Ca2+ and Calmodulin-Dependent Phosphorylation of Endogenous Synaptic Vesicle Tubulin by a Vesicle-Bound Calmodulin Kinase System

Endogenous synaptic vesicle alpha- and beta-tubulin were shown to be the major substrates for a Ca2+-calmodulin-regulated protein kinase system in enriched synaptic vesicle preparations from rat cortex as determined by two-dimensional gel electrophoresis and peptide mapping. The activation of this endogenous tubulin kinase system was dependent on Ca2+ and the Ca2+ binding protein, calmodulin. Under maximally stimulated conditions, approximately 40% of the tubulin present in enriched synaptic vesicles was phosphorylated within less than 50 s by the vesicle Ca2+-calmodulin kinase. Evidence is presented indicating that the Ca2+-calmodulin tubulin kinase is an enzyme system distinct from previously described cyclic AMP protein kinases. alpha-Tubulin and beta-tubulin were identified as major components of previously designated vesicle phosphorylation bands DPH-L and DPH-M. The Ca2+-calmodulin tubulin kinase is very labile and specialized isolation procedures were necessary to retain activity. Ca2+-activated synaptic vesicle tubulin phosphorylation correlated with vesicle neurotransmitter release. Depolarization-dependent Ca2+ uptake in intact synaptosomes simultaneously stimulated the release of neurotransmitters and the phosphorylation of synaptic vesicle alpha- and beta-tubulin. The results indicate that regulation of the synaptic vesicle tubulin kinase by Ca2+ and calmodulin may play a role in the functional utilization of synaptic vesicle tubulin and may mediate some of the effects of Ca2+ on vesicle function and neurosecretion.     

Identification of Endogenous Calmodulin-Dependent Kinase and Calmodulin-Binding Proteins in Cold-Stable Microtubule Preparations from Rat Brain

Calmodulin-dependent kinase activity was investigated in cold-stable microtubule fractions. Calmodulin-dependent kinase activity was enriched approximately 20-fold over cytosol in cold-stable microtubule preparations. Calmodulin-dependent kinase activity in cold-stable microtubule preparations phosphorylated microtubule-associated protein-2, alpha- and beta-tubulin, an 80,000-dalton doublet, and several minor phosphoproteins. The endogenous calmodulin-dependent kinase in cold-stable microtubule fractions was identical to a previously purified calmodulin-dependent kinase from rat brain by several criteria including (1) subunit molecular weights, (2) subunit isoelectric points, (3) calmodulin-binding properties, (4) subunit autophosphorylation, (5) calmodulin-binding subunit composition on high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (6) isolation of kinase on calmodulin affinity resin, (7) kinetic parameters, (8) phosphoamino acid phosphorylation sites on beta-tubulin, and (9) phosphopeptide mapping. Endogenous cold-stable calmodulin-dependent kinase activity was isolated from the microtubule fraction by calmodulin affinity resin column chromatography and specifically eluted with EGTA. This kinase fraction contained the calmodulin-binding, autophosphorylating rho and sigma subunits of the previously purified kinase. The rho and sigma subunits of this kinase represented the major calmodulin-binding proteins in the cold-stable microtubule fractions as assessed by denaturing and non-denaturing procedures. These results indicate that calmodulin-dependent kinase is a major calmodulin-binding enzyme system in cold-stable microtubule fractions and may play an important role in mediating some of the effects of calcium on microtubule and cytoskeletal dynamics.     

Identification of the Major Postsynaptic Density Protein as Homologous with the Major Calmodulin-Binding Subunit of a Calmodulin-Dependent Protein Kinase

The major postsynaptic density protein (mPSDp), comprising greater than 50% of postsynaptic density (PSD) protein, is an endogenous substrate for calmodulin-dependent phosphorylation as well as a calmodulin-binding protein in PSD preparations. The results in this investigation indicate that mPSDp is highly homologous with the major calmodulin-binding subunit (p) of tubulin-associated calmodulin-dependent kinase (TACK), and that PSD fractions also contain a protein homologous with the sigma-subunit of TACK. Homologies between mPSDp and a 63,000 dalton PSD protein and the rho- and sigma-subunits of TACK were established by the following criteria: (1) identical apparent molecular weights; (2) identical calmodulin-binding properties; (3) manifestation of Ca2+-calmodulin-stimulated autophosphorylation; (4) identical isoelectric points; (5) identical calmodulin binding and autophosphorylation patterns on two-dimensional gels; (6) homologous two-dimensional tryptic peptide maps; and (7) similar phosphoamino acid-specific phosphorylation of tubulin. The results suggest that mPSDp is a calmodulin-binding protein involved in modulating protein kinase activity in the postsynaptic density and that a tubulin kinase system homologous with TACK exists in a membrane-bound form in the PSD.     

Phosphorylation of the GABAa/Benzodiazepine Receptor α Subunit by a Receptor-Associated Protein Kinase

Abstract: Partially purified preparations of GABA_a/benzodiazepine receptor from rat brain were found to contain high levels of a protein kinase activity that phosphorylated a small number of proteins in the receptor preparations, including a 50-kilodalton (kD) phosphoprotein that comigrated on two-dimensional electrophoresis with purified, immunolabeled, and photolabeled receptor α subunit. Further evidence that the comigrating 50-kD phosphoprotein was, in fact, the receptor α subunit was obtained by peptide mapping analysis: the 50-kD phosphoprotein yielded one-dimensional peptide maps identical to those obtained from iodinated, purified α subunit. Phosphoamino acid analysis revealed that the receptor α subunit is phosphorylated on serine residues by the protein kinase activity present in receptor preparations. Preliminary characterization of the receptor-associated protein kinase activity suggested that it may be a second messenger-independent protein kinase. Protein kinase activity was unaltered by cyclic AMP, cyclic GMP, calcium plus calmodulin, calcium plus phosphatidylserine, and various inhibitors of these protein kinases. Examination of the substrate specificity of the receptor-associated protein kinase indicated that the enzyme preferred basic proteins as substrates. Endogenous phosphorylation experiments indicated that the receptor α subunit may also be phosphorylated in crude membranes by a protein kinase activity present in those membranes. As with phosphorylation of the receptor in purified preparations, its phosphorylation in crude membranes also appeared to be unaffected by activators and inhibitors of second messenger-dependent protein kinases. These findings raise the possibility that the phosphorylation of the α subunit of the GABA_a/ benzodiazepine receptor by a receptor-associated protein kinase plays a role in modulating the physiological activity of the receptor in vivo.     

Phosphorylation of Tubulin by Casein Kinase II Regulates Its Binding to a Neuronal Protein (NP185) Associated with Brain Coated Vesicles

We recently described a new protein associated exclusively with neuronal clathrin-coated vesicles (CCVs), and characterized two monoclonal antibodies that react with it (S-8G8 and S-6G7). In this report, the association of neuronal protein of 185 kilodaltons (NP185) with CCV kinases and its interaction with tubulin are described. The affinity of NP185 for tubulin is significantly enhanced when tubulin is phosphorylated by CCV-associated casein kinase II. In contrast, phosphorylation of tubulin by a kinase activity associated with purified brain tubulin decreases its affinity for NP185. Together, these data suggest that the interaction of NP185 with tubulin is modulated by protein phosphorylation. Recent evidence has suggested that tubulin is phosphorylated by casein kinase II during neurite development. The enhanced affinity of NP185 for tubulin phosphorylated by casein kinase II could be important for proper intracellular sorting of this protein in the developing neuron.     

Ca2+, calmodulin-dependent regulation of microtubule formation via phosphorylation of microtubule-associated protein 2, tau factor, and tubulin, and comparison with the cyclic AMP-dependent phosphorylation

Abstract: Isolated microtubule-associated protein 2 (MAP2), τ factor, and tubulin were phosphorylated by a purified Ca²⁺, calmodulin-dependent protein kinase (640K enzyme) from rat brain. The phosphorylation of MAP2 and τ factor separately induced the inhibition of microtubule assembly, in accordance with the degree. Tubulin phosphorylation by the 640K enzyme induced the inhibition of microtubule assembly, whereas the effect of tubulin phosphorylation by the catalytic subunit was undetectable. The effects of tubulin and MAPs phosphorylation on microtubule assembly were greater than that of either tubulin or MAPs phosphorylation. Because MAP2, τ factor, and tubulin were also phosphorylated by the catalytic subunit of type-II cyclic AMP-dependent protein kinase from rat brain, the kinetic properties and phosphorylation sites were compared. The amount of phosphate incorporated into each microtubule protein was three to five times higher by the 640K enzyme than by the catalytic subunit. The K _m values of the 640K enzyme for microtubule proteins were four to 24 times lower than those of the catalytic subunit. The peptide mapping analysis showed that the 640K enzyme and the catalytic subunit incorporated phosphate into different sites on MAP2, τ factor, and tubulin. Investigation of phosphoamino acids revealed that only the seryl residue was phosphorylated by the catalytic subunit, whereas both seryl and threonyl residues were phosphorylated by the 640K enzyme. These data suggest that the Ca²⁺, calmodulin system via phosphorylation of MAP2, τ factor, and tubulin by the 640K enzyme is more effective than the cyclic AMP system on the regulation of microtubule assembly.     

A Casein Kinase-Like Kinase Phosphorylates β-Tubulin and May Be a Microtubule-Associated Protein

The hypothesis that casein kinase II (CKII) is a microtubule-associated protein kinase was investigated using a neuronal cell line and bovine brain. Heparin, an inhibitor of CKII, inhibited the phosphorylation of a PC12 cytosolic protein whose molecular weight was similar to that of beta-tubulin. Partially purified PC12 CKII was immunoreactive to an antibody directed against bovine CKII and was able to phosphorylate purified beta-tubulin in a heparin-inhibitable manner when the concentration of tubulin was less than 50 micrograms/ml. To better determine if CKII is a microtubule-associated protein kinase, bovine brain tubulin was chromatographed on FPLC Mono Q and phosphocellulose columns. Several tubulin casein kinase (TCK) activities were apparent. All TCK activities phosphorylated tubulin and casein, but none was able to phosphorylate the CKII-specific synthetic peptide RRREEETEEE. One of these TCK fractions was immunoreactive to the antibody directed against CKII, and this antibody labeled a 50-kDa molecular mass band that had a molecular mass distinctly different from those of the subunits of CKII. Thus, we suggest that a CKII-like protein, but not CKII, might be a microtubule-associated protein.     

Identification and Characterization of Protein Kinase FA/ Glycogen Synthase Kinase 3 in Clathrin-Coated Brain Vesicles

Abstract: Mg-ATP-dependent protein phosphatase activating factor [kinase F_A/glycogen synthase kinase 3 (GSK-3)] has been identified in highly purified clathrin-coated vesicles (CCVs) isolated from pig brain. Kinase F_A was found to exist in an inactive state but can be activated by 1% Triton X-100 or [ M /Tris-HC] extraction in brain CCVs. Activation of kinase F_A in CCVs is due to disassociation of the kinase from CCVs as demonstrated on sucrose density-gradient ultracentrifugation and Sepharose CL-4B gel filtration. Using purified brain CCVs as substrates, kinase F_A enhanced the endogenous phosphorylation of assembly protein complexes in the molecular weight range of 100,000-130,000 severalfold, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. Comparisons with well-defined brain CCV-associated endogenous protein kinases such as pp50 kinase/AP50 and casein kinase 2 provide evidence that kinase F_A/GSK-3 represents a third potent and unique CCV-associated protein kinase distinctly different from the previously described CCV protein kinases, suggesting the possible involvement of kinase FA in the regulation of CCV functions in the brain. The results also support the notion that protein kinase F_A is involved in cell surface signal transduction in the CNS.     

Characterization of Ca2+/Calmodulin-Dependent Protein Kinase Associated with Rat Cerebral Synaptic Junction: Substrate Specificity and Effect of Autophosphorylation

The Ca2+/calmodulin (CaM)-dependent protein kinase associated with rat cerebral synaptic junction (SJ) was characterized, using the SJ fraction as the enzyme preparation, to clarify the functional significance of the enzyme in situ. The protein kinase was greatly activated in the presence of micromolar concentrations of both Ca2+ and calmodulin (EC50 for Ca2+, 1.0 microM; that for CaM, 100 nM). The Km for ATP was 150 microM. SJ proteins were phosphorylated without a lag time, and the phosphorylation reached its maximum within 2-10 min at 25 degrees C. The endogenous substrates consisted of four major (160K, 120K, 60K, and 51K Mr) and 10 minor proteins. Compared with the endogenous substrate phosphorylation, the phosphorylation of exogenously added proteins (myosin light chains from chicken muscle, casein, arginine-rich histone, microtubule-associated protein-2, tau-protein, and tubulin) was weak, although they are expected to be good substrates for the soluble form of the Ca2+/CaM-dependent protein kinase. Autophosphorylation of the enzyme in SJ inhibited its activity and did not alter the subcellular distribution of the enzyme.     

Resolution of cyclic AMP stimulated protein kinase from polymerization-purified brain microtubules.

Polymerization-deploymerization purified microtubules from mouse brain contain, in addition to tubulin, several minor proteins, including protein kinase activity. The protein kinase copurifies with microtubules in constant proportion to tubulin through two, three, or four cycles of polymerization; it can be resolved from tubulin by gel filtration chromatography and has an apparent molecular weight of 280,000. Its activity is stimulated 7-fold by cyclic AMP, and resembles the soluble brain protein kinase described by Miyamoto $\text{et al.}$ (1). The microtubule preparation serves as an endogenous substrate for this protein kinase; both 6S and 30S tubulin are substrates for phosphorylation to the extent of about 0.10 ± 0.05 moles/mole.     

Dephosphorylation of Microtubule Proteins by Brain Protein Phosphatases 1 and 2A, and Its Effect on Microtubule Assembly

Protein phosphatase C was purified 140-fold from bovine brain with 8% yield using histone H1 phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase (cyclic AMP-kinase). Brain protein phosphatase C was considered to consist of 10 and 90%, respectively, of the catalytic subunits of protein phosphatases 1 and 2A on the basis of the effects of ATP and inhibitor-2. Protein phosphatase C dephosphorylated microtubule-associated protein 2 (MAP2), tau factor, and tubulin phosphorylated by a multifunctional Ca2+/calmodulin-dependent protein kinase (calmodulin-kinase) and the catalytic subunit of cyclic AMP-kinase. The properties of dephosphorylation of MAP2, tau factor, and tubulin were compared. The Km values were in the ranges of 1.6-2.7 microM for MAP2 and tau factor. The Km value for tubulin decreased from 25 to 10-12.5 microM in the presence of 1.0 mM Mn2+. No difference in kinetic properties of dephosphorylation was observed between the substrates phosphorylated by the two kinases. Protein phosphatase C did not dephosphorylate the native tubulin, but universally dephosphorylated tubulin phosphorylated by the two kinases. The holoenzyme of protein phosphatase 2A from porcine brain could also dephosphorylate MAP2, tau factor, and tubulin phosphorylated by the two kinases. The phosphorylation of MAP2 and tau factor by calmodulin-kinase separately induced the inhibition of microtubule assembly, and the dephosphorylation by protein phosphatase C removed its inhibitory effect. These data suggest that brain protein phosphatases 1 and 2A are involved in the switch-off mechanism of both Ca2+/calmodulin-dependent and cyclic AMP-dependent regulation of microtubule formation.     

Glycolipids Isolated from Aplysia kurodaiCan Activate Cyclic Adenosine 3', 5'-Monophosphate-Dependent Protein Kinase from Rat Brain

Abstract: Cyclic AMP (cAMP)-dependent protein kinase (cAMP-kinase) partially purified from the membrane fractions of rat brains was stimulated by novel phosphonogly-cosphingolipids (glycolipids) derived from the skin and nerve fibers of Aplysia kurodai. Among various glycolipids tested, a major glycolipid from the skin, 3-O-MeGalβ 1→3GalNAcα 1→3 [6'- O -(2-aminoethylphosphonyl) Galα1→2] (2-aminoethylphosphonyl→6) Glcβ 1→4GICβ1→1ceramide (SGL-II), was most potent, giving half-maximal activation at 32.2 μ M. Activation of cAMP-kinase was maximal with 250 μ M SGL-II using kemptide as substrate. The effect of SGL-II was additive on kinase activity at submaximal concentrations of cAMP. The kinase activity activated with SGL-II was inhibited by the addition of protein kinase inhibitor peptide, a specific peptide inhibitor for cAMP-kinase. Its inhibitory pattern was similar to that for the catalytic subunit. Of the various substrates tested, the glycolipid-stimulated cAMP-kinase could phosphorylate microtubule-associated protein 2, synapsin I, and myelin basic protein but not histone H1 and casein. The regulatory subunit strongly inhibited the activity of purified catalytic subunit of cAMP-kinase. This inhibition was reversed by addition of SGL-II, as observed for cAMP. SGL-II was capable of partially dissociating cAMP-kinase, which was observed by gel filtration column chromatography. However, the binding activity of cAMP to the holoenzyme was not inhibited with SGL-II. These results demonstrate that the glycolipids can directly activate cAMP-kinase in a manner similar, but not identical, to that of cAMP.     

A calmodulin-dependent protein kinase in Rous sarcoma virus-transformed rat cells and normal liver

A calmodulin-dependent protein kinase has been purified extensively from a Rous sarcoma virus-transformed rat cell line (RR1022) and from normal rat liver. The calmodulin-dependent protein kinase activity was manifested by in vitro phosphorylation of a single Mr 57 000 endogenous phosphoprotein (pp57) present in both the virally transformed cells and normal rat liver. The calmodulin-dependent protein kinase from transformed cells fractionated with the viral src gene product, pp60v-src, through a 650-fold purification of the oncogene product. However, purification of the calmodulin-dependent protein kinase from normal liver demonstrated that the calmodulin-dependent kinase was distinct from pp60v-src. Phosphorylation of pp57 by the kinase purified from the transformed cell line required Ca2+ and calmodulin, was inhibited by EDTA and was unaffected by cAMP or the heat- and acid-stable protein inhibitor of cAMP-dependent protein kinase. Troponin C did not substitute for calmodulin. A virtually identical calmodulin-dependent protein kinase activity was purified from rat liver by affinity chromatography on calmodulin-Sepharose. Phosphorylation of pp57 by the affinity-purified liver protein kinase was also observed, and required Ca2+ and calmodulin. EGTA and trifluoroperazine inhibited pp57 phosphorylation. The calmodulin-dependent protein kinase reported here did not phosphorylate substrates of known calmodulin-dependent protein kinases in vitro (myosin light chain, phosphorylase b, glycogen synthase, microtubule-associated proteins, tubulin, alpha-casein). Because none of these proteins served as substrates in vitro and pp57 was the only endogenous substrate found, the properties of this enzyme appear to be different from any previously described calmodulin-dependent protein kinase.     

Phosphorylation of Tyrosine Hydroxylase by Cyclic GMP-Dependent Protein Kinase

Tyrosine hydroxylase purified from rat pheochromocytoma was phosphorylated and activated by purified cyclic GMP-dependent protein kinase as well as by cyclic AMP-dependent protein kinase catalytic subunit. The extent of activation was correlated with the degree of phosphate incorporated into the enzyme. Comparable stoichiometric ratios (0.6 mol phosphate/mol tyrosine hydroxylase subunit) were obtained at maximal concentrations of either cyclic AMP-dependent or cyclic GMP-dependent protein kinases. The enzymes appeared to mediate the phosphorylation of the same residue based on the observation that incorporation was not increased when both enzymes were present. The major tryptic phosphopeptide obtained from tyrosine hydroxylase phosphorylated by each protein kinase exhibited an identical retention time following HPLC. The purified phosphopeptides also exhibited identical isoelectric points. These data provide support for the notion that the protein kinases are phosphorylating the same residue of tyrosine hydroxylase.     

Autophosphorylation of Calmodulin-Kinase II in Synaptic Junctions Modulates Endogenous Kinase Activity

Previous studies have purified from brain a Ca2+/calmodulin-dependent protein kinase II (designated CaM-kinase II) that phosphorylates synapsin I, a synaptic vesicle-associated phosphoprotein. CaM-kinase II is composed of a major Mr 50K polypeptide and a minor Mr 60K polypeptide; both bind calmodulin and are phosphorylated in a Ca2+/calmodulin-dependent manner. Recent studies have demonstrated that the 50K component of CaM-kinase II and the major postsynaptic density protein (mPSDp) in brain synaptic junctions (SJs) are virtually identical and that the CaM-kinase II and SJ 60K polypeptides are highly related. In the present study the photoaffinity analog [alpha-32P]8-azido-ATP was used to demonstrate that the 60K and 50K polypeptides of SJ-associated CaM-kinase II each bind ATP in the presence of Ca2+ plus calmodulin. This result is consistent with the observation that these proteins are phosphorylated in a Ca2+/calmodulin-dependent manner. Experiments using 32P-labeled peptides obtained by limited proteolysis of 60K and 50K polypeptides from SJs demonstrated that within each kinase polypeptide the same peptide regions contain both autophosphorylation and 125I-calmodulin binding sites. These results suggested that the autophosphorylation of CaM-kinase II could regulate its capacity to bind calmodulin and, thus, its capacity to phosphorylate substrate proteins. By using 125I-calmodulin overlay techniques and sodium dodecyl sulfate-polyacrylamide gel electrophoresis we found that phosphorylated 50K and 60K CaM-kinase II polypeptides bound more calmodulin (50-70%) than did unphosphorylated kinase polypeptides. Levels of in vitro CaM-kinase II activity in SJs were measured by phosphorylation of exogenous synapsin I. SJs containing highly phosphorylated CaM-kinase II displayed greater activity in phosphorylating synapsin I (300% at 15 nM calmodulin) relative to control SJs that contained unphosphorylated CaM-kinase II. The CaM-kinase II activity in phosphorylated SJs was indistinguishable from control SJs at saturating calmodulin concentrations (300-1,000 nM). These findings show that the degree of autophosphorylation of CaM-kinase II in brain SJs modulates its in vitro activity at low and possibly physiological calmodulin concentrations; such a process may represent a mechanism of regulating this kinase's activity at CNS synapses in situ.     

Calmodulin Kinase II in Pure Cultured Astrocytes

Calcium- and calmodulin-dependent protein kinase activity was studied in pure neuronal and glial cultures. The addition of calcium and calmodulin stimulated 32P incorporation into several neuronal proteins including two in the 50- and 60-kilodalton (kD) region which comigrated with purified forebrain calmodulin kinase II subunits (CaM kinase II). In mature astrocytes, CaM kinase activity was also present, and was inhibited by trifluoroperazine and diazepam. Again in homogenates of these cells, two phosphoproteins of apparent molecular masses of 50 and 60 kD comigrated with purified CaM kinase. CaM kinase activity was absent in immature mixed glia and oligodendrocytes. The presence of CaM kinase in neurons and mature astrocytes was confirmed using monoclonal antibodies specific for the 50-kD subunit of the enzyme. No immunoreactivity was observed in oligodendrocytes. The presence of CaM kinase in astrocytes suggests a more ubiquitous role of this enzyme in regulating cellular processes than was previously recognized.     

Localization and Characterization of Tubulin-Like Proteins Associated with Brain Mitochondria: The Presence of a Membrane-Specific Isoform

A mitochondrial fraction, purified from pig brain, was found to contain associated polypeptides with the same electrophoretic migration and isoelectric points as the alpha- and beta-tubulin subunits present in brain microtubules. When analyzed by Western blotting these polypeptides reacted specifically with purified tubulin antibodies. The tubulin-like proteins were then visualized in mitochondrial membranes by protein A-gold complexes after the incubation of purified mitochondria with tubulin antibodies. When membrane and microtubule proteins were compared by isoelectric focussing and two-dimensional gel electrophoresis, differences were observed in the patterns of tubulin isoforms. An additional polypeptide, with the electrophoretic migration of beta-tubulin but the isoelectric point of alpha-tubulin, was found to be enriched in the mitochondrial fraction. This peptide had several Staphylococcus aureus V8 protease peptides in common with alpha-tubulin and may result from a posttranslational modification of that subunit.     

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.     

Direct Visualization of the Calmodulin Subunit of Phosphorylase Kinase via Electron Microscopy Following Subunit Exchange

Calmodulin is a tightly bound, intrinsic subunit (delta) of the hexadecameric phosphorylase-b kinase holoenzyme, (alphabetagammadelta)4. To introduce specifically labeled calmodulin into the phosphorylase-b kinase complex for its eventual visualization by electron microscopy, we have developed a method for rapidly exchanging exogenous calmodulin for the intrinsic delta subunit. This method exploits previous findings that low concentrations of urea in the absence of Ca(2+) ions cause the specific dissociation of only the delta subunit from the holoenzyme [Paudel, H. K., and Carlson, G. M. (1990) Biochem. J. 268, 393-399]. In the current study, phosphorylase-b kinase was incubated with excess exogenous calmodulin and a threshold concentration of urea to promote exchange of its delta subunit with the exogenous calmodulin. Size exclusion HPLC was then used to remove the excess calmodulin from the holoenzyme containing exchanged delta subunits. Using metabolically labeled [35S]calmodulin to allow quantification and optimization of exchange conditions, we achieved exchange of approximately 10% of all delta subunits within 1 h, with the exchanged holoenzyme retaining full catalytic activity. Calmodulins derivatized with Nanogold for visualization by scanning transmission electron microscopy were then exchanged for delta, which for the first time allowed localization of the delta subunit within the bridged, bilobal phosphorylase b kinase holoenzyme complex. The delta subunits were determined to be near the edge of the lobes, just distal to the interlobal bridges and proximal to a previously identified region of the enzyme's catalytic gamma subunit.     

Purification and Characterization of a Ca2+- and Calmodulin-Dependent Protein Kinase from Rat Brain

Abstract: A Ca²⁺- and calmodulin-dependent protein kinase was purified from rat brain cytosol fraction to apparent homogeneity at approximately 800-fold and with a 5% yield. The purified enzyme had a molecular weight of 640,000 as determined by gel filtration analysis on Sephacryl S-300 and a sedimentation coefficient of 15.3 S by sucrose density gradient centrifugation, and resulted in a single protein band of MW 49,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that the native enzyme has a large molecular weight and consists of 11 to 14 identical subunits. The purified enzyme exhibited K _m values of 109 and 30 μM for ATP and chicken gizzard myosin light chain, respectively, and K _a values of 12 n M and 1.9 μM for brain calmodulin and Ca²⁺, respectively. In addition to myosin light chain, myelin basic protein, casein, arginine-rich histone, microtubule protein, and synaptosomal proteins were phosphorylated by the enzyme in a Ca²⁺- and calmodulin-dependent manner. The purified enzyme was phosphorylated without the addition of the catalytic subunit of cyclic AMP-dependent protein kinase. Our findings indicate that there is a multifunctional Ca²⁺- and calmodulin-dependent protein kinase in the brain and that this enzyme may regulate the reactions of various endogenous proteins.