Antagonistic roles of neurofilament subunits NF-H and NF-M against NF-L in shaping dendritic arborization in spinal motor neurons

Dendrites play important roles in neuronal function. However, the cellular mechanism for the growth and maintenance of dendritic arborization is unclear. Neurofilaments (NFs), a major component of the neuronal cytoskeleton, are composed of three polypeptide subunits, NF-H, NF-M, and NF-L, and are abundant in large dendritic trees. By overexpressing each of the three NF subunits in transgenic mice, we altered subunit composition and found that increasing NF-H and/or NF-M inhibited dendritic arborization, whereas increasing NF-L alleviated this inhibition. Examination of cytoskeletal organization revealed that increasing NF-H and/or NF-M caused NF aggregation and dissociation of the NF network from the microtubule (MT) network. Increasing NF-H or NF-H together with NF-M further reduced NFs from dendrites. However, these changes were reversed by elevating the level of NF-L with either NF-H or NF-M. Thus, NF-L antagonizes NF-H and NF-M in organizing the NF network and maintaining a lower ratio of NF-H and NF-M to NF-L is critical for the growth of complex dendritic trees in motor neurons.     

Phosphorylation of myristoylated alanine-rich protein kinase C substrate by mitogen-activated protein kinase in cultured rat hippocampal neurons following stimulation of glutamate receptors

Glutamate-induced phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) was investigated in cultured rat hippocampal neurons. In 32P-labeled hippocampal neurons, exposure to 10 microM glutamate induced a long lasting increase in phosphorylation of MARCKS. The long lasting increase in MARCKS phosphorylation mainly required activation of the N-methyl-D-aspartate receptor. Unexpectatively, the MARCKS phosphorylation after the 10-min incubation with glutamate was not inhibited by treatment with calphostin C, a potent inhibitor for protein kinase C (PKC), or down-regulation of PKC but was largely prevented by PD098059, a selective inhibitor for mitogen-activated protein (MAP) kinase kinase. In contrast, the phosphorylation following the short exposure to glutamate was prevented by a combination of PD098059 and calphostin C. The phosphopeptide mapping and immunoblotting analyses confirmed that PKC-dependent phosphorylation of MARCKS was transient and the MAP kinase-dependent phosphorylation was relatively persistent. Investigations of the functional properties also showed that the MARCKS phosphorylation by MAP kinase regulates its calmodulin-binding ability and its interaction with F-actin as seen in the PKC-dependent phosphorylation. These results suggest that glutamate causes a long lasting increase in MARCKS phosphorylation through activation of the N-methyl-D-aspartate receptor and subsequent activation of MAP kinase in the hippocampal neurons.     

Change of ganglioside accessibility at the plasma membrane surface of cultured neurons, following protein kinase C activation

While the mechanism of signal transduction across the plasma membrane from the exo- to the endoplasmic side has been extensively investigated, the possible return of messages back to the outer layer is less known. We studied the effect of protein kinase C activation on the ganglioside accessibility at the exoplasmic face of intact rat cerebellar granule cells in culture, using the enzyme sialidase as the probing molecule. Under the experimental conditions (1 milliunit/mL enzyme, 2 min incubation at 37 degreesC), only GT1b and GD1a gangliosides were partially affected by the enzyme (28.6 and 25.7% hydrolysis, respectively). After cell treatment with phorbol 12-myristate 13-acetate, inducing protein kinase C activation, GT1b and GD1a ganglioside susceptibility to sialidase was strongly decreased (8.6 and 15.9% hydrolysis, respectively). A reduction of ganglioside hydrolysis was also observed when protein kinase C activation was induced by cell treatment for 15 min with 100 microM glutamate. On the contrary, accessibility did not vary when protein kinase C translocation was not effective (either in the absence of Ca2+ in the medium or using 1 microM glutamate) or when the kinase activity was inhibited by staurosporine. These data suggest that following PKC activation, a key step of inbound transmembrane signaling, cell may dispatch outbound messages to the plasma membrane outer layer, changing the selective recognition and crypticity of glycolipids at the cell surface, possibly through a modulation of their segregation state.     

Calcium-evoked dendritic exocytosis in cultured hippocampal neurons. Part II: mediation by calcium/calmodulin-dependent protein kinase II

Calcium-evoked dendritic exocytosis (CEDE), demonstrated in cultured hippocampal neurons, is a novel mechanism that could play a role in synaptic plasticity. A number of forms of neuronal plasticity are thought to be mediated by calcium/calmodulin-dependent protein kinase II (CaMKII). Here, we investigate the role of CaMKII in CEDE. We find that the developmental time course of CEDE parallels the expression of alphaCaMKII, a dominant subunit of CaMKII. An inhibitor of this enzyme, KN-62, blocks CEDE. Furthermore, 7 d in vitro neurons (which normally do not express alphaCaMKII nor show CEDE) can undergo CEDE when infected with a recombinant virus producing alphaCaMKII. Expression of a constitutively active CaMKII produces dendritic exocytosis in the absence of calcium stimulus, and this exocytosis is blocked by nocodazole, an inhibitor of microtubule polymerization that also blocks CEDE. These results indicate that CEDE is mediated by the activation of CaMKII, consistent with the view that CEDE plays a role in synaptic plasticity.     

Activation of protein kinase C triggers its ubiquitination and degradation

Treatment of cells with tumor-promoting phorbol esters results in the activation but then depletion of phorbol ester-responsive protein kinase C (PKC) isoforms. The ubiquitin-proteasome pathway has been implicated in regulating the levels of many cellular proteins, including those involved in cell cycle control. We report here that in 3Y1 rat fibroblasts, proteasome inhibitors prevent the depletion of PKC isoforms alpha, delta, and epsilon in response to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Proteasome inhibitors also blocked the tumor-promoting effects of TPA on 3Y1 cells overexpressing c-Src, which results from the depletion of PKC delta. Consistent with the involvement of the ubiquitin-proteasome pathway in the degradation of PKC isoforms, ubiquitinated PKC alpha, delta, and epsilon were detected within 30 min of TPA treatment. Diacylglycerol, the physiological activator of PKC, also stimulated ubiquitination and degradation of PKC, suggesting that ubiquitination is a physiological response to PKC activation. Compounds that inhibit activation of PKC prevented both TPA- and diacylglycerol-induced PKC depletion and ubiquitination. Moreover, a kinase-dead ATP-binding mutant of PKC alpha could not be depleted by TPA treatment. These data are consistent with a suicide model whereby activation of PKC triggers its own degradation via the ubiquitin-proteasome pathway.     

Activation of protein kinase C delta by the c-Abl tyrosine kinase in response to ionizing radiation

The c-Abl protein tyrosine kinase is activated by ionizing radiation (IR) and certain other DNA-damaging agents. The present studies demonstrate that c-Abl associates constitutively with protein kinase C delta (PKCdelta). The results show that the SH3 domain of c-Abl interacts directly with PKCdelta. c-Abl phosphorylates and activates PKCdelta in vitro. We also show that IR treatment of cells is associated with c-Abl-dependent phosphorylation of PKCdelta and translocation of PKCdelta to the nucleus. These findings support a functional interaction between c-Abl and PKCdelta in the cellular response to genotoxic stress.     

Regulation of L-type calcium channels by protein tyrosine kinase and protein kinase C in cultured rat and human retinal pigment epithelial cells

The effect of protein tyrosine kinases (PTKs) on L-type calcium channel currents was studied in cultured rat and human retinal pigment epithelial cells. Barium currents through L-type channels were measured in the perforated patch-clamp technique and identified by using the L-type calcium channel opener Bay K8644 (10(-6) M). Application of the PTK blockers genistein (5 x 10(-6) M) or lavendustin A (5 x 10(-6) M) led to a decrease of L-type currents. The inactive genistein analog daidzein (10(-5) M) showed no effect on calcium channels. Intracellular application of pp60(c-src) (30 U/ml) via the patch-pipette during the conventional whole-cell configuration led to an increase of L-type currents. The protein kinase A and protein kinase G blocker H9 (10(-6) M) showed no effect on L-type currents; genistein reduced the current in the presence of H9. The protein kinase C (PKC) blocker chelerythrine (10(-5) M) reduced the L-type current; additional inhibition of PTK by lavendustin showed an additional reduction of currents. Intracellular application of myristoylated PKC substrate (5 x 10(-5) M) for PKC inhibition led to a fast rundown of L-type current amplitudes. Intracellularly applied myristoylated PKC substrate (10(-4) M) together with pp60(c-src) showed no effect on L-type current. Up-regulation of PKC by 10(-6) M phorbol-12-myristate-13-acetate (PMA) had no effect on the L-type current amplitude. However, genistein in cells pretreated with PMA led to an increase of the L-type currents. Intracellular application of pp60(c-src) in PMA-treated cells led to a reduction of L-type currents. We conclude that in the resting cell, PTK and PKC regulate L-type calcium channels in an additive manner. L-type channels appeared as a site of integration of PTK activation and of PKC-dependent pathways. The activity of PKC determines whether PTK decreases or increases L-type channel activity.     

Isoform-specific translocation of protein kinase C following glutamate administration in primary hippocampal neurons

High concentrations of glutamate, the major excitatory neurotransmitter in the mammalian brain, lead to intracellular calcium overload resulting in excitotoxic damage and death of neurons. Since protein kinase C (PKC) is involved in neuronal degeneration resulting from cerebral ischemia and from glutamate excitotoxicity, we investigated the effect of glutamate on changes in the cellular distribution of various PKC isoforms in cultured hippocampal neurons in comparison with the effects elicited by the PKC activator phorbol ester. Out of the expressed PKC isoforms alpha, gamma, epsilon, zeta and lambda only the conventional isoforms PKC alpha and gamma responded to glutamate. Using subcellular fractionation and Western blotting with isoform-specific antibodies and immunocytochemical localization with confocal laser scanning microscopy, we observed that phorbol ester and glutamate have different effects on PKC isoform redistribution: Whereas phorbol ester resulted in translocation of PKC alpha and PKC gamma toward a membrane fraction, the glutamate-mediated rise in intracellular calcium concentration induced a translocation mainly toward a detergent-insoluble, cytoskeletal fraction. Immunocytochemical analysis revealed an isoform-specific translocation following glutamate treatment: PKC gamma was translocated mainly to cytoplasmic, organelle-like structures, whereas PKC alpha redistributed to the plasma membrane and into the cell nucleus. The latter result is of special interest, as it indicates that nuclear PKC may play a role in processes of excitotoxic cell damage.     

Activation of protein kinase C by tumor necrosis factor-alpha in human non-pigmented ciliary epithelium

Previously, we have shown that tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, increases the synthesis and release of endothelin-1 (ET-1), a potent vasoactive peptide from human non-pigmented ciliary epithelial (HNPE) cells, in a protein kinase C (PKC)-dependent manner. Diacylglycerol (DAG) and intracellular calcium ([Ca2+]i) are well known activators of PKC. Some cytokines induce PKC activation by stimulating phospholipase C that hydrolyzes phosphatidylinositol bisphosphate (PIP2) into IP3 (intracellular calcium mobilizer) and DAG. In this study, the existence of a similar pathway was evaluated in HNPE cells treated with TNF-alpha, using intracellular calcium ([Ca2+]i) measurements, PKC translocation assays and thin-layer chromatography (TLC) for quantification of DAG. Incubation times for agonists and inhibitors ranged from 1-30 minutes. The increase in DAG levels with TNF-alpha treatment was consistent with the observed translocation of the calcium-dependent PKC alpha isoform from the cytosol to the plasma membrane. However, these observations were not accompanied by a concomitant increase in [Ca2+]i. Similar translocation responses were observed with phorbol ester (phorbol 12-myristate 13-acetate) treatment. Our results indicate that TNF-alpha-induced PKC activation in HNPE cells occurs as a result of elevated DAG levels and is not due to an increase in intracellular calcium. Activated PKC, could enhance the pro-inflammatory responses of TNF-alpha in part by increasing the production of endothelins in the eye.     

Activation of protein kinase C induces cortical granule exocytosis in a Ca(2+)-independent manner, but not the resumption of cell cycle in porcine eggs

N-Acetylneuraminic acid (1) is a common sugar in many biological recognition processes. Neuraminidase enzymes recognize and cleave terminal sialic acids from cell surfaces. Viral entry into host cells requires neuraminidase activity, thus inhibition of neuraminidase is a useful strategy for development of drugs for viral infections. A recent crystal structure for influenza viral neuraminidase with sialic acid bound shows that the sialic acid is in a boat conformation [Prot Struct Funct Genet 14: 327 (1992)]. Our studies seek to determine if structural pre-organization can be achieved through the use of sialyllactones. Determination of whether siallylactones are pre-organized in a binding conformation requires conformational analysis. Our inability to find a systematic study comparing the results obtained by various computational methods for carbohydrate modeling led us to compare two different conformational analysis techniques, four different force fields, and three different solvent models. The computational models were compared based on their ability to reproduce experimental coupling constants for sialic acid, sialyl-1,4-lactone, and sialyl-1,7-lactone derivatives. This study has shown that the MM3 forcefield using the implicit solvent model for water implemented in Macromodel best reproduces the experimental coupling constants. The low-energy conformations generated by this combination of computational methods are pre-organized toward conformations which fit well into the active site of neuraminidase.     

Dopamine fails to stimulate protein kinase C activity in renal proximal tubules of spontaneously hypertensive rats

We have previously reported that dopamine-1 receptor-mediated activation of phospholipase C is diminished in renal cortical slices of spontaneously hypertensive rats. The present study was carried out to examine the effect of dopamine on protein kinase C (PKC), which is one of the enzymes involved in the signal-transduction pathway leading to dopamine-induced inhibition of Na+/K(+)-ATPase in the renal proximal tubule. Renal proximal tubule suspensions were obtained from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats of 10-12 weeks old. The tubules were incubated with dopamine in the presence or absence of DA-1 receptor antagonist SCH 23390. The PKC activity was measured by using a specific fluorescent peptide substrate (sequence, PKSRTLSVAAK). We found that dopamine produced a concentration-dependent increase in protein kinase C activity in the WKY rats, however, it failed to stimulate PKC activity in the SHR. Peak stimulation of 3.828 +/- 0.35 (ng/micrograms) protein in the WKY rats was observed at dopamine concentration of 1 microM, which was blocked in a concentration-dependent manner by SCH 23390 (0.25 microM). These results provide evidence that dopamine directly stimulates PKC activity via activation of DA-1 receptors in WKY rats. Furthermore, we discovered that dopamine fails to stimulate PKC activity in the SHR. This phenomenon may be responsible for the failure of dopamine to inhibit Na+/K(+)-ATPase activity in the hypertensive animals.     

Modulation of AMPA/kainate receptors in cultured murine hippocampal neurones by protein kinase C

1. The patch clamp technique, together with intracellular perfusion of the catalytic fragment of protein kinase C (PKCM), was employed to investigate the role of this enzyme in the intracellular regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptors in cultured hippocampal neurones. 2. The responses evoked by near-maximal concentrations of kainate (250 microM) and AMPA (100 microM) were potentiated by the introduction of PKCM, whilst co-application of the inhibitory peptide fragment PKCI(19-36) prevented this action. 3. Modulation of kainate responses by PKCM was dependent upon the concentration of agonist applied. Currents evoked by kainate were potentiated at concentrations above those which caused 50% of the maximal response (EC50) and depressed at lower concentrations. Furthermore, okadaic acid, a specific inhibitor of phosphatases 1 and 2A, had a similar effect upon concentration-response relationships when currents activated by kainate were recorded using the perforated patch technique. 4. In addition, the mean amplitude and/or time constant of decay of miniature excitatory synaptic currents (mediated by AMPA/kainate receptors) was increased by the intracellular injection of PKCM. 5. These observations suggest that the function of postsynaptic excitatory amino acid receptors can be modulated by the activity of PKC as well as by endogenous phosphatases. This regulation may contribute to some forms of synaptic plasticity within the central nervous system.     

Differential expression of MARCKS and other calmodulin-binding protein kinase C substrates in cultured neuroblastoma and glioma cells

Expression of the protein kinase C substrate MARCKS and other heat-stable myristoylated proteins have been studied in four cultured neural cell lines. Amounts of MARCKS protein, measured by [3H]myristate labeling and western blotting, were severalfold higher in rat C6 glioma and human HTB-11 (SK-N-SH) neuroblastoma cells than in HTB-10 (SK-N-MC) or mouse N1E-115 neuroblastoma cells. Higher levels of MARCKS mRNA were also detected in the former cell lines by S1 nuclease protection assay. At least two additional 3H-myristoylated proteins of 50 and 40-45 kDa were observed in cell extracts heated to > 80 degrees C or treated with perchloric acid. The 50-kDa protein, which bound to calmodulin in the presence of Ca2+, was more prominent in cells (N1E-115 and HTB-10) with less MARCKS, whereas neuromodulin (GAP-43) was detected in N1E-115 and HTB-11 cells only. Heating resulted in a fourfold increase in the detection of MARCKS by western blotting; this was not paralleled by a similar increase in [3H]myristate-labeled MARCKS and may be due to a conformational change affecting the C-terminal epitope or enhanced rechange of the protein on nitrocellulose. Addition of beta-12-O-tetradecanoylphorbol 13-acetate resulted in three- to fourfold increased phosphorylation of MARCKS in HTB-11 cells, with little increase noted in HTB-10 cells. These results indicate that MARCKS, neuromodulin, and other calmodulin-binding protein kinase C substrates exhibit distinct levels of expression in cultured neurotumor cell lines. Of these proteins, only MARCKS appears to be correlated with phorbol ester stimulation of phosphatidylcholine turnover in these cells.     

Dysfunction of nitric oxide induces protein kinase C activation resulting in vasospasm after subarachnoid hemorrhage

We hypothesize that the interaction between protein kinase C (PKC) and nitric oxide (NO) plays a role in the modulation of cerebral vascular tone, and the disturbance of this interaction following subarachnoid hemorrhage (SAH) results in vasospasm. To prove this hypothesis with direct evidence, PKC activities of smooth muscle cells of canine basilar arteries in the control and in the SAH groups were measured by an enzyme immunoassay method. N omega-nitro-L arginine (L-NA), an inhibitor of NO production, enhanced PKC activity. This enhancement was inhibited neither by 8-bromo-guanosine 3',5'-cyclic monophosphate (8-bromo-cGMP) nor SIN-1, a NO releasing agent. PKC activity in the SAH was significantly higher than in the control; however, no further enhancement was produced with L-NA. In the SAH, PKC activity was not inhibited either by 8-bromo-cGMP or SIN-1. We conclude that NO maintains an appropriate vascular tone through inactivation of PKC, and that this effect is disturbed following SAH, resulting in PKC-dependent vascular contraction, such as vasospasm. On the other hand, once PKC has been activated, NO precursors do not inhibit PKC. These facts indicate NO inactivates PKC through the inhibition of phosphatidylinositol breakdown.     

Activation of the mitogen-activated protein kinase pathway in U937 leukemic cells induces phosphorylation of the amino terminus of the TATA-binding protein

Quality-adjusted life-years (QALYs) and willingness to pay (WTP) are two preference-based measures of health-related outcomes. In this article, we compare these two measures in eliciting individuals' preferences for health outcomes associated with shingles. To collect the necessary preference data, we administered computer-interactive interviews to a sample of 65- to 70-year-olds. We found no significant correlation between QALYs and WTP across individuals. We discuss our findings and argue that our results raise questions about whether QALYs and WTP are equivalent preference-based measures of health outcomes.     

Modulation by protein kinase C of nitric oxide and cyclic GMP poffation in cultured cerebellar granule cells

The possible modulation of nitric oxide (NO) synthase (NOS) activity by protein kinase C (PKC) was investigated in primary cultures of rat cerebellar neurons. Incubation of the cells with L-arginine and nicotinamide-adenine dinucleotide phosphate (NADPH) produced detectable levels of NO, as quantified by photometric assay [0.14 +/- 0.03 nmol/h/dish (2.5 x 10(6) cells)]. The NO producing activity was paralleled by concomitant accumulation of cyclic GMP (cGMP) (0.12 +/- 0.02 pmol/dish). Downregulation of PKC by prolonged treatment with phorbol esters or inhibition of the kinase by treatment with 4taurosporine raised the basal levels of NO and cGMP five fold. When granule cells were incubated in the absence of extracellular Mg2+, N-methyl-D-aspartate and to a lesser extent, glutamate became effective in enhancing NO formation and cGMP accumulation with respect to the control. The NO and cGMP increases induced by the two agonists were almost doubled by treatment of the cells with staurosporine or depletion of PKC. Calphostin C. an inhibitor of the regulatory domain of PKC, was as effective as staurosporine in increasing the formation of NO in both resting and excited cells. These results indicate that downregulation or inhibition of PKC increase NOS activity in cerebellar neurons, and suggest that phosphorylation of NOS by PKC negatively modulates the catalytic activity of the enzyme in these cells.     

Activation of protein kinase C by intracellular free calcium in the motoneuron cell line NSC-19

Clotrimazole (CLT), a member of the antifungal imidazole family of compounds, has been found to inhibit both calcium (Ca2+)-activated 86Rb and potassium (K) fluxes of human red cells and to inhibit red cell binding of 125I-charybdotoxin (ChTX) [11]. We have now used patch-clamp techniques to demonstrate reversible inhibition of whole cell KCa2+ currents in murine erythroleukemia (MEL) cells by submicromolar concentrations of CLT. Inhibition was equivalent whether currents were elicited by bath application of the Ca2+ ionophore A23187 or by dialyzing cells with a pipette solution containing micromolar concentrations of free Ca2+. The extent of inhibition of whole cell MEL KCa2+ currents was voltage-dependent, decreasing with increasing test potential. We also determined the single channel basis of the CLT inhibition in MEL cells by demonstrating the inhibition of a calcium-activated, ChTX-sensitive K channel by CLT in outside-out patches. The channel was also blocked by the des-imidazolyl metabolite of CLT, 2-chlorophenyl-bisphenyl-methanol (MET II) [15], thus demonstrating that the imidazole ring is not required for the inhibitory action of CLT. Single KCa2+ channels were also evident in inside-out patches of MEL cells. Block of K current by CLT was not unique to MEL cells. CLT also inhibited a component of the whole cell K current in PC12 cells. Channel specificity of block by CLT was determined by examining its effects on other types of voltage-sensitive currents. CLT block showed the following rank order of potency: K currents in PC12 cells > Ca2+ currents in PC12 cells > Na currents in sympathetic neurons. These results demonstrate that direct inhibition of single KCa2+ by CLT can be dissociated from inhibition of cytochrome P-450 in MEL cells.     

Adducin is an in vivo substrate for protein kinase C: phosphorylation in the MARCKS-related domain inhibits activity in promoting spectrin-actin complexes and occurs in many cells, including dendritic spines of neurons

Adducin is a heteromeric protein with subunits containing a COOH-terminal myristoylated alanine-rich C kinase substrate (MARCKS)-related domain that caps and preferentially recruits spectrin to the fast-growing ends of actin filaments. The basic MARCKS-related domain, present in alpha, beta, and gamma adducin subunits, binds calmodulin and contains the major phosphorylation site for protein kinase C (PKC). This report presents the first evidence that phosphorylation of the MARCKS-related domain modifies in vitro and in vivo activities of adducin involving actin and spectrin, and we demonstrate that adducin is a prominent in vivo substrate for PKC or other phorbol 12-myristate 13-acetate (PMA)-activated kinases in multiple cell types, including neurons. PKC phosphorylation of native and recombinant adducin inhibited actin capping measured using pyrene-actin polymerization and abolished activity of adducin in recruiting spectrin to ends and sides of actin filaments. A polyclonal antibody specific to the phosphorylated state of the RTPS-serine, which is the major PKC phosphorylation site in the MARCKS-related domain, was used to evaluate phosphorylation of adducin in cells. Reactivity with phosphoadducin antibody in immunoblots increased twofold in rat hippocampal slices, eight- to ninefold in human embryonal kidney (HEK 293) cells, threefold in MDCK cells, and greater than 10-fold in human erythrocytes after treatments with PMA, but not with forskolin. Thus, the RTPS-serine of adducin is an in vivo phosphorylation site for PKC or other PMA-activated kinases but not for cAMP-dependent protein kinase in a variety of cell types. Physiological consequences of the two PKC phosphorylation sites in the MARCKS-related domain were investigated by stably transfecting MDCK cells with either wild-type or PKC-unphosphorylatable S716A/S726A mutant alpha adducin. The mutant alpha adducin was no longer concentrated at the cell membrane at sites of cell-cell contact, and instead it was distributed as a cytoplasmic punctate pattern. Moreover, the cells expressing the mutant alpha adducin exhibited increased levels of cytoplasmic spectrin, which was colocalized with the mutant alpha adducin in a punctate pattern. Immunofluorescence with the phosphoadducin-specific antibody revealed the RTPS-serine phosphorylation of adducin in postsynaptic areas in the developing rat hippocampus. High levels of the phosphoadducin were detected in the dendritic spines of cultured hippocampal neurons. Spectrin also was a component of dendritic spines, although at distinct sites from the ones containing phosphoadducin. These data demonstrate that adducin is a significant in vivo substrate for PKC or other PMA-activated kinases in a variety of cells, and that phosphorylation of adducin occurs in dendritic spines that are believed to respond to external signals by changes in morphology and reorganization of cytoskeletal structures.     

Activation and interaction with protein kinase C of a cytoplasmic tyrosine kinase, Itk/Tsk/Emt, on Fc epsilon RI cross-linking on mast cells

Cross-linking of the high-affinity IgE receptor (Fc epsilon RI) on mast cells induces rapid phosphorylation on serine, threonine, and tyrosine residues and increases the enzymatic activity, of a Tec subfamily tyrosine kinase, Itk/Tsk/Emt (Emt). The pleckstrin homology domain of Emt at its amino-terminal interacts directly with multiple isoforms of protein kinase C (PKC) in vitro. In addition, a portion of Emt is physically associated with multiple isoforms of PKC in intact mast cells. PKC phosphorylates a bacterial fusion protein containing the pleckstrin homology domain of Emt in vitro. Coexpression of Emt in COS-7 cells with Ca(2+)-dependent PKC isoforms (alpha, beta I, or beta II) induces an enhancement in tyrosine phosphorylation of Emt. In vivo inhibition of PKC expression or activity attenuates tyrosine phosphorylation and enzymatic activity of Emt induced upon Fc epsilon RI cross-linking. These data collectively suggest that PKC phosphorylates Emt and activates its autophosphorylating activity. Alternatively, PKC could activate another tyrosine kinase that phosphorylates Emt, or PKC-mediated phosphorylation of Emt may render it a target for another tyrosine kinase. In any case, PKC appears to play a major role in the activation of Emt induced upon Fc epsilon RI cross-linking.