Louise-Anne Pradel

In Vitro Phosphorylation of Annexin 2 Heterotetramer by Protein Kinase C COMPARATIVE PROPERTIES OF THE UNPHOSPHORYLATED AND PHOSPHORYLATED ANNEXIN 2 ON THE AGGREGATION AND FUSION OF CHROMAFFIN GRANULE MEMBRANES

Heterotetrameric annexin 2 phosphorylated “in vitro” by rat brain protein kinase C is purified and obtained devoid of unphosphorylated protein; it contains 2 mol of phosphate/mol of heterotetramer. The aggregative and binding properties of the phosphorylated annexin 2 toward purified chromaffin granules are compared with those of the unphosphorylated annexin 2. Annexin 2 binds to chromaffin granules with high affinity. Phosphorylation of annexin 2 decreases the affinity of this binding without affecting the maximum binding capacity. The binding curves are strongly cooperative. It is suggested that a surface oligomerization of the proteins may take place upon binding. Besides, phosphorylation of annexin 2 is followed by a dissociation of the light chains from the heavy chains in the heterotetramer. Whereas annexin 2 induces the aggregation of chromaffin granules at μM calcium concentration, the phosphorylated annexin 2 does not induce aggregation at any concentration of calcium either at pH 6 or 7. The phosphorylation of annexin 2 by protein kinase C, MgATP, and 12-O-tetradecanoylphorbol-13-acetate on chromaffin granules induces a fusion of chromaffin granules membranes observed in electron microscopy. The fusion requires the activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate. Given these results and since annexin 2 is phosphorylated by protein kinase C under stimulation of chromaffin cells, it is suggested that phosphorylated annexin 2 may be implicated in the fusion step during exocytosis of chromaffin granules.

Interaction between microtubule-associated protein tau and spectrin

Tau factor, one of the microtubule-associated proteins (MAPs), is shown here to bind to spectrin. Evidence for an interaction between these two proteins is provided by spectrin affinity chromatography of brain MAPs, gel overlay of electrophoresed MAPs with 125I-labelled spectrin, incorporation of tau factor in human erythrocyte ghosts, and demonstration that tau inhibits the F-actin cross-linking activity of tetrameric spectrin. The wide distribution of both tau and spectrin-like proteins in eukaryotic cells is in favor of the possible biological significance of this interaction. The results suggest that tau could be one of the proteins involved in the concerted regulation of microtubule and actin networks in the membrane vicinity.

Translocation of cytosolic annexin 2 to a Triton‐insoluble membrane subdomain upon nicotine stimulation of chromaffin cultured cells

To gain a better understanding of the function of annexin 2, we have investigated the subcellular distribution of the monomeric and heterotetrameric forms of annexin 2 and their relationship to the cytoskeleton upon stimulation of chromaffin cells. Quantitative immunoblotting has revealed that in resting cells a large amount of annexin 2 is monomeric and cytosolic. Upon nicotine stimulation 80% of total annexin 2 becomes associated with a Triton‐X100‐insoluble fraction where the monomeric and the heterotetrameric forms are found. The translocation of monomeric annexin 2 is Ca2+‐dependent and complete at 1 μM free Ca2+. We have shown that about 66% of the annexin 2 associated with the Triton‐X100‐insoluble fraction is soluble in octylglucoside while the remnants are insoluble in the detergent and remain likely associated with actin filaments and associated cytoskeleton proteins. The octylglucoside‐soluble fraction contains integral proteins from the plasma membrane and from granule membrane, but does not contain caveolin. Moreover, upon nicotine stimulation, a redistribution of proteins was detected in this fraction. These dynamic processes appear concomitantly with the phosphorylation of annexin 2 in this compartment and with catecholamine release. It is suggested that the soluble octylglucoside fraction may represent a special lipidic membrane compartment where the NSF attachment proteins and the cytosolic proteins like annexin 2 and rab3a may become concentrated upon stimulation of the cell. The presence of annexin 2 is consistent with its proposed function on granule and target membrane proteins required for the close apposition of two distinct membranes and supports its functional role in the regulated exocytosis/endocytosis process.

Biochemical characterization of annexins I and II isolated from pig nervous tissue.

Abstract: Five proteins having molecular masses of 90, 67, 37, 36, and 32 kDa (p90, p67, p37, p36, and p32, respectively) were identified in the participate fractions of pig brain cortex and pig spinal cord prepared in the presence of 0.2 mM Ca2+ and further purified using a protocol previously described for the purification of calpactins. Proteins p90, p37, and p36 are related to annexins I and II. Annexin II, represented by p90, is found as an heterotetramer, composed of two heavy chains of 36 kDa and two light chains of 11 kDa, and as a monomer of 36 kDa. Protein p37, which differs immunologically from p36, is a monomer and could be related to annexin I. All three proteins are Ca2+‐dependent phospholipid‐ and F‐actin‐binding proteins; they are phosphorylated on a serine and on a tyrosine residue by protein kinases associated with synaptic plasma membranes. Purified p36 monomer and p36 heterotetramer proteins bind to actin at millimolar Ca2+ concentrations. The stoichiometry of p36 binding to F‐actin at saturation is 1:2, corresponding to one tetramer or monomer of calpactin for two actin monomers (KD, 3 × 10−6M). Synaptic plasma membranes supplemented with the monomeric or tetrameric forms of p36 phosphorylate the proteins on a serine residue. The monomer is phosphorylated on a serine residue by a Ca2+‐independent protein kinase, whereas the heterotetramer is phosphorylated on a serine residue and a tyrosine residue by Ca2+‐dependent protein kinases. Antibodies to brain p37 and p36 together with antibodies to lymphocytes lipocortins 1 and 2 were used to follow the distribution of these proteins in nervous tissues. Polypeptides of 37, 34, and 36 kDa cross‐react with these antibodies. Anti‐p37 and antilipocortin 1 cross‐react on the same 37‐ and 34‐kDa polypeptides; anti‐p36 and antilipocortin 2 cross‐react only on the 36‐kDa polypeptides.

Biochemical evidence for a low molecular weight protein (profilin-like protein) in hog thyroid gland and its involvement in actin polymerisation

Actin was characterized ubiquitously in muscle fibers and in non-muscle cells from the most primitive to the most advanced organisms. However, in nonmuscle cells, this protein is found together in microtilamentous and monomeric states (Fand G-actin, respectively). It has been suggested that cell constituents in non-muscle cells interact with G-actin to maintain it in non-polymerized form. In this context, it has been shown that a low molecular weight protein named profilin prevents the actin polymerisation [ 11. In thyroid cells actin microfilaments appear to be involved in the endocytotic process [2]. The organization of these microfilaments depends on the physiological state of the gland. The bundles of actin filaments in the resting gland are transformed into a net-. work in response to thyrotropin stimulation; concomitantly, an increase of the ratio G/F actin is observed [ 31. Here, we have initiated a search for effecters which may have an influence on the state of actin polymerisation in hog thyroid cells.

Phosphorylation by Protein Kinase C of Annexin 2 in Chromaffin Cells Stimulated by Nicotine

Abstract: Annexin 2 phosphorylated in vitro by protein kinase C has been shown to restore partially catecholamine secretion in streptolysin O‐permeabilized chromaffin cells depleted of their protein kinase C activity. This result suggested a phosphorylation of annexin 2 in stimulated cells. Nicotine stimulation induced an increase of 32P incorporation in annexin 2 heavy chain concomitant with catecholamine release. This incorporation results from phosphorylation by protein kinase C because (a) serine was the only phosphorylated residue, (b) 32P incorporation was inhibited by the protein kinase inhibitors H7, GF 109203X, and staurosporine, and (c) activators of this enzyme, 12‐O‐tetradecanoylphorbol 13‐acetate and 1,2‐dioctanoylglycerate, increased the incorporation of radioactivity. The phosphorylated heavy chain had an electrophoretic mobility lower than that of the unmodified one, thus allowing determination of the fraction of phosphorylated protein. In the resting state, a significant fraction of annexin 2 heavy chain was phosphorylated, and nicotine stimulation resulted in an activation of both phosphorylation and dephosphorylation. Phosphorylation was largely increased in the presence of okadaic acid, indicating the involvement of type 1 and 2A phosphatases.

Annexin 1 is present in different molecular forms in rat cerebral cortex

Previously we have purified annexin 1 [J. Neurochem. 56 (1991) 1985‐1986] from pig cerebral cortex as a monomeric protein of 37 kDa. Here, the localization of annexin 1 was investigated in subcellular fractionations of rat cerebral cortex using immunodetection by a specific antibody. In contrast to synaptophysin, a specific synaptic vesicle integral membrane protein, annexin 1 is located in the synaptic plasma membrane fraction where it appears on SDS‐PAGE as a polypeptide of 74 kDa. Annexin 1 is extracted also as a 74 kDa polypeptide from the purified synaptic plasma membranes. These results suggest for the 74 kDa molecular form an enzymatic dimerization of annexin 1 when associated to the membrane.

Spatial proximity of a tyrosyl and a lysyl residue in the active site region of yeast 3-phosphoglycerate kinase.

The effect of 7-chloro-4-nitrobenzofurazan on yeast 3-phosphoglycerate kinase causes a modification of one tyrosyl residue concomitantly with a total loss of activity of the enzyme. The modification is not accompanied by any significant conformational change. A total protection against inactivation is observed with the substrates : furthermore, AMP, tripolyphosphate and pyrophosphate afford an effective protection. At pH 9, a shift in the absorbance spectrum of the tyrosine O-nitrobenzofurazan derivative of 3-phosphoglycerate kinase is observed. It can be related to the transfer of the reagent from tyrosine to lysine. The N-nitrobenzofurazan derivative is also completely inactive. It is concluded that a lysine residue is located close to the essential tyrosyl residue.