A novel particulate form of Ca(2+)/calmodulin-dependent [correction of Ca(2+)/CaMKII-dependent] protein kinase II in neurons.

Cytoskeletal and postsynaptic density (PSD) fractions from forebrain contain discrete spherical structures that are immunopositive for Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Spherical structures viewed by rotary shadow electron microscopy have an average diameter of approximately 100 nm and, in distinction to postsynaptic densities, do not immunolabel for PSD-95. These structures were purified to near homogeneity by extraction with the detergent N-lauryl sarcosinate. Biochemical analysis revealed that CaMKII accounts for virtually all of the protein in the purified preparation, suggesting that spherical structures are clusters of self-associated CaMKII. Exposure of cultured hippocampal neurons to a mitochondrial uncoupler in glucose-free medium promotes the formation of numerous CaMKII-immunopositive structures identical in size and shape to the CaMKII clusters observed in subcellular fractions. Clustering of CaMKII would reduce its kinase function by preventing its access to fixed substrates. On the other hand, clustering would not affect the ability of the large cellular pool of CaMKII to act as a calmodulin sink, as demonstrated by the Ca(2+)-dependent binding of gold-conjugated calmodulin to CaMKII clusters. We propose that the observed clustering of CaMKII into spherical structures is a protective mechanism preventing excessive protein phosphorylation upon loss of Ca(2+) homeostasis, without compromising calmodulin regulation.

Inhibition of phosphatase activity facilitates the formation and maintenance of NMDA-induced calcium/calmodulin-dependent protein kinase II clusters in hippocampal neurons.

The majority of hippocampal neurons in dissociated cultures and in intact brain exhibit clustering of calcium/calmodulin-dependent protein kinase II (CaMKII) into spherical structures with an average diameter of 110 nm when subjected to conditions that mimic ischemia and excitotoxicity [Neuroscience 106 (2001) 69]. Because clustering of CaMKII would reduce its effective concentration within the neuron, it may represent a cellular strategy to prevent excessive CaMKII-mediated phosphorylation during episodes of Ca2+ overload. Here we employ a relatively mild excitatory stimulus to promote sub-maximal clustering for the purpose of studying the conditions for the formation and disappearance of CaMKII clusters. Treatment with 30 microM N-methyl-D-aspartic acid (NMDA) for 2 min produced CaMKII clustering in approximately 15% of dissociated hippocampal neurons in culture, as observed by pre-embedding immunogold electron microscopy. These CaMKII clusters could be labeled with antibodies specific to the phospho form (Thr286) of CaMKII, suggesting that at least some of the CaMKII molecules in clusters are autophosphorylated. To test whether phosphorylation is involved in the formation and maintenance of CaMKII clusters, the phosphatase inhibitors calyculin A (5 nM) or okadaic acid (1 microM) were included in the incubation medium. With inhibitors more neurons exhibited CaMKII clusters in response to 2 min NMDA treatment. Furthermore, 5 min after the removal of NMDA and Ca2+, CaMKII clusters remained and could still be labeled with the phospho-specific antibody. In contrast, in the absence of phosphatase inhibitors, no clusters were detected 5 min after the removal of NMDA and Ca2+ from the medium. These results suggest that phosphatases type 1 and/or 2A regulate the formation and disappearance of CaMKII clusters.

Calcium/calmodulin-dependent protein kinase II clusters in adult rat hippocampal slices.

We have previously reported the formation of calcium/calmodulin-dependent protein kinase II (CaMKII) clusters approximately 110 nm in diameter in hippocampal neurons in culture and in the intact adult brain, under conditions that simulate ischemic stress and increase [Ca(2+)](i) [Dosemeci et al. (2000) J. Neurosci. 20, 3076-3084; Tao-Cheng et al. (2001) Neuroscience 106, 69-78]. These observations suggest that ischemia-like conditions that prevail during the dissection of brain tissue for the preparation of hippocampal slices could lead to the formation of CaMKII clusters. We now show by pre-embedding immuno-electron microscopy that, indeed, CaMKII clusters are present in the CA1 pyramidal neurons in hippocampal slices from adult rats fixed immediately after dissection, and that the number of CaMKII clusters increases with the delay time between decapitation and fixation. Moreover, CaMKII clusters are typically localized near the endoplasmic reticulum. When acute slices are allowed to recover in oxygenated medium for 2 h, CaMKII clusters mostly disappear, indicating that clustering is reversible. Also, the postsynaptic density, another site for CaMKII accumulation under excitatory conditions, becomes thinner upon recovery. Treatment of recovered slices with high potassium for 90 s causes the re-appearance of CaMKII clusters in nearly all CA1 pyramidal cells examined. On the other hand, when dissociated hippocampal neurons in primary culture are exposed to the same depolarizing conditions, only approximately 25% of neurons exhibit CaMKII clusters, indicating a difference in the susceptibility of the neurons in culture and in acute slices to excitatory stimuli. Altogether these observations indicate that the effect of CaMKII clustering should be considered when interpreting experimental results obtained with hippocampal slices.

Sustained elevation of calcium induces Ca(2+)/calmodulin-dependent protein kinase II clusters in hippocampal neurons.

Treatment of cultured hippocampal neurons with the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) in the absence of glucose mimics ischemic energy depletion and induces formation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) clusters, spherical structures with diameters of 75-175 nm [Dosemeci et al., J. Neurosci. 20 (2000) 3076-3084]. The demonstration that CaMKII clustering occurs in the intact, adult rat brain upon interruption of blood flow indicates that clustering is not confined to cell cultures. Application of N-methyl-D-aspartate (250 microM, 15 min) to hippocampal cultures also induces cluster formation, suggesting a role for Ca(2+). Indeed, intracellular Ca(2+) monitored with Fluo3-AM by confocal microscopy reaches a sustained high level within 5 min of CCCP treatment. The appearance of immunolabeled CaMKII clusters, detected by electron microscopy, follows the onset of the sustained increase in intracellular Ca(2+). Moreover, CaMKII does not cluster when the rise in intracellular Ca(2+) is prevented by the omission of extracellular Ca(2+) during CCCP treatment, confirming that clustering is Ca(2+)-dependent. A lag period of 1-2 min between the onset of high intracellular Ca(2+) levels and the formation of CaMKII clusters suggests that a sustained increase in Ca(2+) level is necessary for the clustering. CaMKII clusters disappear within 2 h of returning the cultures to normal incubation conditions, at which time no significant cell death is detected. These results indicate that pathological conditions that promote sustained episodes of Ca(2+) overload result in a transitory clustering of CaMKII into spherical structures. CaMKII clustering may represent a cellular defense mechanism to sequester a portion of the CaMKII pool, thereby preventing excessive protein phosphorylation.

Demonstration by phase-partitioning in Triton X-114 solutions that phosphoprotein B-50 (F-1) from rat brain is an integral membrane protein.

The Triton X-114 phase separation technique was employed to fractionate phosphoproteins present in membrane fragments from rat brain. Membranes were labelled with [gamma-32P]ATP in media containing Ca2+, Ca2+ plus calmodulin or cyclic AMP, and then treated with Triton X-114. Phosphoproteins recovered in the detergent-insoluble fraction, aqueous and detergent phases were detected by SDS-polyacrylamide gel electrophoresis and autoradiography. Of the proteins solubilised by the detergent, a known substrate of protein kinase C, the B-50 phosphoprotein (45 kD; also known as F-1), partitioned quantitatively into the detergent-rich phase, making it very probable that this phosphoprotein is an integral membrane protein. The detergent-rich phase also contained an 80 kD phosphoprotein, which probably corresponds to the widespread acidic 87 kD substrate of protein kinase C.

Bovine neurofilament-enriched preparations contain kinase activity similar to casein kinase I--neurofilament phosphorylation by casein kinase I (CKI).

Neurofilament (NF)-enriched preparations from bovine spinal cord contain regulator-independent kinase activities that phosphorylate NF subunits as well as alpha-casein. CKI-7 (N-2-amino ethyl, 5-chloroisoquinoline, 8-sulfonamide), a specific inhibitor of casein kinase I (CKI), inhibits the phosphorylation of NF subunits in the neurofilament preparation. This inhibition occurs at a concentration range identical to concentrations where CKI-7 inhibits rabbit reticulocyte CKI phosphorylation of alpha-casein. Heparin, a specific inhibitor of casein kinase II (CKII), produced only 20% inhibition of 32P incorporation into NF subunits, and only at concentrations 5 to 10-fold higher than those required to inhibit CKII from reticulocytes. CKI from rabbit reticulocytes phosphorylated all three NF subunits (NF-H, NF-M and NF-L). Comparison of the tryptic phosphopeptide maps of NF-M, phosphorylated by the NF-associated kinase and CKI, indicates that the casein kinase I phosphorylates many of the peptides phosphorylated by the NF-associated kinase and this phosphorylation occurs at the carboxy terminal tail domain of the NF-M subunit. These studies suggest that the major independent kinase activity associated with NFs is CKI.

Effects of CaMKII inhibitor tatCN21 on activity-dependent redistribution of CaMKII in hippocampal neurons.

TatCN21 is a membrane permeable calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor derived from the inhibitor protein CaMKIIN. TatCN21 has been used to demonstrate the involvement of CaMKII in a variety of physiological and pathological phenomena, and it also limits excitotoxic damage in neurons. Here we use preembedding immunogold electron microscopy to examine the effect of tatCN21 on the redistribution of CaMKII in cultured hippocampal neurons. Incubation of cultures with tatCN21 (20 µM for 20 min) prior to exposure to N-methyl-d-asparic acid (NMDA) (50 µM for 2 min) inhibited both the accumulation of CaMKII at postsynaptic densities (PSDs) and CaMKII clustering in the dendrites. Under these conditions, CaMKII also formed morphologically distinct aggregates with polyribosomes near the PSD and in dendrites. Formation of these CaMKII-polyribosome aggregates requires the presence of both tatCN21 and calcium, and was augmented upon exposure to high K(+) or NMDA. CaMKII-polyribosome aggregates formed consistently with 20 µM tatCN21, but minimally or not at all with 5 µM. However, these aggregates are not induced by another CaMKII inhibitor, KN93. Formation of CaMKII-polyribosome aggregates was completely reversible within 1h after washout of tatCN21. Effects of tatCN21 were largely restricted to dendrites, with minimal effect in the soma. The effects of tatCN21 on CaMKII distribution can be used to dissect the mechanism of CaMKII involvement in cellular events.

SynGAP moves out of the core of the postsynaptic density upon depolarization.

SynGAP is a Ras GTPase activating protein present at the postsynaptic density (PSD) in quantities matching those of the core scaffold protein PSD-95. SynGAP is reported to inhibit synaptic accumulation of AMPA receptors. Here, we characterize by immunogold electron microscopy the distribution of SynGAP at the PSD under basal and depolarizing conditions in rat hippocampal neuronal cultures. The PSD core, extending up to 40 nm from the postsynaptic membrane, typically shows label for SynGAP, while half of the synapses exhibit additional labeling in a zone 40-120 nm from the postsynaptic membrane. Upon depolarization with high K(+), labeling for SynGAP significantly decreases at the core of the PSD and concomitantly increases at the 40-120 nm zone. Under the same depolarization conditions, label for PSD-95, the presumed binding partner of SynGAP, does not change its localization at the PSD. Depolarization-induced redistribution of SynGAP is reversible and also occurs upon application of N-methyl-d-aspartic acid (NMDA). Activity-induced movement of SynGAP could vacate sites in the PSD core allowing other elements to bind to these sites, such as transmembrane AMPA receptor regulatory proteins (TARPs), and simultaneously facilitate access of SynGAP to CaMKII and Ras, elements of a regulatory cascade.

Activity induced changes in the distribution of Shanks at hippocampal synapses.

Dendritic spines contain a family of abundant scaffolding proteins known as Shanks, but little is known about how their distributions might change during synaptic activity. Here, pre-embedding immunogold electron microscopy is used to localize Shanks in synapses from cultured hippocampal neurons. We find that Shanks are preferentially located at postsynaptic densities (PSDs) as well as in a filamentous network near the PSD, extending up to 120 nm from the postsynaptic membrane. Application of sub-type specific antibodies shows that Shank2 is typically concentrated at and near PSDs while Shank1 is, in addition, distributed throughout the spine head. Depolarization with high K+ for 2 min causes transient, reversible translocation of Shanks towards the PSD that is dependent on extracellular Ca2+. The amount of activity-induced redistribution and subsequent recovery is pronounced for Shank1 but less so for Shank2. Thus, Shank1 appears to be a dynamic element within the spine, whose translocation could be involved in activity-induced, transient structural changes, while Shank2 appears to be a more stable element positioned at the interface of the PSD with the spine cytoplasm.

Rapid turnover of spinules at synaptic terminals.

Spinules found in brain consist of small invaginations of plasma membranes which enclose membrane evaginations from adjacent cells. Here, we focus on the dynamic properties of the most common type, synaptic spinules, which reside in synaptic terminals. In order to test whether depolarization triggers synaptic spinule formation, hippocampal slice cultures (7-day-old rats, 10-14 days in culture) were exposed to high K+ for 0.5-5 min, and examined by electron microscopy. Virtually no synaptic spinules were found in control slices representing a basal state, but numerous spinules appeared at both excitatory and inhibitory synapses after treatment with high K+. Spinule formation peaked with approximately 1 min treatment at 37 degrees C, decreased with prolonged treatment, and disappeared after 1-2 min of washout in normal medium. The rate of disappearance of spinules was substantially slower at 4 degrees C. N-methyl-D-aspartic acid (NMDA) treatment also induced synaptic spinule formation, but to a lesser extent than high K+ depolarization. In acute brain slices prepared from adult mice, synaptic spinules were abundant immediately after dissection at 4 degrees C, extremely rare in slices allowed to recover at 28 degrees C, but frequent after high K(+) depolarization. High pressure freezing of acute brain slices followed by freeze-substitution demonstrated that synaptic spinules are not induced by chemical fixation. These results indicate that spinules are absent in synapses at low levels of activity, but form and disappear quickly during sustained synaptic activity. The rapid turnover of synaptic spinules may represent an aspect of membrane retrieval during synaptic activity.

Regulation of the phosphorylation state of the AMPA receptor GluR1 subunit in the postsynaptic density.

1. Changes in the phosphorylation state of AMPA-type glutamate receptors are thought to underlie activity-dependent synaptic modification. It has been established that the GluR1 subunit is phosphorylated on two distinct sites, Ser-831 and Ser-845, by CaMKII and by PKA, respectively, and that phosphorylation by either kinase correlates with an increase in the AMPA receptor-mediated current. GluR1 is concentrated in postsynaptic densities and it is expected that this particular receptor pool is involved in synaptic modification. The present study describes the regulation of the phosphorylation state of GluR1 in isolated postsynaptic densities. 2. Addition of Ca2+/calmodulin to the postsynaptic density fraction promotes phosphorylation of GluR1, and under these conditions, dephosphorylation is prevented by the inclusion of phosphatase type 1 inhibitors, microcystin-LR and Inhibitor-1. CaMKII and phosphatase type 1 are also found to be enriched in the PSD fraction compared to the parent fractions. 3. On the other hand, the addition of cAMP, either by itself or with exogenous PKA, does not change the phosphorylation state of GluR1. Prior incubation of PSDs under dephosphorylating conditions results in only a small PKA-mediated phosphorylation of GluR1. 4. These results support the hypothesis that PSDs contain the molecular machinery to promote the phosphorylation as well as the dephosphorylation of GluR1 on Ser-831, while Ser-845, the site phosphorylated by PKA, appears to be mostly occluded. Thus, it is possible that a large pool of PSD-associated GluR1 is regulated through modification of the phosphorylation state of the Ser-831 site only.

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.

Ca(2+)-independent autophosphorylation of postsynaptic density-associated Ca2+/calmodulin-dependent protein kinase.

A major protein in the postsynaptic density fraction is alpha-CAM kinase II, the alpha-subunit of the Ca2+/calmodulin-dependent protein kinase. Autophosphorylation of the postsynaptic density-associated CaM kinase II is likely to be a crucial event in the induction of activity-dependent synaptic modification. This study focuses on the regulation and consequences of Ca(2+)-independent autophosphorylation of the enzyme. In isolated postsynaptic densities, a sub-stochiometric level of autophosphorylation in the presence of Ca2+ is sufficient to trigger maximal Ca(2+)-independent autophosphorylation of alpha-CaM Kinase II. A major fraction of the sites phosphorylated in the absence of Ca2+ can be dephosphorylated by the endogenous phosphatase activity in the preparation. Ca(2+)-independent autophosphorylation is correlated with a drastic decrease in calmodulin binding to postsynaptic densities. This may represent a physiological mechanism that lower the calmodulin trapping capacity of the organelle, thus increasing the availability of calmodulin to other elements within a spine.

Identification of novel phosphorylation sites on postsynaptic density proteins.

Phosphorylation of the components of the postsynaptic density (PSD), a protein complex lining the postsynaptic membrane, may regulate synaptic structure and function. We carried out mass spectrometric analyses to identify phosphorylation sites on PSD proteins. Phosphopeptides were isolated from the total tryptic digest of a PSD fraction by immobilized metal affinity chromatography and analyzed by liquid chromatography and tandem mass spectrometry. The phosphorylated residues detected following in vitro phosphorylation in the presence of Ca2+/calmodulin included S-1058 on SynGAP and S-1662 and S-1668 on Shank3. Other phosphorylated residues were identified in control samples, presumably reflecting phosphorylation in the intact cell. These included the homologous residues, S-295 on PSD-95 and S-365 on PSD-93, located between the PDZ2 and PDZ3 domains of these proteins; and S-367 located on the actin-binding domain of beta-CaMKII. The sequence RXXSPV emerged as a common phosphorylation motif of three specialized PSD scaffolding proteins, PSD-95, PSD-93, and Shank3. Phosphorylated serine residues in several of the identified phosphorylation sites were followed by prolines, suggesting prominent involvement of proline directed kinases in the regulation of PSD components.

Identification of a major autophosphorylation site on postsynaptic density-associated Ca2+/calmodulin-dependent protein kinase.

One of the most abundant proteins in postsynaptic densities is identical or very similar to the alpha-subunit of the Ca2+/calmodulin-dependent protein kinase II. Autophosphorylation of this protein in isolated postsynaptic densities was studied under various conditions, following inhibition of endogenous phosphatase activity with microcystin-LR. Phosphorylation accompanied by a shift in the enzyme's electrophoretic mobility was observed upon incubation with Ca2+ and calmodulin at 37 degrees C. Brief incubation with Ca2+ and calmodulin at 0 degrees C resulted in a low level of phosphorylation and no change in mobility. Following this limited Ca(2+)-dependent phosphorylation, however, a high level of phosphorylation could be achieved in the absence of Ca2+, upon incubation at 37 degrees C. Comparison of reverse-phase HPLC phosphopeptide elution profiles obtained following phosphorylation at 37 degrees C, in the presence and absence of Ca2+, as described above, showed differences, suggesting that certain distinct sites may be phosphorylated under each condition. A major phosphopeptide peak, however, with the amino acid sequence Met-Leu-Thr(P)-Ile-Asn-Pro-Ser-Lys was identified under both conditions. This sequence is identical to the predicted sequence containing Thr-253 of the Ca2+/calmodulin-dependent protein kinase II. The results suggest that phosphorylation at Thr-253 requires an initial Ca(2+)-dependent phosphorylation, which may be at a different site, but does not depend on the continued presence of Ca2+ to proceed. The observed mode of regulation of autophosphorylation at Thr-253 appears to be unique to the postsynaptic density-associated enzyme.

Acceptors for cyclic AMP-dependent and calcium ion-dependent protein kinases in rat brain cytosol fractions: a comparison of occluded (synaptosomal) cytosol with non-occluded cytosol.

Endogenous protein phosphorylation patterns were compared in occluded and non-occluded cytosol fractions prepared from rat forebrain. The occluded fraction was taken as representative of synaptosomal cytosol. One- and two-dimensional autoradiographs revealed the presence in non-occluded cytosol of a substrate for cAMP- and Ca2+/calmodulin-dependent protein kinase activities of Mr 300kD, corresponding to phosphorylated microtubule-associated protein-2 (MAP-2); this protein was absent in occluded cytosol. In contrast, a major substrate for protein kinase C was observed exclusively in occluded cytosol after phosphorylation under basal conditions. However, after phosphorylation in the presence of exogenous lipids, approximately equal amounts of the 82kD substrate were detected in both fractions, suggesting that protein kinase C in the occluded fraction was present in a partially activated state. Other minor differences in phosphorylation patterns between the two fractions were observed.

Inhibition of endogenous phosphatase in a postsynaptic density fraction allows extensive phosphorylation of the major postsynaptic density protein.

The major postsynaptic density protein, proposed to be a calcium/calmodulin-dependent protein kinase, becomes phosphorylated when a postsynaptic density preparation from rat cerebral cortex is incubated in medium containing calcium and calmodulin. Upon longer incubation, however, the level of phosphorylation declines, suggesting the presence of a phosphatase activity. When Microcystin-LR, a phosphatase inhibitor, is included in the phosphorylation medium, the decline in phosphorylation is prevented and a higher maximal level of phosphorylation can be achieved. Under these conditions, the maximal phosphorylation of major postsynaptic density protein is accompanied by a nearly complete shift in its electrophoretic mobility from 50 kDa to 54 kDa, similar to that described for the alpha subunit of the soluble calcium/calmodulin-dependent protein kinase II. Of the four major groups of serine/threonine protein phosphatases, the enzyme responsible for the dephosphorylation of major postsynaptic density protein is neither type 2C, which is insensitive to Microcystin-LR, nor type 2B, which is calcium-dependent. As Microcystin-LR is much more potent than okadaic acid in inhibiting the dephosphorylation of major postsynaptic density protein, it is likely that the postsynaptic density-associated phosphatase is a type 1. The above results indicate that the relatively low level of phosphorylation of the major postsynaptic density protein observed in preparations containing postsynaptic densities is not due to a difference between the cytoplasmic and postsynaptic density-associated calcium/calmodulin-dependent kinases as previously proposed, but to a phosphatase activity, presumably belonging to the type 1 group.

Effect of calpain on the composition and structure of postsynaptic densities.

Endogenous calcium-activated proteases, the calpains, are thought to play a role in the regulation of postsynaptic function. Here we characterize some biochemical and morphological effects of calpain on isolated postsynaptic densities (PSDs). When a PSD preparation from rat forebrain was treated with exogenous calpain, many proteins, including spectrin, tubulin and the alpha-subunit of calcium calmodulin-dependent protein kinase (alpha-CaM kinase), were proteolyzed at varying rates, while another major protein, actin, remained intact. The selectivity of calpain action became more apparent in experiments designed to achieve limited proteolysis by using a lower calpain-to-protein ratio; it was possible to obtain extensive breakdown of spectrin with no decrease in the levels of either tubulin, alpha-CaM kinase, or actin. Electron microscopy of freeze-substituted preparations showed that limited calpain action caused a partial unraveling of the PSD, in which the characteristic central dense lamina became wider and less dense. We interpret these changes as due to calpain-mediated breakdown of cross-bridging elements, leading to a partial unraveling of the central PSD lamina. Opening up of the PSD structure following limited calpain action could facilitate exposure of previously occluded functional sites within the PSD and contribute to the modification of the synaptic function.

Association of cyclic-AMP-dependent protein kinase with neurofilaments.

Neurofilament preparations isolated from bovine spinal cord contain cyclic-AMP-dependent protein kinase (PKA) activity. Treatment of this preparation with cyclic AMP, to dissociate the regulatory subunit of the kinase from the catalytic subunit, resulted in retention of the kinase activity but loss of cyclic AMP regulation. This suggests that PKA is associated via its catalytic subunit with the neurofilament preparation. The association of exogenous PKA from bovine heart with the neurofilament preparation and with neurofilaments reconstituted from purified neurofilament proteins was also investigated. Either the free catalytic subunit or combinations of the catalytic and regulatory subunits of PKA were incubated with the preparations, and the degree of association was determined as the level of kinase activity that co-sediments with neurofilaments. The results indicate that the free catalytic subunit of PKA co-sediments with neurofilaments reconstituted from purified proteins. The regulatory subunit of PKA from bovine heart, when pre-mixed with the catalytic subunit, decreased the level of kinase that co-sediments with the neurofilament fraction in a dose-dependent manner. This effect of the regulatory subunit was reversed by inclusion of cyclic AMP in the incubation medium before centrifugation. The above findings suggest that the regulatory subunit, when attached to the catalytic subunit, has an inhibitory effect on its association with neurofilaments, with the implication that the association may be a cyclic-AMP-regulated event.