The participation of annexin II (calpactin I) in calcium-evoked exocytosis requires protein kinase C

Permeabilized adrenal chromaffin cells secrete catecholamines by exocytosis in response to micromolar calcium concentrations. Recently, we have demonstrated that chromaffin cells permeabilized with digitonin progressively lose their capacity to secrete due to the release of certain cytosolic proteins essential for exocytosis (Sarafian T., D. Aunis, and M. F. Bader. 1987. J. Biol. Chem. 34:16671-16676). Here we show that one of the released proteins is calpactin I, a calcium-dependent phospholipid-binding protein known to promote in vitro aggregation of chromaffin granules at physiological micromolar calcium levels. The addition of calpactin I into digitonin- or streptolysin-O-permeabilized chromaffin cells with reduced secretory capacity as a result of the leakage of cytosolic proteins partially restores the calcium-dependent secretory activity. This effect is specific of calpactin I since other annexins (p32, p37, p67) do not stimulate secretion at similar or higher concentrations. Calpactin I requires the presence of Mg-ATP, suggesting that a phosphorylating step may regulate the activity of calpactin. Calpactin is unable to restore the secretory activity in cells which have completely lost their cytosolic protein kinase C or in cells having their protein kinase C inhibited by sphingosine or downregulated by long-term incubation with TPA. In contrast, calpactin I prephosphorylated in vitro by purified protein kinase C is able to reconstitute secretion in cells depleted of their protein kinase C activity. This stimulatory effect is also observed with thiophosphorylated calpactin I which is resistant to cellular phosphatases or with phosphorylated calpactin I introduced into cells in the presence of microcystin, a phosphatase inhibitor. These results suggest that calpactin I is involved in the exocytotic machinery by a mechanism which requires phosphorylation by protein kinase C.     

Structure and chromosome assignment of the murine p36 (calpactin I heavy chain) gene

p36 is a major substrate of both viral and growth factor receptor associated protein kinases. This protein has recently been named calpactin I heavy chain since it is the large subunit of a Ca2(+)-dependent phospholipid and actin binding heterotetramer. The primary structure of p36 has been determined from analysis of cloned cDNA. The protein contains 338 amino acids, has an approximate molecular weight of 39,000, and is comprised of several distinct domains, including four 75 amino acid repeats. From two overlapping cosmid clones isolated from different mouse genomic liver libraries, the complete intron/exon structure of the p36 gene was determined and the 5' and 3' noncoding regions of the gene were analyzed. The coding and 3' untranslated region of the p36 gene contains 12 exons which range in size from 48 to 322 base pairs (bp) with an average size of 107 bp. The repeat structures found at the protein level are not delineated by single exons, but the N-terminal p11-binding domain is encoded by a single exon. Structural mapping of the gene demonstrated that the lengths of the first two introns in the coding region are together approximately 6 kilobases (kb), while the other introns range in size from 600 to 3600 bp with an average size of 1650 bp. The p36 gene is at least 22 kb in length and has a coding sequence of approximately 1 kb, representing only 4.5% of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycosylation of annexin I and annexin II.

Human placental annexin I and annexin II were shown to be glycosylated by one-dimensional affinity blotting with the lectin concanavalin A, which recognizes D-mannose and D-glucose residues. Further evidence that annexin I and annexin II are glycosylated was provided by the finding that these proteins incorporated D-[2,6-3H]mannose and D-[6-3H]glucose when they were biosynthesized by the human squamous carcinoma cell line SqCC/Y1. Annexin I and annexin II could be rapidly purified from a human placental membrane extract by concanavalin A-Sepharose, which indicated that these proteins contain two biantennary mannosyl residues.     

The cDNA sequence for the protein-tyrosine kinase substrate p36 (calpactin I heavy chain) reveals a multidomain protein with internal repeats

We have isolated and sequenced a full-length cDNA clone for the protein-tyrosine kinase substrate p36 (calpactin I heavy chain). This sequence predicts a 339 amino acid (Mr 38,493) protein containing an N-terminal region of 20 amino acids, known to interact with a 10 kd protein (light chain), and a C-terminal region, found to contain two Ca2+/phospholipid-binding sites, that can be aligned as four 70 amino acid repeats. A single p36 gene was detected in the mouse genome, and a major p36 mRNA of 1.6 kb was found to be expressed in different mouse tissues. Unexpectedly, p36 and the phospholipase A2 inhibitor lipocortin I were found to be 50% identical in sequence over the C-terminal 300 residues. The function of p36 and its relation to other proteins are discussed.     

The growth-regulated gene 1B6 is identified as the heavy chain of calpactin I

The expression of 1B6, a growth-regulated sequence isolated from a Syrian hamster fibroblast cDNA library, was studied in BALB/c 3T3 cells. The level of cytoplasmic 1B6 mRNA (1600 bases) was low in quiescent cells and plateaued in mid/late G1 after the cells were stimulated with 15% fetal calf serum (FCS). Protein synthesis was not required for the induction of 1B6 mRNA; therefore, the expression of 1B6 is a primary response to serum stimulation. The induction of 1B6 mRNA was also observed after stimulation with insulin, epidermal growth factor, and fibroblast growth factor but not with platelet-derived growth factor. When quiescent cells were serum-stimulated, the percentage of cells that became committed to enter DNA synthesis was proportional to the length of their incubation with serum. To determine if 1B6 expression was also correlated with the time of exposure to serum, quiescent cells were stimulated with a pulse of 15% FCS and the abundance level of 1B6 induced by that pulse was determined. The amount of 1B6 mRNA increased with increasing time of exposure to serum and paralleled the increase in the percentage of nuclei that were induced into DNA synthesis by the serum pulse. Comparison of the nucleotide sequence of the p1B6 cDNA to the GenBank database revealed a striking identity of 1B6 to the 3' end of p36, the heavy chain of calpactin I. The previous characterization of p36 as a substrate for tyrosine kinases suggests a possible role for 1B6/p36 in cell proliferation.     

cDNA sequence of human p11 calpactin I light chain.

The cDNA encoding full-length human p11 calpactin I light chain has been cloned and subjected to DNA sequencing. The open reading frame specifies a 97-amino-acid residue protein that surprisingly is identical to the p11 sequences of two mammalian ungulate species, cow and pig. However, the previously reported p11 polypeptide sequences of mouse and rat exhibited 8-9% nonidentity to human p11. These mammalian sequence comparison results are unexpected in view of current molecular cladistic theories that suggest a closer relationship between primates and rodents, rather than primates and ungulates. The mouse p11 gene has been previously mapped to chromosome 3 at a position syntenic with a centromeric-proximal region on human chromosome 1, and the human p11 cDNA clone is likely to be useful in physical mapping on chromosome 1.     

Association of the S-100-related calpactin I light chain with the NH2-terminal tail of the 36-kDa heavy chain

Calpactin I, a Ca2+- and phospholipid-binding cytoskeletal protein, which serves as a major substrate of protein-tyrosine kinases, was isolated from bovine intestine and lung as a species containing two 36-kDa heavy chains and two 10-kDa light chains. The heavy chain is comprised of two distinct domains which can be identified by limited proteolysis: a COOH-terminal 33-kDa core, which contains the Ca2+- and phospholipid-binding sites, and an NH2-terminal tail, which contains the major site of phosphorylation by pp60v-src. To determine the site of association of the light chain on the heavy chain, we analyzed the association states of the light chain, core, and tail by sucrose gradient centrifugation after limited chymotryptic digestion. The core was not detected in higher Mr complexes with the light chain, and the tail cosedimented with a light chain dimer. The tail, isolated from chymotryptic digests and radiolabeled with 125I, was found to form a specific complex with the light chain, but not the core. The authentic tail and a synthetic peptide corresponding to residues 1-29 of the calpactin I heavy chain were both able to specifically inhibit the reassociation between heavy and light chain, whereas a synthetic peptide corresponding to residues 15-33 was inactive. These results suggest that the tail may serve as a site of regulation by light chain or phosphorylation.     

Mitotic arrest caused by the amino terminus of Xenopus cyclin B2.

Progression through mitosis requires the inactivation of the protein kinase activity of the p34cdc2-cyclin complex by a mechanism involving the degradation of cyclin. We have examined the stability in Xenopus egg extracts of radiolabeled Xenopus or sea urchin B-type cyclins synthesized in reticulocyte lysates. Xenopus cyclin B2 and sea urchin cyclin B were stable in metaphase extracts from unfertilized eggs but were specifically degraded following addition of Ca2+ to the extracts. The degradation of either cyclin was inhibited by the addition of an excess of unlabeled Xenopus cyclin B2 but not by the addition of a number of control proteins. A truncated protein containing only the amino terminus of Xenopus cyclin B2, including sequences known to be essential for cyclin degradation in other species, also inhibited cyclin degradation, even though the truncated protein was stable in extracts following Ca2+ addition. The addition of the truncated protein did not stimulate histone H1 kinase activity in extracts but prevented the loss of H1 kinase activity that normally follows Ca2+ addition to metaphase extracts. When the amino-terminal fragment was added to extracts capable of several cell cycles in vitro, progression through the first mitosis was inhibited and elevated histone H1 kinase activity was maintained. These results indicate that although the amino terminus of cyclin does not contain all of the information necessary for cyclin destruction, it is capable of interacting with components of the cyclin destruction pathway and thereby preventing the degradation of full-length cyclins.     

Phosphorylation of the heavy chain of skeletal muscle myosin by casein kinase II: localization of the phosphorylation site to the amino terminus

While the heavy chain of rabbit skeletal muscle myosin is not phosphorylatable by casein kinase II, it turned out to be phosphorylatable after removal of all of the light chains. The phosphorylation site for the kinase was determined to be Ser-1 and/or Ser-2 at the amino terminus.     

Annexin II (calpactin I) in the mouse mammary gland: immunolocalisation by light- and electron microscopy

Summary To elucidate the putative role of annexin II (calpactin I) in the secretory function of mammary tissue its immunolocalisation in the mammary gland of pregnant and lactating mice was investigated by light- and electron microscopy using the immunoperoxidase technique. A low level of fairly uniform annexin II staining was evident throughout the gland despite its mixed composition during pregnancy. In lactating tissue it was revealed that apparently mature alveoli contained a concentration of annexin II staining outlining their epithelium. The staining was localised by immuno-electron microscopy to the apical membrane of these alveolar epithelial cells and their microvillar extentions. There was also an apparent association of annexin II with vesicles of a range of sizes located near, or actually fused with, the apical membrane. Many of the small, stained vasicles could clearly be identified as casein-containing vesicle while the large vesicles were apparently associated with either casein granules or possibly lipid. The appearance of a selective concentration of annexin II in apparently actively secreting mammary epithelial cells, as revealed in this study, is consistent with a possible structural and/or functional role for this protein at the membranes participating in the secretion of protein and possibly lipid from these secretory cells.     

The 4F2 antigen heavy chain induces uptake of neutral and dibasic amino acids in Xenopus oocytes.

The 4F2 cell surface antigen is a disulfide-linked heterodimer induced during the process of cellular activation and expressed widely in mammalian tissues (Parmacek, M. S., Karpinski, B. A., Gottesdiener, K. M., Thompson, C. B., and Leiden, J. M. (1989) Nucleic Acids Res. 17, 1915-1931). The human heavy chain component, a type II membrane glycoprotein, has 29% identity to the amino acid transport-related protein encoded by the recently cloned rat D2 cDNA. We have demonstrated that Xenopus oocytes injected with in vitro transcribed cRNA from D2 take up cystine and dibasic and neutral amino acids (Wells, R. G., and Hediger, M. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5596-5600). In the present study, we examine the role of the human 4F2 heavy chain in amino acid transport. In vitro transcribed 4F2 cRNA was injected into Xenopus oocytes which were assayed for the uptake of radiolabeled amino acids. Our results show that cRNA from 4F2 stimulates the uptake of dibasic and neutral amino acids into oocytes at levels up to 3-fold higher than for water-injected control oocytes. There is no demonstrable uptake of cystine. Uptake is saturable, with characteristics of high affinity transport, and inhibition data suggest that uptake occurs via a single transporter. Dibasic amino acids are taken up by both 4F2 and D2 cRNA-injected oocytes in a sodium-independent manner. In contrast, 4F2-induced but not D2-induced neutral amino acid uptake has a significant component of sodium dependence. Likewise, neutral amino acids in excess inhibit the 4F2-induced uptake of radiolabeled arginine but not leucine in a sodium-dependent manner. The 4F2-induced uptake we observe most likely represents the activity of a single transport system with some characteristics of systems y+, b0,+, and B0,+. We suggest that 4F2 and D2 represent a new family of proteins which induce amino acid transport with distinct characteristics, possibly functioning as transport activators or regulators.     

Analysis of the amino terminus of maize branching enzyme II by polymerase chain reaction random mutagenesis

Maize endosperm branching enzyme II (mBEII) plays a pivotal role in determining the quality of starch by catalyzing the synthesis of the alpha-1,6-branch points. While the central (alpha/beta)8-barrel and the C-terminal domains of mBEII have been analyzed previously, the possible role of its amino terminus in catalysis is still poorly understood. Because the amino terminus of mBEII shares very little sequence homology with other amylolytic enzymes, the Met1-Gly276 region of mBEII was randomly mutagenized under error-prone PCR conditions. Subsequent screening by a heterologous complementation system, utilizing an Escherichia coli strain devoid of the endogenous glycogen branching enzyme (glgB-), led to the recovery of mBEII mutants with altered iodine-staining patterns and reduced branching enzyme activities. The NR-625 mutant enzyme, which lacks the N-terminal 39 residues of mBEII due to a frameshift mutation introduced during the random mutagenesis, retained more than 70% of the wild-type activity. The chain transfer pattern and substrate preference of the truncated enzyme were almost identical to those of the wild-type mBEII. It appears that the N-terminal 39 residues of mBEII are neither required for catalysis nor involved in chain transfer. On the other hand, the Gln-to-Arg substitution at position 270 of mBEII resulted in the loss of more than 90% of branching activity. The Gln270 of mBEII, located at the beginning of the (alpha/beta)8-barrel domain, may be required for maximum enzyme activity.     

Purification of annexin I and annexin II from human placental membranes by high-performance liquid chromatography.

Annexin I and annexin II were extracted from human placental membranes with ethylene glycol bis(beta-amino-ethyl ether)-N,N'-tetraacetic acid (EGTA) and purified by high-performance liquid chromatography by measuring their ability to inhibit phospholipase A2 activity in vitro. Neither protein was capable of binding to a DEAE-5PW HPLC column at neutral pH; however, they were resolved through binding to a Mono S column and passage through size-exclusion HPLC columns. Annexin I and its covalently linked dimer (36 and 66 kDa, respectively, by sodium dodecyl sulfate (SDS)-gel electrophoresis) reacted in one-dimensional immunoblots with monoclonal antibodies to annexin I and calpactin II, and with monoclonal and polyclonal antibodies to lipocortin I, confirming that annexin I, calpactin II, and lipocortin I are the same or closely related proteins. Milligram amounts of monomeric annexin I containing negligible amounts of the cross-linked dimeric annexin I were selectively isolated from placental membranes by using buffers containing the sulfhydryl reagent iodoacetic acid. Milligram amounts of cross-linked annexin I were selectively isolated when placental membranes were initially treated with buffers that did not contain iodoacetic acid and then extracted with Triton X-100, suggesting that sulfhydryl-dependent transglutaminase activity contributes to the selective isolation of this protein. A third phospholipase A2-inhibitory protein (35 kDa by SDS-gel electrophoresis) that reacted in immunoblots with monoclonal antibodies to calpactin I and annexin II, indicating their similar identity, was isolated. The procedure employed allows the rapid purification of annexins I and II in milligram amounts from placental membranes within 2 days.     

Molecular cloning and expression in Escherichia coli of the cDNA coding for rat lipocortin I (calpactin II)

Lipocortins (LC) are a family of proteins that were initially described to be induced by glucocorticosteroids and to inhibit phospholipase A2 (PLA2). Using oligodeoxynucleotide probes corresponding to partial amino acid (aa) sequences of rat lipocortin I (LCI), we have isolated a cDNA clone for rat LCI from a cDNA library prepared from poly(A)+RNA of peritoneal cells of dexamethasone-treated rat. The cDNA insert (1355 bp) had an open reading frame of 1038 bp that encoded a 346-aa polypeptide (Mr 38,784). The nucleotide sequence and the amino acid sequence deduced from it showed high homology with the reported sequences of human LCI. A plasmid containing the trc promoter and cDNA sequence for 346 aa residues of the rat LCI was constructed and expressed in Escherichia coli. Antibody to human LCI crossreacted with the recombinant rat LCI, and the recombinant protein had characteristics of natural rat LCI including PLA2 inhibitory activity in vitro.     

Characterization of the interaction between calpactin I and fodrin (non-erythroid spectrin)

Calpactin I, a calcium-binding protein associated with the membrane cytoskeleton, has been reported to bind to a calcium-dependent manner to fodrin, to certain phospholipids, and to F-actin. We have investigated the interaction between calpactin I and fodrin. Using a gel filtration assay, we observed one or more calpactin I molecules were bound calcium-dependently only at high concentrations of calpactin (greater than 1 microM), indicating that the interaction is of only moderate affinity. At higher concentrations of calpactin I, the calpactin coprecipitated with fodrin in a calcium-dependent manner. The molar ratio of calpactin to fodrin tetramer in the precipitate was greater than 25:1, indicating that the calpactin binds to a large number of sites. Moreover, the monomeric form of calpactin I (p36), which did not induce precipitation of fodrin, showed no evidence of saturation in its binding to fodrin even when more than 30 mol of p36 were bound per mole of fodrin tetramer. Several proteins other than fodrin, including clathrin, alpha-actinin, and neurofilament-H, also interacted calcium-dependently with calpactin I in the gel filtration assay. These results demonstrate that the interaction between calpactin and fodrin is not of high affinity, is not readily saturated, and is not specific for fodrin. Our results suggest that calpactin's interaction with fodrin is a particular example of a calcium-dependent, but promiscuous, binding of calpactin to proteins.