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.     

Dephosphorylation of Microtubule-Associated Protein 2, τ Factor, and Tubulin by Calcineurin

Calcineurin dephosphorylated microtubule-associated protein 2 (MAP2) and tau factor phosphorylated by cyclic AMP-dependent and Ca2+, calmodulin-dependent protein kinases from the brain. Tubulin, only phosphorylated by the Ca2+, calmodulin-dependent protein kinase, served as substrate for calcineurin. The concentrations of calmodulin required to give half-maximal activation of calcineurin were 21 and 16 nM with MAP2 and tau factor as substrates, respectively. The Km and Vmax values were in ranges of 1-3 microM and 0.4-1.7 mumol/mg/min, respectively, for MAP2 and tau factor. The Km value for tubulin was in a similar range, but the Vmax value was lower. The peptide map analysis revealed that calcineurin dephosphorylated MAP2 and tau factor universally, but not in a site-specific manner. The autophosphorylated Ca2+, calmodulin-dependent protein kinase was not dephosphorylated by calcineurin. These results suggest that calcineurin plays an important role in the functions of microtubules via dephosphorylation.     

Phosphorylation of P400 Protein by Cyclic AMP-Dependent Protein Kinase and Ca2+/Calmodulin-Dependent Protein Kinase II

Purified P400 protein was phosphorylated by both purified Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and the catalytic subunit of cyclic AMP-dependent protein kinase (A-kinase). Because P400 protein was suggested to function as an integral membrane protein, we investigated the phosphorylation of P400 protein using crude mitochondrial and microsomal fractions (P2/P3 fraction). Incubation of the P2/P3 fraction from mouse cerebellum with cyclic AMP or the catalytic subunit of A-kinase stimulated the phosphorylation of P400 protein. The phosphorylation of P400 protein was not observed in the P2/P3 fraction from mouse forebrain. Cyclic AMP and A-kinase enhanced the phosphorylation of several proteins, including P400 protein, suggesting that P400 protein is one of the best substrates for A-kinase in the P2/P3 fraction. Although endogenous and exogenous CaM kinase II stimulated the phosphorylation of some proteins in the P2/P3 fraction, the phosphorylation of P400 protein was weak. Immunoprecipitation with the monoclonal antibody to P400 protein confirmed that the P400 protein itself was definitely phosphorylated by the catalytic subunit of A-kinase and CaM kinase II. A-kinase phosphorylated only the seryl residue in P400 protein. Immunoblot analysis of the cells in primary culture of mouse cerebellum confirmed the expression of P400 protein, which migrated at the same position on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as that in the P2/P3 fraction. Incubation of the cultured cerebellar cells with [32P]orthophosphate resulted in the labeling of P400 protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ischemia-Induced Translocation of Ca2+/Calmodulin-Dependent Protein Kinase II: Potential Role in Neuronal Damage

The activities of Ca2+/calmodulin (CaM)-dependent, Ca2+/phospholipid-dependent, and cyclic AMP-dependent protein kinases (CaM-KII, PKC, and PKA, respectively) were determined in rat brains after global ischemia. Both CaM-KII and PKC activities were significantly depressed in both hippocampal and cerebral cortical regions of ischemic animals, whereas no change was detected in PKA activity. The loss of CaM-KII activity was more dramatic and more sustained than the loss of PKC activity and correlated with the duration of ischemia. These decreases in enzyme activity were found in both supernatant and pellet fractions from crude homogenates. When the supernatant and pellet were analyzed for the amount of CaM-KII 50-kDa protein, a significant decrease was detected in supernatant fractions that paralleled a gain in the amount of CaM-KII in the pellet. Thus, the loss of CaM-KII activity in the supernatant can be explained by translocation of the enzyme to the pellet. Whether inactivation of CaM-KII occurs during or after the enzyme translocates from the supernatant to the pellet is unknown. Our results indicate that loss in CaM-KII activity parallels neuronal damage associated with ischemia; down-regulation of CaM-KII activity coincided with translocation of the enzyme to the particulate fraction, and it is proposed that this may be, in fact, a mechanism for controlling excessive CaM-KII phosphorylation.     

Inactivation and Reactivation of the Multifunctional Calmodulin-Dependent Protein Kinase from Brain by Autophosphorylation and Dephosphorylation: Involvement of Protein Phosphatases from Brain

The multifunctional calmodulin-dependent protein kinase (calmodulin-kinase) from rat brain was autophosphorylated in a Ca2+- and calmodulin-dependent manner. The activity of the autophosphorylated enzyme was independent of Ca2+ and calmodulin. Calmodulin-kinase was dephosphorylated by protein phosphatase C from bovine brain, which is the catalytic subunits of protein phosphatases 1 and 2A. The holoenzyme of protein phosphatase 2A was also involved in the dephosphorylation of the enzyme. The autophosphorylated sites of calmodulin-kinase were universally dephosphorylated by protein phosphatase C. Calmodulin-kinase was inactivated and reactivated by autophosphorylation and dephosphorylation, respectively. Furthermore, the regulation of calmodulin-kinase by autophosphorylation and dephosphorylation was observed using calmodulin-kinase from canine heart. These results suggest that the activity of calmodulin-kinase is regulated by autophosphorylation and dephosphorylation, and that the regulation is the universal phenomenon for many other calmodulin-kinases in various tissues.     

Decreased protein phosphatase 2A activity in hippocampal long-term potentiation

Using autophosphorylated Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) as substrate, we now find that long-term potentian (LTP) induction and maintenance are also associated with a significant decrease in calyculin A-sensitive protein phosphatase (protein phosphatase 2A) activity, without changes in Mg2+-dependent protein phosphatase (protein phosphatase 2C) activity. This decrease in protein phosphatase 2A activity was prevented when LTP induction was inhibited by treatment with calmidazolium or D-2-amino-5-phosphonopentanoic acid. In addition, the application of high-frequency stimulation to 32P-labeled hippocampal slices resulted in increases in the phosphorylation of a 55-kDa protein immunoprecipitated with anti-phosphatase 2A antibodies. Use of a specific antibody revealed that the 55-kDa protein is the B'alpha subunit of protein phosphatase 2A. Following purification of brain protein phosphatase 2A, the B'alpha subunit was phosphorylated by CaM kinase II, an event that led to the reduction of protein phosphatase 2A activity. These results suggest that the decreased activity in protein phosphatase 2A following LTP induction contributes to the maintenance of constitutively active CaM kinase II and to the long-lasting increase in phosphorylation of synaptic components implicated in LTP.     

Inhibition of Endogenous Phosphatase in a Postsynaptic Density Fraction Allows Extensive Phosphorylation of the Major Postsynaptic Density Protein

Abstract: 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 a 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.     

Protein Phosphatase 2A Is the Major Enzyme in Brain that Dephosphorylates τ Protein Phosphorylated by Proline-Directed Protein Kinases or Cyclic AMP-Dependent Protein Kinase

Abstract: The paired helical filament (PHF), which makes up the major fibrous component of the neurofibrillary lesions of Alzheimer's disease, is composed of hyperphosphorylated and abnormally phosphorylated microtubule-associated protein τ. Previous studies have identified serine and threonine residues phosphorylated in PHF-τ and have shown that τ can be phosphorylated at several of these sites by proline-directed protein kinases and cyclic AMP-dependent protein kinase. Here we have investigated which protein phosphatase activities can dephosphorylate recombinant τ phosphorylated with mitogen-activated protein kinase, glycogen synthase kinase-3β, neuronal cdc2-like kinase, or cyclic AMP-dependent protein kinase. We show that protein phosphatase 2A is by far the major protein phosphatase activity in brain that dephosphorylates τ phosphorylated in this manner.     

Kinetics of Protein Phosphorylation in Microvessels Isolated from Rat Brain: Modulation by Second Messengers

The role of second messengers in the regulation of protein phosphorylation was studied in microvessels isolated from rat cerebral cortex. The phosphoproteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the kinetics of 32P incorporation into specific protein substrates were evaluated by computer-aided x-ray film densitometry. With the use of this method, Ca2+-calmodulin (CAM)-, Ca2+/phospholipid (PK C)-, cyclic GMP (cGMP)-, and cyclic AMP (cAMP)-dependent protein kinases were detected. CAM-dependent protein kinase proved to be the major phosphorylating enzyme in the microvascular fraction of the rat cerebral cortex; the activity of cGMP-dependent protein kinase was much higher than that of the cAMP-dependent one. Autophosphorylation of both the alpha- and beta-subunits of CAM-dependent protein kinase and the proteolytic fragment of the PK C enzyme was also detected. The kinetics of phosphorylation of the individual polypeptides indicate the presence in the cerebral endothelium of phosphoprotein phosphatases. The phosphorylation of proteins in the cerebral capillaries was more or less reversible; the addition of second messengers initiated a very rapid increase in 32P incorporation, followed by a slow decrease. Because the intracellular signal transducers like Ca2+ and cyclic nucleotides are frequently regulated by different vasoactive substances in the endothelial cells, the modified phosphorylation evoked by these second messengers may be related in vivo to certain changes in the transport processes of the blood-brain barrier.     

Calcium/Calmodulin-Dependent Protein Kinase II in Squid Synaptosomes

The Ca2+/calmodulin (CaM)-dependent protein kinase II system in squid nervous tissue was investigated. The Ca2+/CaM-dependent protein kinase II was found to be very active in the synaptosome preparation from optic lobe, where it was associated with the high-speed particulate fraction. Incubation of the synaptosomal homogenate with calcium, calmodulin, magnesium, and ATP resulted in partial and reversible conversion of the Ca2+/CaM-dependent protein kinase II from its calcium-dependent form to a calcium-independent species. The magnitude of this conversion reaction could be increased by inclusion of the protein phosphatase inhibitor NaF or by substitution of adenosine 5'-O-(3-thiotriphosphate) for ATP. When [gamma-32P]ATP was used, proteins of 54 and 58 kilodaltons (kDa) as well as proteins greater than 100 kDa were rapidly 32P-labeled in a calcium-dependent manner. Major 125I-CaM binding proteins in the synaptosome membrane fraction were 38 and 54 kDa. The Ca2+/CaM-dependent protein kinase II was purified from the squid synaptosome and was shown to consist of 54- and 58-60-kDa subunits. The purified kinase, like Ca2+/CaM-dependent protein kinase II from rat brain, catalyzed autophosphorylation associated with formation of the calcium-independent form. These studies, characterizing the Ca2+/CaM-dependent protein kinase II in squid neural tissue, are supportive of the putative role of this kinase in regulating calcium-dependent synaptic functions.     

Phosphatase Resistance of ERK2 Brain Kinase PK40erk2

Abstract: We have previously shown that a brain protein kinase, termed PK40, catalyzes the multiple phosphorylation of the KSP-repeat site of neurofilaments (NFs) and also can transform τ proteins into the paired helical filament-like state as found in Alzheimer's disease (AD) brains. Protein sequence analysis suggests that PK40 is a form of the extracellular signal-regulated kinase ERK2. A subpopulation of ERK2 species in soluble brain fractions can be efficiently phosphorylated and activated in cell-free systems, simply by adding Mg²⁺-ATP. Two phosphoisoforms of PK40^erk2 are formed in this process, which have a reduced gel mobility, very much like the ERK2 form obtained in cell culture by stimulation with growth factors. One of these low-mobility forms cannot be inactivated with protein phosphatase 2A (PP2A) or with tyrosine phosphatases. The second form can be slowly inactivated by PP2A. In this case two Ser/Thr phosphates are removed at different rates during inactivation: One phosphate is very quickly removed to result in the formation of a high-mobility 39-kDa ERK2 species without consequence for activity; the other, slowly removed Ser/Thr phosphate controls the activity but has no effect on the gel mobility of ERK2. These results show that forms of ERK2 exist with properties different from the previously characterized ERK2 (p42^mapk) from stimulated cell cultures. The active ERK2 forms produced in the presence of Mg²⁺-ATP alone could provide an explanation for the existence of constitutive ERK2-like NF phosphorylation in vivo. Excessive formation of an ERK2 species resistant to inactivation by PP2A might be relevant to the persistent pathological τ hyperphosphorylation in AD.     

Coordination of Protein Phosphorylation and Dephosphorylation in Synaptic Plasticity

A central theme in nervous system function is equilibrium: synaptic strengths wax and wane, neuronal firing rates adjust up and down, and neural circuits balance excitation with inhibition. This push/pull regulatory theme carries through to the molecular level at excitatory synapses, where protein function is controlled through phosphorylation and dephosphorylation by kinases and phosphatases. However, these opposing enzymatic activities are only part of the equation as scaffolding interactions and assembly of multi-protein complexes are further required for efficient, localized synaptic signaling. This review will focus on coordination of postsynaptic serine/threonine kinase and phosphatase signaling by scaffold proteins during synaptic plasticity.     

Persistent Translocation and Inhibition of Ca2+/Calmodulin-Dependent Protein Kinase II in the Crude Synaptosomal Fraction of the Vulnerable Hippocampus Following Hypoglycemia

Abstract: Alterations in the levels and activity of Ca²⁺/calmodulin-dependent protein kinase II (CaM-kinase II) were studied in the rat hippocampus during and after insulin-induced hypoglycemic coma. A permanent loss of CaM-kinase II immunohistostaining in the neuronal layer begins at 10 min of isoelectricity in the tip of the dentate gyrus and at 30-min isoelectricity in the CA1 region. The reduction in immunohistostaining in the neurites is less pronounced. Immunoreactivity of CaM-kinase II on western blots increases in the crude synaptosomal fractions and decreases in cytosolic fraction, indicative of a translocation of CaM-kinase II. The translocation persists for at least 1 day of recovery after 30 min of isoelectricity in the vulnerable hippocampus (dorsomedial hippocampus) but not in the resistant hippocampus (dorsolateral hippocampus). Calmodulin binding to western blots shows changes similar to the immunoblots. Ca²⁺/calmodulin-dependent activity of CaM-kinase II in the crude synaptosomal fraction is elevated immediately before isoelectricity and is then inhibited during and after 30 min of isoelectricity, despite the increase of CaM-kinase II immunoreactivity. This was seen in the vulnerable hippocampus. The data indicate that stimulus of translocation and inhibition of CaM-kinase II persist during the recovery phase, preceding neuronal degeneration in the vulnerable hippocampus. This may be of significance for hypoglycemia-induced neuronal death.     

Staurosporine: An Effective Inhibitor for Ca2+/Calmodulin-Dependent Protein Kinase II

We investigated the effect of staurosporine on Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) purified from rat brain. (a) Staurosporine (10-100 nM) inhibited the activity of CaM kinase II. The half-maximal and maximal inhibitory concentrations were 20 and 100 nM, respectively. (b) The inhibition with staurosporine was of the noncompetitive type with respect to ATP, calmodulin, and phosphate acceptor (beta-casein). (c) Staurosporine suppressed the auto-phosphorylation of alpha- and beta-subunits of CaM kinase II at concentrations similar to those at which the enzyme activity was inhibited. (d) Staurosporine also attenuated the Ca2+/calmodulin-independent activity of the autophosphorylated CaM kinase II. These results suggest that staurosporine inhibits CaM kinase II by interacting with the catalytic domain, distinct from the ATP-binding site or substrate-binding site, of the enzyme and that staurosporine is an effective inhibitor for CaM kinase II in the cell system.     

Ca2+– and Calmodulin-Dependent Phosphorylation of Microtubule-Associated Protein 2 and t Factor, and Inhibition of Microtubule Assembly

Microtubule-associated proteins (MAPs) were phosphorylated by a Ca2+- and calmodulin-dependent protein kinase from rat brain cytosol. The maximal amount of phosphate incorporated into MAPs was 25 nmol of phosphate/mg protein. A Ka value of the enzyme for calmodulin was 57.0 nM, with MAPs as substrates. Among MAPs, MAP2 and tau factor were phosphorylated in a Ca2+- and calmodulin-dependent manner. The phosphorylation of MAPs led to an inhibition of microtubule assembly in accordance with its degree. This reaction was dependent on addition of the enzyme, Ca2+, and calmodulin, and had a greater effect on the initial rate of microtubule assembly rather than on the final extent. The critical tubulin concentration for microtubule assembly was unchanged by the MAPs phosphorylation. Therefore assembly and disassembly of brain microtubule are regulated by the Ca2+- and calmodulin-dependent protein kinase that requires only a nanomolar concentration of calmodulin for activation.     

Activation of Ca2+/Calmodulin-Dependent Protein Kinase II by Extracellular Calcium in Cultured Hippocampal Pyramidal Neurons

Abstract: The ability of various stimuli to convert Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) into a Ca²⁺-independent (autonomous) form was examined in cultured embryonic rat hippocampal pyramidal neurons. The most effective stimulation by far was observed when cells were equilibrated in buffer containing low extracellular [Ca²⁺] ([Ca²⁺]_o) (～50 nM) and then shifted to normal [Ca²⁺]_o (～1.26 mM) by addition of CaCl₂ (referred to as “Ca²⁺ stimulation”). Virtually complete (>90%) conversion of the kinase to the autonomous form occurred within 30–50 s, with a return to baseline within 5 min. By contrast, depolarization of cells with high [K⁺] or treatment with glutamate or a Ca²⁺ ionophore caused insignificant increases (<10%) in levels of the autonomous form. The Ca²⁺-stimulated increase in CaMKII autonomy coincided with a two- to threefold increase in kinase subunit phosphorylation. In the first 40 s of Ca²⁺ stimulation, ³²P incorporation into the immunoprecipitated subunits of CaMKII occurred exclusively on threonine residues, including Thr²⁸⁶Thr²⁸⁷ of the α/β subunits. Longer incubation of cells resulted in a decline of phosphothreonine content, whereas levels of phosphoserine-containing peptides showed a significant increase. The activation of CaMKII by Ca²⁺ stimulation was accompanied by only a small rise in intracellular [Ca²⁺]. Inhibitor studies showed that Na⁺-dependent action potentials and Ca²⁺ influx through glutamate receptors or voltage-sensitive Ca²⁺ channels did not contribute to the activation. Moreover, CaMKII was not activated by extracellular addition of other cations, including Mn²⁺, Mg²⁺, Co²⁺, or Gd³⁺. Although the mechanism of Ca²⁺ stimulation is presently unclear, it may involve either activation of extracellular calcium receptors or capacitative calcium entry. The dramatic rise in CaMKII autonomy and the Ca²⁺ selectivity of the response suggest a direct and specific relationship between [Ca²⁺]_o and the state of activation of the kinase in intact neurons.     

Tyrosine Hydroxylase Dephosphorylation by Protein Phosphatase 2A in Bovine Adrenal Chromaffin Cells

This study was undertaken to characterise the protein phosphatases in bovine adrenal chromaffin cells acting on tyrosine hydroxylase. Cells were pre-labelled with ³²P_i and permeabilized with digitonin. The extent of dephosphorylation of Ser-8, Ser-19, Ser-31 and Ser-40 on tyrosine hydroxylase was found to be 30%, 38%, 37% and 71% respectively over 5 min. For Ser-19, Ser-31 and Ser-40 the dephosphorylation was entirely due to protein phosphatase 2A, as the dephosphorylation could be completely blocked by microcystin, but not by the protein phosphatase 1 inhibitory peptide. Permeabilization did not change the distribution of protein phosphatase 2A or tyrosine hydroxylase, or the activity of PP2A, from that occurring in intact cells. The dephosphorylation of Ser-8 was not altered by any inhibitor, suggesting the involvement of other protein phosphatases. The method developed here can be used to determine the protein phosphatases acting on substrates in conditions closely approximating those in situ, including the endogenous state of substrate phosphorylation and phosphatase location.     

Widespread Distribution of the 14-3-3 Protein in Vertebrate Brains and Bovine Tissues: Correlation with the Distributions of Calcium-Dependent Protein Kinases

A highly specific antiserum was prepared against bovine brain 14-3-3 protein, a protein kinase-dependent activator of tyrosine and tryptophan hydroxylases. The immunoassay using this antiserum proved the presence of 14-3-3 protein in various bovine tissues and in brains of various vertebrate species. The quantitative analysis indicated that the tissue distribution of 14-3-3 protein is more closely related to the known distributions of the Ca2(+)-dependent protein kinases, i.e., Ca2+/calmodulin- and Ca2+/phospholipid-dependent protein kinases, rather than those of tyrosine and tryptophan hydroxylases. This result, together with the available data on this protein, suggests potential roles of the 14-3-3 protein in more diverse kinase-mediated processes than the predicted role in monoamine synthesis.     

Differential Activities of Protein Phosphatase Types 1 and 2A in Cytosolic and Participate Fractions from Rat Forebrain

Abstract: The activities and concentrations of protein phosphates type 1 (PP1) and type 2A (PP2A) were compared in cytosol and particulate fractions of rat forebrain. Although the activity of PP2A was highest in the cytosol, immunoblot analysis with a PP2A-specific antibody showed that there were significant levels of the enzyme in the particulate fraction. There was no significant difference between the concentration of PP2A in the cytosol and particulate fractions such that the low activity of PP2A in the particulate fraction represents an inactivation of this form of the enzyme. Similar analysis in skeletal muscle, heart, and liver showed this finding was unique to the brain. Similarly, the majority of PP1 activity was recovered in the cytosol, but most PP1 enzyme was associated with the particulate fraction. Comparison with other tissues showed that the activities of PP1 in the particulate fractions were similar but that the forebrain contained significantly more enzyme than the other tissues. Thus, like PP2A it appears that the specific activity of PP1 in the particulate fraction of rat forebrain is much lower than that of the cytosol and of the particulate fractions of other tissues. Elution of PP1 and PP2A from membranes with 0.5 M NaCl plus 0.3% Triton X-100 resulted in severalfold activation of both enzymes. That the majority of PP1 and PP2A in rat forebrain are associated with membrane structures but in a low activity state suggests that novel regulatory mechanisms exist that have considerable and unique potential for activation of protein dephosphorylation.     

Spinophilin is phosphorylated by Ca2+/calmodulin-dependent protein kinase II resulting in regulation of its binding to F-actin

Spinophilin is a protein phosphatase-1- and actin-binding protein that modulates excitatory synaptic transmission and dendritic spine morphology. We have recently shown that the interaction of spinophilin with the actin cytoskeleton depends upon phosphorylation by protein kinase A. We have now found that spinophilin is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in neurons. Ca(2+)/calmodulin-dependent protein kinase II, located within the post-synaptic density of dendritic spines, is known to play a role in synaptic plasticity and is ideally positioned to regulate spinophilin. Using tryptic phosphopeptide mapping, site-directed mutagenesis and microsequencing analysis, we identified two sites of CaMKII phosphorylation (Ser-100 and Ser-116) within the actin-binding domain of spinophilin. Phosphorylation by CaMKII reduced the affinity of spinophilin for F-actin. In neurons, phosphorylation at Ser-100 by CaMKII was Ca(2+) dependent and was associated with an enrichment of spinophilin in the synaptic plasma membrane fraction. These results indicate that spinophilin is phosphorylated by multiple kinases in vivo and that differential phosphorylation may target spinophilin to specific locations within dendritic spines.