一种新的人CNA1基因转录本的克隆和功能鉴定

钙调神经磷酸酶（calcineurin,CN）是唯一依赖于Ca2+和钙调蛋白（calmodulin,CaM）的丝氨酸/苏氨酸型蛋白磷酸酶,由1个催化亚基CNA和1个调节亚基CNB组成. CNA 有3种亚型,最常见的是由CNA1基因编码的α亚型（CNAα）. 在克隆CNA1基因cDNA的过程中,发现了1种新的人CNA1转录本-CNAα4. 与CNA1基因的其它转录本相比,CNAα4缺失第2外显子,其编码蛋白质由454个氨基酸组成,具有比其它3种CNAα亚型更短的磷酸酶催化结构域. CNAα4具有与CNAα1相似的CaM亲和力,但是其激活活化T细胞核因子（nuclear factor of activated T cells,NFAT）的活性明显强于CNAα1,提示CNAα4所缺失的氨基酸序列（Ala20 Thr86）并非CNA催化结构域所必需,相反,Ala²⁰-Thr⁸⁶缺失可能有助于其酶活性中心与NFAT的结合并发挥作用.     

The cardiac-specific nuclear delta(B) isoform of Ca2+/calmodulin-dependent protein kinase II induces hypertrophy and dilated cardiomyopathy associated with increased protein phosphatase 2A activity.

The delta isoform of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) predominates in the heart. To investigate the role of CaMKII in cardiac function, we made transgenic (TG) mice that express the nuclear delta(B) isoform of CaMKII. The expressed CaMKIIdelta(B) transgene was restricted to the myocardium and highly concentrated in the nucleus. Cardiac hypertrophy was evidenced by an increased left ventricle to body weight ratio and up-regulation of embryonic and contractile protein genes including atrial natriuretic factor, beta-myosin heavy chain, and alpha-skeletal actin. Echocardiography revealed ventricular dilation and decreased cardiac function, which was also observed in hemodynamic measurements from CaMKIIdelta(B) TG mice. Surprisingly, phosphorylation of phospholamban at both Thr(17) and Ser(16) was significantly decreased in the basal state as well as upon adrenergic stimulation. This was associated with diminished sarcoplasmic reticulum Ca(2+) uptake in vitro and altered relaxation properties in vivo. The activity and expression of protein phosphatase 2A were both found to be increased in CaMKII TG mice, and immunoprecipitation studies indicated that protein phosphatase 2A directly associates with CaMKII. Our findings are the first to demonstrate that CaMKII can induce hypertrophy and dilation in vivo and indicate that compensatory increases in phosphatase activity contribute to the resultant phenotype.     

Use of alkaline phosphatase fusions to study protein secretion in Bacillus subtilis.

We have constructed a vector designed to facilitate the study of protein secretion in Bacillus subtilis. This vector is based on a translational fusion between the expression elements and signal sequence of Bacillus amyloliquefaciens alkaline protease and the mature coding sequence for Escherichia coli alkaline phosphatase (phoA). We show that export of alkaline phosphatase from B. subtilis depends on a functional signal sequence and that alkaline phosphatase activity depends upon secretion. The vector design facilitates the insertion of heterologous coding sequences between the signal and phoA to generate three-part translational fusions. Such phoA fusions are easily analyzed by monitoring alkaline phosphatase activity on agar plates or in culture supernatants or by immunological detection. Exploitation of this methodology, which has proven to be extremely useful in the study of protein secretion in E. coli, has a variety of applications for studying protein secretion in B. subtilis.     

Tissue-specific expression of an 11 beta-hydroxysteroid dehydrogenase with a truncated N-terminal domain. A potential mechanism for differential intracellular localization within mineralocorticoid target cells.

The enzyme 11 beta-hydroxysteroid dehydrogenase (11-HSD) is thought to confer specificity on the nonselective Type I adrenocorticoid receptor by converting glucocorticoids to receptor-inactive metabolites in mineralocorticoid target tissues. S1 nuclease analyses using a rat liver 11-HSD probe demonstrated tissue-specific expression of the 5' region of the 11-HSD gene in the liver, lung, and kidney not evident in previous studies. Renal tissue contained a unique protected species which mapped to a position within the coding region, consistent with a divergence in liver and kidney protein sequences. Screening of a rat kidney cDNA library resulted in the isolation of several clones (11-HSD1B) noncolinear in their 5' regions with the liver sequence (11-HSD1A). Nucleic acid sequence analysis showed that the divergent clones code for a protein lacking a 26-amino acid NH2-terminal putative membrane-spanning signal peptide. The deletion of the leader sequence from the microsomal 11-HSD1A protein may result in a nuclear localization of the 11-HSD1B isoform. The renal 11-HSD1A and 11-HSD1B species increased coordinately during ontogeny and in parallel with the developmental surge in glucocorticoids. At least three alternate sites of polyadenylation were found to be utilized by the 11-HSD gene. Southern blot analysis showed the presence of a single gene in the rat. This study shows the expression of a kidney-specific 11-HSD isoform which may protect the Type I adrenocorticoid receptor from occupation by glucocorticoids in the nucleus of a mineralocorticoid target cell.     

Secretion of Bacillus subtilis alpha-amylase in the periplasmic space of Escherichia coli

The Bacillus subtilis alpha-amylase structural gene (amyE) lacking its own signal peptide coding sequence was joined to the end of the Escherichia coli alkaline phosphatase (phoA) signal peptide coding sequence by using the technique of oligonucleotide-directed site-specific deletion. On induction of the phoA promoter, the B. subtilis alpha-amylase was expressed and almost all the activity was found in the periplasmic space of E. coli. The sequence of the five amino-terminal amino acids of the secreted polypeptide was Glu-Thr-Ala-Asn-Lys-, and thus the fused protein was correctly processed by the E. coli signal peptidase at the end of the phoA signal peptide.     

Changes in Microtubule-Associated Protein MAP1B Phosphorylation During Rat Brain Development

Microtubule-associated protein MAP1B from neonatal rat brain was separated on sodium dodecyl sulfate-containing polyacrylamide gels into two isoforms (high and low MAP1B), both of which were recognized by a panel of monoclonal and polyclonal antibodies against MAP1B. In addition, SMI31, a monoclonal antibody directed against phosphorylated epitopes of the neurofilament proteins, showed phosphatase-sensitive reactivity against the high isoform of MAP1B. The antigenic relationship between the phosphorylated isoform of MAP1B and neurofilaments was confirmed by the reactivity of SMI31 with the immunoprecipitated MAP1B protein. After dephosphorylation of MAP1B with alkaline phosphatase, the higher-molecular-weight isoform of MAP1B was no longer detectable with phosphate-insensitive anti-MAP1B antibodies, whereas there was a significant increase in the immunoreactivity of the lower-molecular-weight MAP1B isoform. These data suggest that the structural microheterogeneity of MAP1B is due to differences in phosphorylation. The two isoforms were present in all brain regions of the young rat. During brain development, the general decrease in MAP1B levels was accompanied by changes in the relative amount of the two isoforms. In particular, the phosphorylated isoform of MAP1B decreased dramatically to almost undetectable levels in adult brain. This conclusion was further supported by immunoblotting analysis that showed the disappearance of phosphorylated epitopes of MAP1B early during brain development. In addition, dephosphorylation experiments demonstrated the phosphatase sensitivity of the phosphorylated isoform throughout development.     

Inhibition of protein phosphatases by Michael adducts of ascorbic acid analogues with alpha,beta-unsaturated carbonyl compounds

By investigating the stereospecific Michael reaction of derivatives of ascorbic acid with acrolein we obtained a novel class of protein Ser/Thr phosphatase inhibitors. The inhibitory effect of the Michael adducts was examined using the canonical protein phosphatases type 1, 2A and 2B. Of the isozymes examined the type 1 isoform was strongly inhibited.     

Regulation of releasable vesicle pool sizes by protein kinase A-dependent phosphorylation of SNAP-25

Protein kinase A (PKA) is a key regulator of neurosecretion, but the molecular targets remain elusive. We combined pharmacological manipulations of kinase and phosphatase activities with mutational studies on the exocytotic machinery driving fusion of catecholamine-containing vesicles from chromaffin cells. We found that constitutive PKA activity was necessary to maintain a large number of vesicles in the release-ready, so-called primed, state, whereas calcineurin (protein phosphatase 2B) activity antagonized this effect. Overexpression of the SNARE protein SNAP-25a mutated in a PKA phosphorylation site (Thr-138) eliminated the effect of PKA inhibitors on the vesicle priming process. Another, unidentified, PKA target regulated the relative size of two different primed vesicle pools that are distinguished by their release kinetics. Overexpression of the SNAP-25b isoform increased the size of both primed vesicle pools by a factor of two, and mutations in the conserved Thr-138 site had similar effects as in the a isoform.     

The uncovering of a novel regulatory mechanism for PLD2: formation of a ternary complex with protein tyrosine phosphatase PTP1B and growth factor receptor-bound protein GRB2

The regulation of PLD2 activation is poorly understood at present. Transient transfection of COS-7 with a mycPLD2 construct results in elevated levels of PLD2 enzymatic activity and tyrosyl phosphorylation. To investigate whether this phosphorylation affects PLD2 enzymatic activity, anti-myc immunoprecipitates were treated with recombinant protein tyrosine phosphatase PTP1B. Surprisingly, lipase activity and PY levels both increased over a range of PTP1B concentrations. These increases occurred in parallel to a measurable PTP1B-associated phosphatase activity. Inhibitor studies demonstrated that an EGF-receptor type kinase is involved in phosphorylation. In a COS-7 cell line created in the laboratory that stably expressed myc-PLD2, PTP1B induced a robust (>6-fold) augmentation of myc-PLD2 phosphotyrosine content. The addition of growth factor receptor-bound protein 2 (Grb2) to cell extracts also elevated PY levels of myc-PLD (>10-fold). Systematic co-immunoprecipitation-immunoblotting experiments pointed at a physical association between PLD2, Grb2, and PTP1B in both physiological conditions and in overexpressed cells. This is the first report of a demonstration of the mammalian isoform PLD2 existing in a ternary complex with a protein tyrosine phosphatase, PTP1b, and the docking protein Grb2 which greatly enhances tyrosyl phosphorylation of the lipase.     

Identification and characterization of a synaptojanin 2 splice isoform predominantly expressed in nerve terminals

We have previously identified synaptojanin 1, a phosphoinositide phosphatase predominantly expressed in the nervous system, and synaptojanin 2, a broadly expressed isoform. Synaptojanin 1 is concentrated in nerve terminals, where it has been implicated in synaptic vesicle recycling and actin function. Synaptojanin 2A is targeted to mitochondria via a PDZ domain-mediated interaction. We have now characterized an alternatively spliced form of synaptojanin 2 that shares several properties with synaptojanin 1. This isoform, synaptojanin 2B, undergoes further alternative splicing to generate synaptojanin 2B1 and 2B2. Both amphiphysin and endophilin, two partners synaptojanin 1, bind synaptojanin 2B2, whereas only amphiphysin binds synaptojanin 2B1. Sequence similar to the endophilin-binding site in synaptojanin 1 is present only in synaptojanin 2B2, and this sequence was capable of affinity purifying endophilin from rat brain. The Sac1 domain of synaptojanin 2 exhibited phosphoinositide phosphatase activity very similar to that of the Sac1 domain of synaptojanin 1. Site-directed mutagenesis further illustrated its functional similarity to the catalytic domain of Sac1 proteins. Antibodies raised against the synaptojanin 2B-specific carboxyl-terminal region identified a 160-kDa protein in brain and testis. Immunofluorescence showed that synaptojanin 2B is localized at nerve terminals in brain and at the spermatid manchette in testis. Active Rac1 GTPase affects the intracellular localization of synaptojanin 2, but not of synaptojanin 1. These results suggest that synaptojanin 2B has a partially overlapping function with synaptojanin 1 in nerve terminals, with additional roles in neurons and other cells including spermatids.     

The effect of deleting p110delta on the phenotype and function of PTEN-deficient B cells

Control of the intracellular levels of phosphatidylinositol-(3, 4, 5)-trisphosphate by PI3K and phosphatase and tensin homolog (PTEN) is essential for B cell development and differentiation. Deletion of the PI3K catalytic subunit p110delta leads to a severe reduction in B1 and marginal zone (MZ) B cells, whereas deletion of PTEN results in their expansion. We have examined the relationship between these two molecules by generating mice with a B cell-specific deletion of PTEN (PTENB) and a concurrent germline deletion of p110delta. The expanded B1 cell population of PTENB mice was reduced to normal levels in PTENB/p110delta mutant mice, indicating a critical role for the p110delta isoform in the expansion of B1 cells. However, numbers of MZ B cells in the PTENB/p110delta mutants was intermediate between wild-type and PTENB-deficient mice, suggesting an additional role for other PI3K catalytic isoforms in MZ differentiation. Furthermore, the defective class switch recombination in PTENB B cells was only partially reversed in PTENB/p110delta double mutant B cells. These results demonstrate an epistatic relationship between p110delta and PTEN. In addition, they also suggest that additional PI3K catalytic subunits contribute to B cell development and function.     

Molecular cloning and primary structure of a protein phosphatase 2C isoform.

Complementary DNA encoding the isoform of protein phosphatase 2C, termed PP2C2, has been isolated. The cDNA predicts a protein of 390 amino acid residues with a molecular mass of 42,888 Da. The protein displays 76% identity to the PP2C1 isoform.     

Molecular interactions among protein phosphatase 2A, tau, and microtubules. Implications for the regulation of tau phosphorylation and the development of tauopathies.

Hyperphosphorylated forms of the neuronal microtubule (MT)-associated protein tau are major components of Alzheimer's disease paired helical filaments. Previously, we reported that ABalphaC, the dominant brain isoform of protein phosphatase 2A (PP2A), is localized on MTs, binds directly to tau, and is a major tau phosphatase in cells. We now describe direct interactions among tau, PP2A, and MTs at the submolecular level. Using tau deletion mutants, we found that ABalphaC binds a domain on tau that is indistinguishable from its MT-binding domain. ABalphaC binds directly to MTs through a site that encompasses its catalytic subunit and is distinct from its binding site for tau, and ABalphaC and tau bind to different domains on MTs. Specific PP2A isoforms bind to MTs with distinct affinities in vitro, and these interactions differentially inhibit the ability of PP2A to dephosphorylate various substrates, including tau and tubulin. Finally, tubulin assembly decreases PP2A activity in vitro, suggesting that PP2A activity can be modulated by MT dynamics in vivo. Taken together, these findings indicate how structural interactions among ABalphaC, tau, and MTs might control the phosphorylation state of tau. Disruption of these normal interactions could contribute significantly to development of tauopathies such as Alzheimer's disease.