Multiple subcellular localizations of PCTAIRE-1 in brain.

We developed a selective antibody to a synthetic peptide corresponding to an N-terminal sequence of the PCTAIRE-1 protein. In rodent brain extracts it recognized only the protein doublet characteristic of PCTAIRE-1, and this signal is completely abolished by preincubation of the antibody with the immunopeptide. Immunolabeling experiments done with this PCTAIRE-1-specific antibody reveal that the protein is widely distributed in the rodent brain as are the mRNAs visualized using an antisense riboprobe corresponding to the entire PCTAIRE-1 open reading frame. Two types of PCTAIRE-1 protein localizations were observed: first a diffuse labeling of almost all brain regions, particularly intense in the molecular layer of the cerebellum and the mossy fiber region of the hippocampus, and second a spot-like localization in the nuclei of large neurons such as cerebellar Purkinje cells and pyramidal cells of the hippocampus. Colocalization with the B23 protein allows one to identify these compartments as nucleoli. Our results suggest a nucleolar function of PCTAIRE-1 in large neurons and a role in regions containing important granule cell projections.

Characterization of brain PCTAIRE-1 kinase immunoreactivity and its interactions with p11 and 14-3-3 proteins.

An antibody directed against the C-terminal part of PCTAIRE-1 recognized three proteins in rodent brain. The high-molecular-mass band is most abundant in the cerebellum, hippocampus and cortex. It migrated at the same apparent molecular mass as recombinant PCTAIRE-1 and interacted, like recombinant PCTAIRE-1, with p11 and 14-3-3 proteins. Combination of p11 or 14-3-3 affinity resins with immunoprecipitation and peptide elution allowed us to obtain a purified full-length PCTAIRE-1 preparation having significant kinase activity. These results suggest that PCTAIRE-1 is an active kinase in brain. The catalytic core region of PCTAIRE-1 which is common for all cyclin-dependent kinases, does not interact with p11 and 14-3-3 proteins in the two-hybrid assay. Full interaction with p11 and 14-3-3 proteins requires both, the N-terminal and C-terminal ends of PCTAIRE-1, suggesting that complex three-dimensional arrangements are responsible for these interactions. A low-molecular-mass protein (migrating at about 30 kDa) that was also recognized by the antibody directed against the carboxy-terminal part of PCTAIRE-1, is abundant and almost homogeneously distributed in all brain areas investigated. Database searches starting with the amino acid sequences of two peptides obtained by tryptic digestion of this protein yielded cDNA and genomic (a gene of about 10 kb on human chromosome 1q24-1q25 and clone 262D12) sequences, allowing us to compose a DNA sequence coding for a putative 26 kDa protein containing both peptides. This protein has no important sequence similarity with any other known protein. But many DNA sequences are found in databases with an almost 100% identity with parts of the 26 kDa protein coding sequence. Our results allow us to attribute these widely distributed cDNA sequences to an existing 26-kDa protein and to localize a gene within two recently published genomic sequences.

The Cdk-like protein PCTAIRE-1 from mouse brain associates with p11 and 14-3-3 proteins.

PCTAIRE-1 is a member of the cyclin-dependent kinase (cdk)-like class of proteins, and is localized mainly in the mammalian brain. Using the yeast two-hybrid system we screened a mouse brain cDNA library with PCTAIRE-1 as bait, and isolated several clones coding for the mouse homologs of the following proteins: p11 (also known as calpactin I light chain) and the eta, theta (also known as tau) and zeta isoforms of 14-3-3 proteins. We confirmed that these four proteins interact with PCTAIRE-1 by demonstrating the biochemical interactions using the pure recombinant proteins. The fact that 14-3-3 proteins are known to interact with many other intracellular proteins (such as C-kinase, Raf, Bcr, P13-kinase) and p11 with annexin II (a major pp60(v-src) and C-kinase substrate) suggests that PCTAIRE-1 might be part of multiple signal transduction cascades and cellular protein networks.

Dephosphorylation of cdc2 on threonine 161 is required for cdc2 kinase inactivation and normal anaphase.

Exit from metaphase of the cell cycle requires inactivation of MPF, a stoichiometric complex between the cdc2 catalytic and the cyclin B regulatory subunits, as well as that of cyclin A-cdc2 kinase. Inactivation of both complexes depends on proteolytic degradation of the cyclin subunit, yet cyclin proteolysis is not sufficient to inactivate the H1 kinase activity of cdc2. Genetic evidence strongly suggests that type 1 phosphatase plays a key role in the metaphase-anaphase transition of the cell cycle. Here we report that inhibition of both type 1 and type 2A phosphatases by okadaic acid allows cyclin degradation to occur, but prevents cdc2 kinase inactivation. Complete inhibition of type 2A phosphatase alone is not sufficient to prevent cdc2 kinase inactivation following cyclin proteolysis. We show further that residue 161 of cdc2 is phosphorylated in active cyclin A or cyclin B complexes at metaphase, whilst unassociated cdc2 is not phosphorylated. Proteolysis of cyclin releases a free cdc2 subunit, which subsequently undergoes dephosphorylation and then migrates more slowly than its Thr161 phosphorylated counterpart in Laemmli gels. Removal of phosphothreonine 161 requires cyclin proteolysis. However, it does not occur even after cyclin proteolysis, when both type 1 and type 2A phosphatases are inhibited. We conclude that both cyclin degradation and dephosphorylation of Thr161 on cdc2, catalysed at least in part by type 1 phosphatase, are required to inactivate either cyclin B- or cyclin A-cdc2 kinases and thus for cells to exit from M phase.

An okadaic acid-sensitive phosphatase negatively controls the cyclin degradation pathway in amphibian eggs.

Inhibition of okadaic acid-sensitive phosphatases released the cyclin degradation pathway from its inhibited state in extracts prepared from unfertilized Xenopus eggs arrested at the second meiotic metaphase. It also switched on cyclin protease activity in a permanent fashion in interphase extracts prepared from activated eggs. Even after cdc2 kinase inactivation, microinjection of okadaic acid-treated interphase extracts pushed G2-arrested recipient oocytes into the M phase, suggesting that the phosphatase inhibitor stabilizes the activity of an unidentified factor which shares in common with cdc2 kinase the maturation-promoting factor activity.

Protein kinase C: properties and possible role in cellular division and differentiation.

Protein kinase C was first described some eight years ago. Recent results indicate that this kinase may have a crucial role in signal transduction for substances involved in cellular differentiation and division. Protein kinase C is activated by attachment to plasma membranes, in the presence of calcium and diacylglycerol. The activator is produced in the membrane following the signal-induced breakdown of phosphoinositides. Tumor promoters, such as phorbol ester, can substitute for diacylglycerol. The recent findings that: tyrosine kinases might be involved in the phosphoinositide turnover and, phosphorylation of growth factor receptors by protein kinase C regulates some of their functions, indicate more and more clearly that this kinase is involved in the control of cell growth division and differentiation. Purification procedures, properties and mechanisms of regulation will be summarized and discussed.

Role of protein synthesis and proteases in production and inactivation of maturation-promoting activity during meiotic maturation of starfish oocytes.

In starfish oocytes, activity of the maturation-promoting factor (MPF) and that of a major cAMP-independent protein kinase dropped at the time of meiotic cleavage, and rose again after the first but not the second meiotic cleavage. Protein synthesis was required before the first meiotic cleavage for both MPF and protein kinase activity to rise again after the first meiotic cleavage. Microinjection of either leupeptin or soybean trypsin inhibitor early enough prior to first polar body emission suppressed both the meiotic cleavage and the associated drop of MPF activity. Microinjection of leupeptin or soybean trypsin inhibitor during the 10-min period before the first meiotic cleavage also suppressed cytokinesis but did not prevent a decrease in MPF activity at the normal time of cytokinesis. The lysosomotropic inhibitor ammonia neither suppressed cytokinesis nor the drop of MPF activity at the time of first meiotic cleavage. Activity of neutral proteases sensitive to leupeptin and soybean trypsin inhibitor was demonstrated in oocyte homogenates prepared at the time of first meiotic cleavage. It is proposed that such proteases might be involved in degradation of protein kinase(s) and in the drop of MPF activity at the time of first meiotic cleavage.

Inositol 1,4,5-triphosphate microinjection triggers activation, but not meiotic maturation in amphibian and starfish oocytes.

Inositol 3,4,5-triphosphate (InsP3) brought about cortical granule exocytosis and elevation of a fertilization membrane, due to a rapid increase of free calcium in cytoplasm, when injected into oocytes of the amphibian Xenopus laevis arrested at second meiotic metaphase. The same result was observed when injection was performed into oocytes of the starfish Marthasterias glacialis arrested either at the first meiotic prophase or after completion of meiosis. Although meiotic maturation was induced in both animals by specific hormones which have been previously shown to release Ca2+ within cytoplasm, InsP3 microinjection into prophase-arrested oocytes did not release them from prophase block.

Metabolic CO2 production by isolated single pieces of rat distal nephron segments.

A method is described which allowed in-vitro measurements of metabolic CO2 production from [U-14C]-substrates by single pieces of kidney tubules. The tubules were isolated by microdissection from collagenase treated rat kidneys. Single pieces of various distal nephrons portions were incubated in 1 microliter of bicarbonate free minimum essential medium containing the required [U-14C]-substrate (about 0.2 mu Ci per sample), and the 14CO2 produced was continuously trapped into a 2-microliter KOH droplet. The KOH droplets were replaced every 30 min. Metabolic CO2 production from the labelled substrate used was calculated as picomoles CO2 per mm of tubular length per minute, by dividing the KOH radioactivity by the specific radioactivity per carbon of the substrate present in the incubate [( U-14C] plus cold substrate concentrations). Under these conditions, it was established that single pieces of tubule could sustain almost constant CO2 production for at least 2 h at 31 degrees C. Experiments testing four different conditions with five to six replicate samples per condition were performed in order to compare oxidative metabolism in medullary (MAL) and cortical (CAL) thick ascending limbs, medullary (MCT) and cortical (CCT) collecting tubules and, in a few instances, proximal convoluted tubules (PCT) and early distal convoluted tubules (DCT).(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma antidiuretic hormone levels and kidney responsiveness to vasopressin in the jerboa, Jaculus orientalis.

The plasma antidiuretic hormone (ADH) concentration and the kidney medulla responsiveness to vasopressin were measured in adult jerboas ( Jaculus orientalis) in different states of hydration. In 15 jerboas adapted to 30 degrees and fed a dry diet, the average ADH concentration in blood plasma was 479 +/- 59 pg/ml, as measured by a radioimmunoassay. About 6 hr after receiving a 5% body wt water load by gavage, the plasma ADH concentration fell to 130 +/- 30 pg/ml in the 5 jerboas still producing hypertonic urine (1022 +/- 267 mosmol/liter) and to 41.5 +/- 8.4 pg/ml in the 6 jerboas producing hypoosmotic urine (157 +/- 6 mosmol/liter). In vitro biochemical experiments were performed on the kidney medullas from two groups of 5 jerboas fed a dry diet (group I) or a water-enriched diet (group II), respectively, for 4 to 7 weeks. Compared to group II, group I animals exhibited (a) higher plasma ADH values, 372 +/- 86 versus 76 +/- 25 pg/ml; (b) higher urine osmolarities (3817 +/- 638 versus 647 +/- 90 mosmol/liter); (c) some decrease in [3H]lysine-vasopressin (LVP) binding capacity to kidney membrane fractions (maximal binding: 0.4 versus 0.6 pmol [3H]LVP bound/mg protein); d) decreased adenylate cyclase responses to arginine-vasopressin, lysine-vasopressin, and oxytocin in kidney membrane fractions; and (e) weaker adenylate cyclase responses to arginine-vasopressin in microdissected pieces of the medullary thick ascending limb of Henle's loop. The values found for (a) the dissociation constant of [3H]lysine-vasopressin binding to membranes (KD); (b) adenylate cyclase sensitivity to the three neurohypophyseal hormones (KA); and (c) adenylate cyclase sensitivity to arginine-vasopressin (KA) in medullary collecting tubules and medullary thick ascending limbs are similar in the two groups of jerboas and roughly comparable to those previously reported for the rat kidney medulla. The reduced maximal adenylate cyclase responses to vasopressin in the jerboas fed a dry diet might indicate some physiological "down regulation" of the number of vasopressin-specific receptors in the kidney as a result of the huge ADH concentration present in blood plasma under these conditions. However, this desensitization is not sufficient to account for the production of hypoosmotic urine in spite of the relatively high ADH plasma levels which persisted after acute overhydration.

Changes in tubular basolateral membrane markers after chronic DOCA treatment.

Chronic administration of DOCA to rabbits is known to increase the surface area of the basolateral membrane and the Na-K-ATPase activity of the cortical collecting tubule (CCT). We attempted to ascertain 1) whether Na-K-ATPase is the only basolateral membrane marker induced by DOCA, and 2) whether CCT is the only nephron segment affected by this steroid. We measured the activity of Na-K-ATPase and adenylate cyclase (AC) and the protein content of nephron segments microdissected from control and DOCA-treated rabbits. Morphogenic effects of DOCA, assessed by 30-60% increases in protein content, were specifically observed in the distal convoluted tubule, CCT, and medullary collecting tubule. When expressed as a function of tubular length, Na-K-ATPase activity rose from 80 to 200% in all these segments, whereas the increments in AC of 40-70%, observed in response to four different hormones, occurred only in some of them. When expressed as a function of protein content, Na-K-ATPase activity increased but AC activity remained unchanged. This study indicates that the morphogenic action resulting from chronic DOCA administration affects the entire rabbit distal nephron. During this action Na-K-ATPase is the preferentially induced enzyme.

Multiple hormonal control of adenylate cyclase in distal segments of the rat kidney.

Using the single tubule adenylate cyclase microassay, we investigated in vitro in three different segments of the rat nephron whether the effects of various hormones are additive when these hormones are tested in combination. In the cortical portion of the thick ascending limb (CAL), no additivity of the effects of glucagon, calcitonin, and PTH was observed. In the medullary portion of the thick ascending limb (MAL), the effects of vasopressin and glucagon were only partly additive, and the effects of vasopressin and calcitonin were fully additive. In the cortical collecting tubule (CCT), the effects of calcitonin and vasopressin were nonadditive in the kidneys in which vasopressin alone induced a high cyclase stimulation, whereas they were fully additive when vasopressin induced a low cyclase stimulation. The data suggest that in each segment, the hormones tested stimulated the same cells: no additivity was observed when cyclase Vmax acted as the limiting factor of the response; partial or full additivity was observed when the number of hormone receptors acted as the limiting factor of the response. As a consequence, calcitonin, glucagon, and PTH should induce the same effects in CAL; vasopressin, glucagon, and calcitonin, the same effects in MAL; and vasopressin and calcitonin, the same effects in CCT.