Nearest neighbor analysis for brain synapsin I. Evidence from in vitro reassociation assays for association with membrane protein(s) and the Mr = 68,000 neurofilament subunit

Synapsin I, a major neuron-specific substrate for cAMP-dependent and Ca2+/calmodulin-dependent protein kinases, associates in in vitro assays with brain integral membrane protein site(s) distinct from secretory vesicles and with the neurofilament Mr = 68,000 subunit. The membrane sites for synapsin involve protein(s) and are likely to have physiological relevance since the binding of 125I-labeled synapsin is abolished by digestion with chymotrypsin, is displaced by unlabeled synapsin, is of high affinity (KD = 10 nM), and has a capacity (42 pmol/mg membrane protein) that is comparable to the amount of synapsin in brain, optimal binding occurs at physiological pH (6.8-7.2) and salt concentrations (50 mM), and synapsin binding to membranes is inhibited by phosphorylation with Ca2+/calmodulin-dependent protein kinase. The brain membrane protein sites for synapsin are not due to synaptic vesicles, since synaptic vesicles do not sediment under the conditions of the binding assay. Association between synapsin and the Mr = 68,000 neurofilament subunit has also been demonstrated. The binding of synapsin with the neurofilament subunit is specific since this binding interaction is saturable, with a 1:1 stoichiometry, the binding involves only certain proteolytically derived domains of synapsin, and is therefore not a simple electrostatic interaction between the basic domains of synapsin and the acidic regions in the neurofilament subunit, and Ca2+/calmodulin-dependent phosphorylation of synapsin inhibits this interaction. Synapsin promotes cross-linking of synaptic vesicles to brain membranes, and these complexes are reduced by phosphorylation of synapsin. This interconnecting function of synapsin may be a general characteristic of synapsin binding, with a membrane (synaptic vesicle or nonsecretory vesicle)-bound synapsin associating with microtubules, neurofilaments, or spectrin.     

Ca2+/calmodulin-dependent microtubule-associated protein 2 kinase: broad substrate specificity and multifunctional potential in diverse tissues

In previous studies, we described a soluble Ca2+/calmodulin-dependent protein kinase which is the major Ca2+/calmodulin-dependent microtubule-associated protein 2 (MAP-2) kinase in rat brain [Schulman, H. (1984) J. Cell Biol. 99, 11-19; Kuret, J. A., & Schulman, H. (1984) Biochemistry 23, 5495-5504]. We now demonstrate that this protein kinase has broad substrate specificity. Consistent with a multifunctional role in cellular physiology, we show that in vitro the enzyme can phosphorylate numerous substrates of both neuronal and nonneuronal origin including vimentin, ribosomal protein S6, synapsin I, glycogen synthase, and myosin light chains. We have used MAP-2 to purify the enzyme from rat lung and show that the brain and lung kinases have nearly indistinguishable physical and biochemical properties. A Ca2+/calmodulin-dependent protein kinase was also detected in rat heart, rat spleen, and in the ring ganglia of the marine mollusk Aplysia californica. Partially purified MAP-2 kinase from each of these three sources displayed endogenous phosphorylation of a 54 000-dalton protein. Phosphopeptide analysis reveals a striking homology between this phosphoprotein and the 53 000-dalton autophosphorylated subunit of the major rat brain Ca2+/calmodulin-dependent protein kinase. The enzymes phosphorylated MAP-2, synapsin I, and vimentin at peptides that are identical with those phosphorylated by the rat brain kinase. This enzyme may be a multifunctional Ca2+/calmodulin-dependent protein kinase with a widespread distribution in nature which mediates some of the effects of Ca2+ on microtubules, intermediate filaments, and other cellular constituents in brain and other tissues.     

Active catalytic fragment of Ca2+/calmodulin-dependent protein kinase II. Purification, characterization, and structural analysis.

We report the purification and characterization of an active catalytic fragment of Ca2+/calmodulin-dependent protein kinase II, derived from autophosphorylation and subsequent limited chymotryptic digestion of the purified rat forebrain soluble kinase. The purified fragment was completely Ca2+/calmodulin-independent, existed as a monomer, and phosphorylated synapsin I at the same sites as does the native form of Ca2+/calmodulin-dependent protein kinase II. Kinetic studies with the purified fragment revealed a more than 10-fold increase in Vmax and a 50% decrease in Km for synthetic peptide substrates, compared with native Ca2+/calmodulin-dependent protein kinase II. No 32P-labeled autophosphorylated residues were detected in the purified active fragment, indicating that the autophosphorylation sites were not contained within this fragment. Comparative studies of this active fragment (30 kDa) and its inactive counterpart (32-kDa fragment) revealed certain structural details of both fragments. Calmodulin-overlay study, immunoblot analysis, and direct amino acid sequencing suggest that both fragments contain the entire NH2-terminal catalytic domain and were generated by distinct cleavage within the regulatory domain. The putative cleavage sites for both fragments are discussed.     

Time-resolved fluorescence study of the neuron-specific phosphoprotein synapsin I. Evidence for phosphorylation-dependent conformational changes

Synapsin I is a major nerve terminal-specific phosphoprotein. It consists of a hydrophobic head region containing one phosphorylation site for either cAMP-dependent protein kinase or Ca2+/calmodulin-dependent protein kinase I and of a basic and elongated tail region containing two phosphorylation sites for Ca2+/calmodulin-dependent protein kinase II. The steady-state emission spectrum of synapsin I was centered at 330 nm and was markedly red shifted upon denaturation, as expected for tryptophan residues segregated from the external aqueous environment in native conditions. Quenching studies showed a low accessibility of synapsin I tryptophans at low ionic strength which was further decreased by exposure to 200 mM NaCl but not significantly affected by phosphorylation. The intrinsic fluorescence of synapsin I was resolved into three major decay components with lifetimes of about 0.2, 3, and 7 ns. Upon phosphorylation of synapsin I on the tail sites, the spectra associated with the intermediate and long lifetimes were shifted to the red region, while the spectrum associated with the short lifetime was shifted to the blue region, in the absence of significant changes of the lifetimes. Phosphorylation of synapsin I on the head site was less effective. The anisotropy decay of synapsin I labeled with the long-living chromophore pyrene on Cys-223 was also analyzed. A shorter rotational correlation time was found for the tail phosphorylated form (corresponding to a Stokes radius of 41-42 A) than for the dephosphorylated or for the head phosphorylated form (corresponding to a Stokes radius of 60-63 A). The data suggest that phosphorylation of the tail sites induces changes in the conformation and hydrodynamic properties of synapsin I which may play a role in the regulation of the molecular interactions of synapsin I within the nerve terminal.     

Kinase activity associated with caldesmon is Ca2+/calmodulin-dependent kinase II.

The relationship of the kinase which co-purifies with caldesmon to Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) was investigated by studying the phosphorylation of bovine brain synapsin I, as well-characterized substrate of CaM-kinase II. Synapsin I is a very good substrate (Km = 90 nM) of the co-purifying kinase, which phosphorylates two sites in synapsin I, both of which are distinct from the single site phosphorylated by cyclic-AMP-dependent protein kinase. Phosphorylation of synapsin I is Ca2(+)- and calmodulin-dependent: half-maximal activation occurs at 0.13 microM-Ca2+ and maximal activity at 0.4 microM-Ca2+. Phosphorylation of the co-purifying kinase slightly enhances the rate, but does not alter the stoichiometry, of subsequent synapsin I phosphorylation; it does, however, circumvent the requirement for Ca2+ and calmodulin. The properties of this kinase therefore closely resemble those of CaM-kinase II, and we conclude that it is probably a smooth-muscle isoenzyme of CaM-kinase II.     

Autophosphorylation of smooth-muscle caldesmon.

Caldesmon, a major actin- and calmodulin-binding protein of smooth muscle, has been implicated in regulation of the contractile state of smooth muscle. The isolated protein can be phosphorylated by a co-purifying Ca2+/calmodulin-dependent protein kinase, and phosphorylation blocks inhibition of the actomyosin ATPase by caldesmon [Ngai & Walsh (1987) Biochem. J. 244, 417-425]. We have examined the phosphorylation of caldesmon in more detail. Several lines of evidence indicate that caldesmon itself is a kinase and the reaction is an intermolecular autophosphorylation: (1) caldesmon (141 kDa) and a 93 kDa proteolytic fragment of caldesmon can be separated by ion-exchange chromatography: both retain caldesmon kinase activity, which is Ca2+/calmodulin-dependent; (2) chymotryptic digestion of caldesmon generates a Ca2+/calmodulin-independent form of caldesmon kinase; (3) caldesmon purified to electrophoretic homogeneity retains caldesmon kinase activity, and elution of enzymic activity from a fast-performance-liquid-chromatography ion-exchange column correlates with caldesmon of Mr 141,000; (4) caldesmon is photoaffinity-labelled with 8-azido-[alpha-32P]ATP; labelling is inhibited by ATP, GTP and CTP, indicating a lack of nucleotide specificity; (5) caldesmon binds tightly to Affi-Gel Blue resin, which recognizes proteins having a dinucleotide fold. Autophosphorylation of caldesmon occurs predominantly on serine residues (83.3%), with some threonine (16.7%) and no tyrosine phosphorylation. Autophosphorylation is site-specific: 98% of the phosphate incorporated is recovered in a 26 kDa chymotryptic peptide. Complete tryptic/chymotryptic digestion of this phosphopeptide followed by h.p.l.c. indicates three major phosphorylation sites. Caldesmon exhibits a high degree of substrate specificity: apart from autophosphorylation, brain synapsin I is the only good substrate among many potential substrates examined. These observations indicate that caldesmon may regulate its own function (inhibition of the actomyosin ATPase) by Ca2+/calmodulin-dependent autophosphorylation. Furthermore, caldesmon may regulate other cellular processes, e.g. neurotransmitter release, through the Ca2+/calmodulin-dependent phosphorylation of other proteins such as synapsin I.     

Synapsin I from human brain. Phosphorylation by Ca2+, phospholipid-dependent protein kinase

Synapsin I has been isolated from human brain by a rapid and efficient purification technique, and its phosphorylation by human brain Ca2+, phospholipid-dependent protein kinase (protein kinase C) has been studied. The inhibitory effect of calmodulin on this process has been demonstrated. It is also found that non-esterified fatty acids and acidic phospholipids are inhibitory for synapsin I phosphorylation by Ca2+, calmodulin-dependent protein kinase II.     

Biochemical basis for the specificity of alloxan inactivation of calmodulin-dependent protein kinase II.

The specificity and biochemical basis of inactivation of calmodulin-dependent protein kinase II by alloxan was studied in dispersed rat brain cells and a partially purified kinase preparation from an insulin-secreting tumor-cell line, RINm5f. When mechanically dispersed rat brain cells were incubated with [32P]-phosphate to label endogenous ATP, depolarization with 44 mM KCl produced a significant (P = 0.03) increase in phosphorylation of endogenous synapsin (132 +/- 8% of basal). Pre-treatment of the brain cells with 1.5 mM alloxan reduced depolarization-sensitive synapsin phosphorylation (109 +/- 5%). Phosphopeptide mapping of depolarization-phosphorylated synapsin showed that alloxan pre-treatment reduced phosphorylation specifically at synapsin sites phosphorylated by calmodulin-dependent protein kinase II. The results demonstrate selective inactivation of calmodulin-dependent protein kinase II activity by alloxan in an intact cell system, which may be useful in the study of the Type II kinase in cells and tissues. Using a partially purified kinase preparation from RINm5f cells, alloxan (100 microM) inactivated 76 +/- 1% calmodulin-dependent protein kinase II activity in 5 min at 37 degrees C. Subsequent incubation with dithiothreitol restored most of the activity. 5,5'-Dithiobis (2-nitrobenzoic acid) (I50 = 2.5 microM) also inactivated the kinase. These results suggested that a sulfhydryl group was involved at the inactivation site. Iodoacetamide (1.0 mM) had no inhibitory effect; however, preincubation with iodoacetamide protected the kinase activity from subsequent inactivation by alloxan. Covalent binding of [14C]-alloxan to calmodulin-dependent protein kinase was demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synapsin I Regulates Glutamate Release from Rat Brain Synaptosomes

Introduction of the dephosphorylated from of synapsin I into rat brain synaptosomes using freeze-thaw (transient) permeabilization significantly decreased the K(+)-induced release of glutamate. In contrast, introduction of synapsin I that had been phosphorylated by Ca2+/calmodulin-dependent protein kinase II was without effect on glutamate release. Addition of dephosphosynapsin I after freeze-thaw treatment also had no effect. Thus, the action of synapsin I was dependent on the phosphorylation state of synapsin I and on its entry into the synaptosomes. Our results implicate synapsin I as an important component in the regulation of neurotransmitter release in the mammalian nervous system.     

Characterization of synapsin I binding to small synaptic vesicles

The binding of synapsin I, a synaptic vesicle-associated phosphoprotein, to small synaptic vesicles has been examined. For this study, synapsin I was purified under nondenaturing conditions from rat brain, using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and characterized. Small synaptic vesicles were purified from rat neocortex by controlled pore glass chromatography as the last purification step, and binding was characterized at an ionic strength equivalent to 40 mM NaCl. After removal of endogenous synapsin I, exogenous dephospho-synapsin I bound with high affinity (Kd, 10 +/- 6 nM) to synaptic vesicles. The binding saturated at 76 +/- 40 micrograms synapsin I/mg of vesicle protein, which corresponded to the amount found endogenously in purified vesicles. Synapsin I binding exhibited a broad pH optimum around pH 7. Other basic proteins, specifically myelin basic protein and histone H2b, did not compete with synapsin I for binding to vesicles. Other membranes purified from rat brain and membranes derived from human erythrocytes did not show the high affinity binding site for synapsin I found in vesicles. The binding of three different forms of phosphosynapsin I to vesicles was investigated. Synapsin I, phosphorylated at sites 2 and 3 by purified calcium/calmodulin-dependent protein kinase II, bound with a 5-fold lower affinity to the vesicles than did dephospho-synapsin I. In contrast, synapsin I, phosphorylated at site 1 by purified catalytic subunit of cAMP-dependent protein kinase, bound with an affinity close to that of dephospho-synapsin I. Synapsin I phosphorylated on all three sites bound to the vesicles with an affinity comparable to that of synapsin I phosphorylated on sites 2 and 3. Under conditions of higher ionic strength (150 mM NaCl equivalent), synapsin I bound with a 5-fold lower affinity to vesicles, and no effect of phosphorylation on binding was observed under these conditions.     

Sequence analysis of phospholamban. Identification of phosphorylation sites and two major structural domains

Phospholamban is a regulatory protein in cardiac sarcoplasmic reticulum that is phosphorylated by cAMP- and Ca2+/calmodulin-dependent protein kinase activities. In this report, we present the partial amino acid sequence of canine cardiac phospholamban and the identification of the sites phosphorylated by these two protein kinases. Gas-phase protein sequencing was used to identify 20 NH2-terminal residues. Overlap peptides produced by trypsin or papain digestion extended the sequence 16 residues to give the following primary structure: Ser-Ala-Ile-Arg-Arg-Ala-Ser-Thr-Ile-Glu-Met-Pro-Gln-Gln-Ala- Arg-Gln-Asn-Leu-Gln-Asn-Leu-Phe-Ile-Asn-Phe-(Cys)-Leu-Ile-Leu-Ile-(Cys)- Leu-Leu-Leu-Ile-. Phospholamban phosphorylated by either cAMP-dependent or Ca2+/calmodulin-dependent protein kinase was cleaved with trypsin, and the major phosphorylated peptide (comprising greater than 70% of the incorporated 32P label) was purified by reverse-phase high performance liquid chromatography. The identical sequence was revealed for the radioactive peptide obtained from phospholamban phosphorylated by either kinase: Arg-Ala-Ser-Thr-Ile-Glu-Met-Pro-Gln-Gln-. The adjacent residues Ser7 and Thr8 of phospholamban were identified as the unique sites phosphorylated by cAMP- and Ca2+/calmodulin-dependent protein kinases, respectively. These results establish that phospholamban is an oligomer of small, identical polypeptide chains. A hydrophilic, cytoplasmically oriented NH2-terminal domain on each monomer contains the unique, adjacent residues phosphorylated by cAMP- and Ca2+/calmodulin-dependent protein kinase activities. Analysis by hydropathic profiling and secondary structure prediction suggests that phospholamban monomers also contain a hydrophobic domain, which could form amphipathic helices sufficiently long to traverse the sarcoplasmic reticulum membrane. A model of phospholamban as a pentamer is presented in which the amphipathic alpha-helix of each monomer is a subunit of the pentameric membrane-anchored domain, which is comprised of an exterior hydrophobic surface and an interior hydrophilic region containing polar side chains.     

Mapping of calmodulin-binding domain of Ca2+/calmodulin-dependent protein kinase II from rat brain

Recent molecular cloning experiments have identified a 25 amino-acid region as the calmodulin-binding domain of the alpha-subunit of rat brain Ca2+/calmodulin-dependent multifunctional protein kinase II (CaM-K II). Synthetic peptides, derived from the deduced amino-acid sequence encompassing this region, were examined for their ability to bind calmodulin in a calcium dependent manner and to inhibit the Ca2+/calmodulin-dependent autophosphorylation of CaM-K II. Comparison of these structure-function relationships highlighted a region of 5 amino-acids, which was essential for calmodulin interaction and inhibition of kinase activity. This region demonstrated some homology with other calmodulin-binding peptides, and may represent a key site of interaction of the kinase with calmodulin. These analyses provide additional insight into the molecular mechanism underlying the Ca2+ regulation of CaM-K II.     

Molecular cloning and sequencing of cDNA encoding the phosphatidylinositol kinase from rat brain.

A complementary DNA (cDNA) clone that encodes phosphatidylinositol 4-kinase (PI 4-kinase) was isolated from a rat brain cDNA library. The deduced amino acid sequence of 697 residues revealed that the protein contains two putative transmembrane sequences and that the N-terminal part of the protein has several sequences representing potential phosphorylation sites for cAMP- and calmodulin-dependent kinase. The C-terminal region is probably a phosphotransferase domain homologous to the kinase region of protein kinase family proteins. Specific antibody against the protein expressed in Escherichia coli successfully immunoprecipitated rat brain PI 4-kinase. The messenger RNA for PI 4-kinase was found predominantly in brain and rat neural cell lines. This PI kinase may play a specific role in neural signal transduction.     

Molecular cloning sequence and distribution of rat calspermin, a high affinity calmodulin-binding protein

Calspermin is a heat-stable, acidic calmodulin-binding protein predominantly found in mammalian testis. The cDNA representing the rat form of this protein has been cloned from a rat testis lambda gt11 library. Sequence analysis of two overlapping clones revealed a 232-nucleotide 5'-nontranslated region, 510 nucleotides of open reading frame, a 148-nucleotide 3'-untranslated region, and a poly(A) tail. Authenticity of the clones was confirmed by comparison of a portion of the deduced amino acid sequence with the sequence of a tryptic peptide obtained from the rat testis protein. The lambda gt11 fusion protein was recognized by affinity purified antibodies to pig testis calspermin and bound 125I-calmodulin in a Ca2+-dependent manner. Calspermin cDNA encodes a 169-residue protein with a calculated Mr of 18,735. The putative calmodulin-binding domain is very close to the amino terminus of the protein. This region shows 46% identity with the calmodulin-binding region of rat brain Ca2+/calmodulin-dependent protein kinase II and 32% identity with the equivalent region of chicken smooth muscle myosin light chain kinase. The 5'-nontranslated region reveals significant homology with a portion of the catalytic region of the calmodulin-dependent protein kinase family. Calspermin contains a stretch of 17 contiguous glutamic acid residues in the central region of the molecule. Computer analysis predicts calspermin to be 81% alpha-helix and 14% random coil. Analysis of genomic DNA indicates calspermin to be the product of a unique gene. Northern blot analysis of rat testis RNA reveals a 1.1-kilobase mRNA. This RNA is restricted to testis among several rat tissues examined and could not be identified in total RNA isolated from testes of other mammals. Analysis of cells isolated from rat testis reveals calspermin mRNA to be predominantly expressed in postmeiotic cells indicating that it may be specific to haploid cells.     

Effect of Digestion with Phospholipase A2 on Endogenous Protein Phosphorylation in Particulate Fractions from Rat Brain Synaptosomes

The endogenous phosphorylation of synapsin 1 in cyclic AMP-containing media was greatly decreased by digestion of synaptic vesicles and synaptosomal membranes with phospholipase A2, suggesting that the system is functionally dependent on the membrane structure. Treatment of the synaptic vesicle fraction with phospholipase A2 also caused a small but significant inhibition of the Ca2+/calmodulin-dependent phosphorylation of the same protein. The Ca2+/calmodulin-dependent phosphorylation of other major acceptors, and the basal phosphorylation of a 52-kD acceptor enriched in the vesicle fraction, remained unchanged after cleavage of the membrane phospholipids with phospholipase A2. The significance of the selective effect of phospholipase A2 treatment on endogenous membrane phosphorylation is discussed.     

Glycosylation Sites Flank Phosphorylation Sites on Synapsin I

Synapsin I is concentrated in nerve terminals, where it appears to anchor synaptic vesicles to the cytoskeleton and thereby ensures a steady supply of fusion-competent synaptic vesicles. Although phosphorylation-dependent binding of synapsin I to cytoskeletal elements and synaptic vesicles is well characterized, little is known about synapsin I's O-linked N-acetylglucosamine (O-GlcNAc) modifications. Here, we identified seven in vivo O-GlcNAcylation sites on synapsin I by analysis of HPLC-purified digests of rat brain synapsin I. The seven O-GlcNAcylation sites (Ser55, Thr56, Thr87, Ser516, Thr524, Thr562, and Ser576) in synapsin I are clustered around its five phosphorylation sites in domains B and D. The proximity of phosphorylation sites to O-GlcNAcylation sites in the regulatory domains of synapsin I suggests that O-GlcNAcylation may modulate phosphorylation and indirectly affect synapsin I interactions. With use of synthetic peptides, however, the presence of an O-GlcNAc at sites Thr562 and Ser576 resulted in only a 66% increase in the Km of calcium/calmodulin-dependent protein kinase II phosphorylation of site Ser566 with no effect on its Vmax. We conclude that O-GlcNAcylation likely plays a more direct role in synapsin I interactions than simply modulating the protein's phosphorylation.     

Phosphorylation and modulation of the enzymic activity of native and protease-cleaved purified hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase by a calcium/calmodulin-dependent protein kinase

A Ca2+/calmodulin-dependent kinase has been purified which catalyzed the phosphorylation and concomitant inactivation of both the microsomal native (100,000 Da) and protease-cleaved purified 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) (53,000 Da) fragments. This low molecular weight brain cytosolic Ca2+/calmodulin-dependent kinase phosphorylates histone H1, synapsin I, and purified HMG-CoA reductase as major substrates. The kinase, purified by sequential chromatography on DEAE-cellulose, calmodulin affinity resin, and high performance liquid chromatography (TSKG 3000 SW) is an electrophoretically homogeneous protein of approximately 110,000 Da. The molecular weight of the holoenzyme, substrate specificity, subunit protein composition, subunit autophosphorylation, subunit isoelectric points, and subunit phosphopeptide analysis suggest that this kinase of Mr 110,000 may be different from other previously reported Ca2+/calmodulin-dependent kinases. Maximal phosphorylation by the low molecular form of Ca2+/calmodulin-dependent kinase of purified HMG-CoA reductase revealed a stoichiometry of approximately 0.5 mol of phosphate/mol of 53,000-Da enzyme. Dephosphorylation of phosphorylated and inactivated native and purified HMG-CoA reductase revealed a time-dependent loss of 32P-bound radioactivity and reactivation of enzyme activity. Based on the results reported here, we propose that HMG-CoA reductase activity may be modulated by yet another kinase system involving covalent phosphorylation. The elucidation of a Ca2+/calmodulin-dependent HMG-CoA reductase kinase-mediated modulation of HMG-CoA reductase activity involving reversible phosphorylation may provide new insights into the molecular mechanisms involved in the regulation of cholesterol biosynthesis.     

Ca2+/calmodulin-dependent protein phosphorylation associated with the cytoskeleton of quiescent rat fibroblast (3Y1) cells.

Endogenous phosphorylation of the crude membrane fraction of cultured 3Y1 fibroblast cells was enhanced by the addition of Ca2+/calmodulin. Both Ca2+/calmodulin-dependent protein kinase activity and its substrate were present in a cytoskeletal fraction, obtained as a pellet after washing of the membrane fraction with 2 mM EGTA, 0.6 M NaCl, and 1% Triton X-100. The phosphorylatable protein in the Triton X-insoluble fraction was identified by immunoblotting as vimentin. This endogenous phosphorylation induced by calmodulin was inhibited by the addition of KN-62, a specific Ca2+/calmodulin-dependent protein kinase II inhibitor, in a dose-dependent manner. However, phosphorylation of the 59 kDa protein (vimentin) in this fraction was not stimulated by adding both phosphatidyl serine and cAMP, thereby suggesting the absence of protein kinase C or of cAMP-dependent protein kinase in this fraction. The protein kinase associated with the Triton X-insoluble fraction phosphorylated the Ca2+/calmodulin-dependent protein kinase II-specific site of synapsin I from the bovine cortex. Two-dimensional phosphopeptide maps of vimentin indicated that a major phosphopeptide phosphorylated by the endogenous calmodulin-dependent kinase also appears to be the same as a major phosphopeptide phosphorylated by the exogenous Ca2+/calmodulin-dependent protein kinase II. Our results suggest that cytoskeleton-associated Ca2+/calmodulin-dependent protein kinase II regulates dynamic cellular functions through the phosphorylation of cytoskeletal elements in non-neural cells.     

Mammalian brain phosphoproteins as substrates for calcineurin

Calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase found in several tissues, is highly concentrated in mammalian brain. In an attempt to identify endogenous brain substrates for calcineurin, kinetic analyses of the dephosphorylation of several well-characterized phosphoproteins purified from brain were performed. The proteins studied were: G-substrate, a substrate for cyclic GMP-dependent protein kinase; DARPP-32, a substrate for cyclic AMP-dependent protein kinase; Protein K.-F., a substrate for a cyclic nucleotide- and Ca2+-independent protein kinase; and synapsin I, a substrate for cyclic AMP-dependent (site I) and a Ca2+/calmodulin-dependent protein kinase (site II). Calcineurin dephosphorylated each of these proteins in a Ca2+/calmodulin-dependent manner. Similar Km values were obtained for each substrate: G-substrate, 3.8 microM; DARPP-32, 1.6 microM; Protein K.-F., approximately 3 microM (S0.5); synapsin I (site I), 7.0 microM; synapsin I (site II), 4.4 microM. However, significant differences were obtained for the maximal rates of dephosphorylation. The kcat values were: G-substrate, 0.41 s-1; DARPP-32, 0.20 s-1; Protein K.-F., 0.7 s-1; synapsin I (site I), 0.053 s-1; synapsin I (site II), 0.040 s-1. Comparisons of the catalytic efficiency (kcat/Km) for each substrate indicated that DARPP-32, G-substrate, and Protein K.-F. are all potential substrates for calcineurin in vivo.