureus V8 protease, and two-dimensional fingerprints of the 125I-labelled proteins digested with a combination of trypsin and chymotrypsin, revealed a close similarity between the two subunits (51 kDa and 53 kDa) of the liver enzyme. Similar identity was observed between the 56-kDa and/or 58-kDa polypeptides of the skeletal muscle calmodulin-dependent protein kinase. The data suggest that the subunits of the liver and muscle protein kinases may be derived by partial proteolysis or by autophosphorylation. The peptide patterns for the 50-kDa and 60-kDa subunits of the brain enzyme confirmed that the two catalytic subunits represented distinct protein products. The comparison of the phosphopeptide maps and the two-dimensional peptide fingerprints, indicated considerable structural homology among the 50-kDa and 60-kDa subunits of the brain calmodulin-dependent protein kinase and the liver and muscle polypeptides. However, a significant number of unique peptides in the liver 51-kDa subunit, skeletal muscle 56-kDa, and the brain 50-kDa and 60-kDa polypeptides were observed and suggest the existence of isoenzyme forms. All calmodulin-dependent protein kinases rapidly phosphorylated synapsin I with a stoichiometry of 3-5 mol phosphate/mol protein. The two-dimensional separation of phosphopeptides obtained by tryptic/chymotryptic digestion of 32P-labelled synapsin I indicated that the same peptides were phosphorylated by all the calmodulin-dependent protein kinases. Such data represent the first structural and immunological comparison of the liver calmodulin-dependent protein kinase with the enzymes isolated from brain and skeletal muscle. The findings indicate the presence of a family of highly conserved calmodulin-dependent multifunctional protein kinases, with similar structural, immunological and enzymatic properties. The individual catalytic subunits appear to represent the expression of distinct protein products or isoenzymes which are selectively expressed in mammalian tissues.     

Isolation of phosphorylated and dephosphorylated forms of the CP43 internal antenna of photosystem II in Hordeum vulgare L

As a consequence of variation in environmental factors, light being the most important one, a number of photosystem II polypeptides may be reversibly phosphorylated by thylakoid-bound kinase(s). Among them, the reaction centre D1 and D2 polypeptides, the PsbH subunit, and the inner antenna CP43. Here, the separation of two forms of CP43 by high-resolution denaturing polyacrylamide gel electrophoresis is reported. By means of immunoblotting with antibody to phosphothreonine-containing proteins and authentic CP43 and limited proteolysis, these two bands could be identified as the phosphorylated and dephosphorylated forms of CP43. Using non-denaturing isoelectrofocusing, a chromatographically derived CP43-enriched fraction could be resolved into three different native forms of CP43. Among them, one was found to be a phosphorylated form, whereas the other two were dephosphorylated forms of the protein. With respect to other methods, the procedure described here allows the isolation, for the first time, of a fully homogeneous population of this chlorophyll-protein complex, opening the way to the study of the role of phopshorylation on functional properties of this core antenna protein.     

RNA polymerase II subunit composition, stoichiometry, and phosphorylation.

RNA polymerase II subunit composition, stoichiometry, and phosphorylation were investigated in Saccharomyces cerevisiae by attaching an epitope coding sequence to a well-characterized RNA polymerase II subunit gene (RPB3) and by immunoprecipitating the product of this gene with its associated polypeptides. The immunopurified enzyme catalyzed alpha-amanitin-sensitive RNA synthesis in vitro. The 10 polypeptides that immunoprecipitated were identical in size and number to those previously described for RNA polymerase II purified by conventional column chromatography. The relative stoichiometry of the subunits was deduced from knowledge of the sequence of the subunits and from the extent of labeling with [35S]methionine. Immunoprecipitation from 32P-labeled cell extracts revealed that three of the subunits, RPB1, RPB2, and RPB6, are phosphorylated in vivo. Phosphorylated and unphosphorylated forms of RPB1 could be distinguished; approximately half of the RNA polymerase II molecules contained a phosphorylated RPB1 subunit. These results more precisely define the subunit composition and phosphorylation of a eucaryotic RNA polymerase II enzyme.     

Identification of Two Distinct Isoforms of the Guanine Nucleotide Binding Protein Go in Neuroblastoma × Glioma Hybrid Cells: Independent Regulation During Cyclic AMP-Induced Differentiation

Three distinct antipeptide antisera generated against synthetic peptides that represent parts of the primary sequence of the alpha-subunit of the (pertussis toxin-sensitive) guanine nucleotide binding protein G0 were used in two-dimensional immunoblots of membranes of neuroblastoma X glioma (NG108-15) cells. Each antiserum identified two distinct polypeptides of some 39 kDa. These had apparent isoelectric points of 5.5 and 5.8. Differentiation of NG108-15 cells in response separately to dibutyryl cyclic AMP (cAMP), 8-bromo cAMP, forskolin, and prostaglandin E1 produced elevated levels of G0 alpha, as has previously been noted in one-dimensional immunoblots. Two-dimensional analysis demonstrated that the cAMP-induced increases in levels of G0 alpha were only of the more acidic isoform. The two isoforms were both substrates for pertussis toxin-catalysed ADP-ribosylation and did not appear to represent differentially phosphorylated forms of the same polypeptide. Separation of the two forms of G0 alpha could be achieved in one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis when 4 M deionized urea was included in the resolving gel. The more slowly migrating band was the acidic form and corresponded exactly in mobility with the major form of G0 from both rat and mouse brain. There was no equivalent in brain of the more rapidly migrating form of G0 from the cells. In agreement with the data from two-dimensional gels, only the more slowly migrating form was expressed in considerably higher amounts following cAMP-induced differentiation of NG108-15 cells. Of these two forms of "G0," the acidic species is equivalent to G0 from brain, but the basic form is not identical with G0*, which has been purified from bovine brain.     

Phosphorylation of Simian Virus 40 Proteins in a Cell-Free System

We have shown previously that all the structural proteins of simian virus 40 (SV40) are phosphoproteins. Virus phosphorylated in vivo could be further phosphorylated with exogenous cellular protein kinases in a cell-free system containing gamma-(32)P-ATP as phosphate donor. In intact infectious virus only polypeptides 1 and 2 (mol wt 49,000 and 40,800, respectively) were further phosphorylated in vitro. However, when infectious SV40 was partially disrupted, treated with nucleases, and then phosphorylated in vitro, all five structural polypeptides accepted additional phosphate groups. Similarly, all polypeptides of intact empty capsids, derived from infected cells, were further phosphorylated in vitro. Phosphorylation of empty capsids and infectious SV40 in vitro was enhanced from 4- to 11-fold after prior treatment of virus with alkali. The phosphate group was linked only to serine residues of the viral polypeptides phosphorylated both in vitro and in vivo.     

Rat brain myo-inositol 3-phosphate synthase is a phosphoprotein

The therapeutic effects of lithium in bipolar disorder are poorly understood. Lithium decreases free inositol levels by inhibiting inositol monophosphatase 1 and myo-inositol 3-phosphate synthase (IPS). In this study, we demonstrate for the first time that IPS can be phosphorylated. This was evident when purified rat IPS was dephosphorylated by lambda protein phosphatase and analyzed by phospho-specific ProQ-Diamond staining and Western blot analysis. These techniques demonstrated a mobility shift consistent with IPS being phosphorylated. Mass spectral analysis revealed that Serine-524 (S₅₂₄), which resides in the hinge region derived from exon 11 of the gene, is the site for phosphorylation. Further, an antibody generated against a synthetic peptide of IPS containing monophosphorylated-S₅₂₄, was able to discriminate the phosphorylated and non-phosphorylated forms of IPS. The phosphoprotein is found in the brain and testis, but not in the intestine. The intestinal IPS isoform lacks the peptide bearing S₅₂₄, and hence, cannot be phosphorylated. Evidences suggest that IPS is monophosphorylated at S₅₂₄ and that the removal of this phosphate does not alter its enzymatic activity. These observations suggest a novel function for IPS in brain and other tissues. Future studies should resolve the functional role of phospho-IPS in brain inositol signaling.     

Microtubule affinity chromatography: a new technique for isolating microtubule-binding proteins from rat pancreas.

We have developed an affinity chromatography method for the isolation of microtubule-associated proteins (MAPs) from soluble cytoplasmic extracts of rat pancreas. Among the ten proteins which copurify with pancreas tubulin on a colchicine derivatives-affinity chromatography, three polypeptides of respectively 58, 55 and 48 kDa strongly bind to the microtubule affinity column. To begin to characterize these proteins, we have generated polyclonal antibodies against tau polypeptides from brains of immature chicken or rat. As judged by immunoblots, the three polypeptides seem to be immunologically related to the tau proteins previously localized in brain.