Cell type-specific upregulation of myristoylated alanine-rich C kinase substrate and protein kinase C-alpha, -beta I, -beta II, and -delta in microglia following kainic acid-induced seizures

Myristoylated alanine-rich C kinase substrate (MARCKS) is a widely distributed protein kinase C (PKC) substrate and has been implicated in actin cytoskeletal rearrangement in response to extracellular stimuli. Although MARCKS was extensively examined in various cell culture systems, the physiological function of MARCKS in the central nervous system has not been clearly understood. We investigated alterations of cellular distribution and phosphorylation of MARCKS in the hippocampus following kainic acid (KA)-induced seizures. KA (25 mg/kg, i.p.) was administered to eight to nine week-old C57BL/6 mice. Behavioral seizure activity was observed for 2 h after the onset of seizures and was terminated with diazepam (8 mg/kg, i.p.). The animals were sacrificed and analyzed at various points in time after the initiation of seizure activity. Using double-labeling immunofluorescence analysis, we demonstrated that the expression and phosphorylation of MARCKS was dramatically upregulated specifically in microglial cells after KA-induced seizures, but not in other types of glial cells. PKC alpha, beta I, beta II and delta, from various PKC isoforms examined, also were markedly upregulated, specifically in microglial cells. Moreover, immunoreactivities of phosphorylated MARCKS were co-localized in the activated microglia with those of the above isoforms of PKC. Taken together, our in vivo data suggest that MARCKS is closely linked to microglial activation processes, which are important in pathological conditions, such as neuroinflammation and neurodegeneration.     

Identification of single and double sites of phosphorylation by ECD FT-ICR/MS in peptides related to the phosphorylation site domain of the myristoylated alanine-rich c kinase protein

A series of phosphorylated test peptides was studied by electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD FT-ICR MS). The extensive ECD-induced fragmentation made identification of phosphorylation sites for these peptides straightforward. The site(s) of initial phosphorylation of a synthetic peptide with a sequence identical to that of the phosphorylation site domain (PSD) of the myristoylated alanine-rich C kinase (MARCKS) protein was then determined. Despite success in analyzing fragmentation of the smaller test peptides, a unique site on the PSD for the first step of phosphorylation could not be identified because the phosphorylation reaction produced a heterogeneous mixture of products. Some molecules were phosphorylated on the serine closest to the N-terminus, and others on one of the two serines closest to the C-terminus of the peptide. Although no definitive evidence for phosphorylation on either of the remaining two serines in the PSD was found, modification there could not be ruled out by the ECD fragmentation data.     

Involvement of protein kinase C pathway in UVC-stimulated phospholipase D2 activity in Vero 76 cells

Phospholipase D (PLD) activity is known to be related to oxidant-induced cellular signaling and membrane disturbance. Previously, an induction of PLD activity in various cell lines by X-ray irradiation was observed. In this study, we examined the effect of UVC radiation on the PLD activity in Vero 76 cells. At a dose of 10 kJ/m2 of UVC irradiation, the PLD activity was stimulated approximately 10-fold over the basal activity. This UVC-induced PLD activity was found to be dependent on the presence of extracellular calcium and was inhibited by catalase as well as amifostine-an intracellular thiol antioxidant. Pretreatments with Ro32-0432-a selective inhibitor of protein kinase C (PKC)-and downregulation of PKC by preincubation of phorbol 12-myristate 13-acetate significantly inhibited the UVC-induced PLD activity. UVC-stimulated PLD activity was observed only in murine PLD2 (mPLD2)-transfected Vero 76 cells and not in human PLD1 (hPLD1)-transfected cells. Transient incorporation of PKC with mPLD2 and the phosphorylation of mPLD2 by a and b forms of PKC by UVC irradiation were observed. These results suggest that the UVC-stimulated PLD activity in Vero 76 cells is mediated through transient phosphorylation of PLD2 by the translocation of PKC to PLD2.     

Shear stress stimulates phosphorylation of protein kinase A substrate proteins including endothelial nitric oxide synthase in endothelial cells

Fluid shear stress plays a critical role in vascular health and disease. While protein kinase A (PKA) has been implicated in shear-stimulated signaling events in endothelial cells, it remains unclear whether and how PKA is stimulated in response to shear stress. This issue was addressed in the present study by monitoring the phosphorylation of endogenous substrates of PKA. Shear stress stimulated the phosphorylation of cAMP responsive element binding protein (CREB) in a PKA-dependent manner. Western blot analysis using the antibody reactive against the consensus motif of PKA substrates detected two proteins, P135 and P50, whose phosphorylation was increased by shear stress. The phosphorylation of P135 was blocked by a PKA inhibitor, H89, but not by a phosphoinositide 3-kinase inhibitor, wortmannin. Expression of a constitutively active PKA subunit stimulated P135 phosphorylation, supporting the potential of P135 as a PKA substrate. P135 was identified as endothelial nitric oxide synthase (eNOS) by immunoprecipitation study. PKA appeared to mediate shear stress-stimulated eNOS activation. Shear stress stimulated intracellular translocation of PKA activity from 'soluble' to 'particulate' fractions without involving cellular cAMP increase. Taken together, this study suggests that shear stress stimulates PKA-dependent phosphorylation of target proteins including eNOS, probably by enhancing intracellular site-specific interactions between protein kinase and substrates.     

80 kDa mouse sperm protein as a substrate of protein kinase C.

Calcium and phospholipid-dependent protein kinase (PKC) activity was detected mainly in the cytosol of the mouse sperm. The PKC in the cytosol fraction was partially purified by ion-exchange chromatography. Using the partially purified PKC, the phosphorylation of PKC substrates was examined in vitro. The phosphorylation of the 80 kDa protein was enhanced by phorbol ester treatment in vitro as well as in vivo. The partial amino acid sequence of this protein was homologous with that of guanosine 5'-cyclic monophosphate (cGMP)-dependent protein kinase and myosin light chain kinase, both of which are related to ligand-receptor-transduction. The present data suggest that the activation of PKC and subsequent specific protein phosphorylation might be involved in the regulation of the zona pellucida-induced acrosome reaction.     

Modulation of the substrate specificity of purified human protein kinase C by its activators

The substrate specificity of purified human protein kinase C was modulated by 12-O-tetradecanoyl-4 beta-phorbol-13-acetate (TPA), dioleoylglycerol, arachidonic acid and lipid A when histone type III-S and myelin basic protein were used as phosphate acceptors. Each activator also showed a distinct pattern in the stimulation of phosphorylation of the kinase itself and of cytosolic placental proteins. The nature of the substrate and the presence of calcium and phospholipid determined the magnitude of the effect observed upon addition of all activators and also the dose dependency of kinase activation by TPA. The apparent Km value for phosphorylation of histone type III-S by the kinase activated by phorbol ester alone and with calcium was 20-30 fold higher than that observed for the enzyme activated by calcium and phospholipid. These observations indicate that the nature and extent of cellular response induced by the activation of C-kinase(s) may be determined by the type of cellular stimulus.     

Phospholipase D is involved in oxidative stress-induced migration of vascular smooth muscle cells via tyrosine phosphorylation and protein kinase C

Oxidative stress has been implicated in mediation of vascular disorders. In the presence of vanadate, H(2)O(2) induced tyrosine phosphorylation of PLD1, protein kinase C-alpha (PKC-alpha), and other unidentified proteins in rat vascular smooth muscle cells (VSMCs). Interestingly, PLD1 was found to be constitutively associated with PKC-alpha in VSMCs. Stimulation of the cells by H(2)O(2) and vanadate showed a concentration-dependent tyrosine phosphorylation of the proteins in PLD1 immunoprecipitates and activation of PLD. Pretreatment of the cells with the protein tyrosine kinase inhibitor, genistein resulted in a dose-dependent inhibition of H(2)O(2)-induced PLD activation. PKC inhibitor and down-regulation of PKC abolished H(2)O(2)-stimulated PLD activation. The cells stimulated by oxidative stress (H(2)O(2)) caused increased cell migration. This effect was prevented by the pretreatment of cells with tyrosine kinase inhibitors, PKC inhibitors, and 1-butanol, but not 3-butanol. Taken together, these results suggest that PLD might be involved in oxidative stress-induced migration of VSMCs, possibly via tyrosine phosphorylation and PKC activation.     

Role of protein kinase C in homologous desensitization of angiotensin II-induced phosphoinositide hydrolysis in rat renal mesangial cells

Conclusions These results indicate that, in rat mesangial cells, angiotensin II induces a homologous desensitization of phospholipase C stimulation. It is proposed that protein kinase C activation plays an important role in the molecular mechanism of desensitization of angiotensin II-stimulated polyphosphoinositide metabolism.

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Die Rolle der Proteinkinase C bei der homologen Desensitisierung der Angiotensin II stimulierten Hydrolyse von Phosphoinositiden

     

Analysis of protein kinase A activity in insulin-secreting cells using a cell-penetrating protein substrate and capillary electrophoresis

A cell-penetrating, fluorescent protein substrate was developed to monitor intracellular protein kinase A (PKA) activity in cells without the need for cellular transfection. The PKA substrate (PKAS) was prepared with a 6×histidine purification tag, an enhanced green fluorescent protein (EGFP) reporter, an HIV-TAT protein transduction domain for cellular translocation and a pentaphosphorylation motif specific for PKA. PKAS was expressed in Escherichia coli and purified by metal affinity chromatography. Incubation of PKAS in the extracellular media facilitated translocation into the intracellular milieu in HeLa cells, βTC-3 cells and pancreatic islets with minimal toxicity in a time and concentration dependent manner. Upon cellular loading, glucose-dependent phosphorylation of PKAS was observed in both βTC-3 cells and pancreatic islets via capillary zone electrophoresis. In pancreatic islets, maximal PKAS phosphorylation (83 ± 6%) was observed at 12 mM glucose, whereas maximal PKAS phosphorylation (86 ± 4%) in βTC-3 cells was observed at 3 mM glucose indicating a left-shifted glucose sensitivity. Increased PKAS phosphorylation was observed in the presence of PKA stimulators forskolin and 8-Br-cAMP (33% and 16%, respectively), with corresponding decreases in PKAS phosphorylation observed in the presence of PKA inhibitors staurosporine and H-89 (40% and 54%, respectively).     

Characterization of phosphorylation of a novel protein kinase in rice cells by capillary electrophoresis.

It was demonstrated that a separation of 20 amino acids constituting a protein and three phosphoamino acids that mostly frequently occur in eukaryotes was achieved within 15 min by capillary electrophoresis coupled with lamp-induced fluorescence detection. Fluorescein isothiocyanate was employed as the fluorescence label to facilitate the fluorescence detection of the 23 amino acid species. The fluorescent derivatization conditions and separation parameters including concentration of electrolyte, surfactant in buffer, applied voltage and sample injection were investigated in detail and optimized. The influence of buffer additives such as methanol, acetone and polyvinylpyrrolidone on separation selectivity and sensitivity were discussed. We showed that addition of 2% polyvinylpyrrolidone into the running buffer could dramatically enhance the separation selectivity of amino acids at the expense of a decrease of sensitivity of phosphoamino acids. Under the optimized conditions, the detection limits (S/N=2) ranged from 1.90 x 10(-8) M to 5.66 x 10(-8) M with an average efficiency of 620,000/m. The method was applied to characterization of the phosphorylation of a novel protein kinase RCaMBP (calcium/calmodulin-binding protein kinase) encoded by a cDNA newly isolated and cloned from rice. We verified that RCaMBP belonged to a type of Ser/Thr kinase, providing insight into its function in signal transduction.     

Recognition of multiple substrate motifs by the c-ABL protein tyrosine kinase

Using a combinatorial peptide library that is based on the one-bead one-peptide approach we identified 14 peptide substrates for the c-ABL protein tyrosine kinase, which define three distinct consensus sequence groups. This is distinct from many serine/threonine kinases, which often phosphorylate only one major consensus sequence. The three consensus sequences accurately predict phosphorylation sites in cellular ABL substrates proven to play a role in cell signaling. Our data suggest that protein tyrosine kinases have evolved to recognize multiple substrate motifs.     

Characterization of the interaction of hypericin with protein kinase C in U-87 MG human glioma cells

A fluorescence imaging technique was used to monitor intracellular localization of protein kinase C (PKC) in U-87 MG human glioma cells in the presence of hypericin (Hyp) and phorbol 12-myristate-13-acetate (PMA). It is shown that PKC localization, which reflects its activity, is influenced by Hyp and this influence is different from that observed for PMA which acts as PKC activator. Fluorescence binding experiments were used to determine the binding constants of Hyp to several isoforms of PKC. The obtained values of K(d)s (approximately 100 nM) suggest that Hyp binds with high affinity to PKC. Finally, molecular modeling was used to compare structural models of the interaction of C1B domain of PKC (PKC isoforms alpha, delta, gamma) with Hyp and our previously published model of the (C1B domain PKCgamma)/PMA complex. The influence of Hyp on PKC translocation in U-87 MG cells in comparison with PMA, colocalization fluorescence pattern of Hyp and PKC, the higher binding affinity of Hyp to PKC in comparison with known binding constants of phorbol esters, as well as the binding mode of Hyp and PMA to the C1B domain of PKC suggested by molecular modeling, support the idea that Hyp and PMA might competitively bind to the regulatory domain of PKC.     

A QM/MM study of the phosphoryl transfer to the Kemptide substrate catalyzed by protein kinase A. The effect of the phosphorylation state of the protein on the mechanism

We present here a theoretical study of the phosphoryl transfer catalytic mechanism of protein kinase A, which is the best known member of the large protein kinase family. We have built different theoretical models of the complete PKA-Mg(2)-ATP-substrate system to explore the two most accepted reaction pathways, using for the first time in a reaction mechanism theoretical study, the heptapeptide substrate Kemptide, which is relevant for its high efficiency and small size. The effect of the protein configuration, as modeled by two different X-ray structures with different phosphorylation states and degrees of flexibility, has been analyzed. The results indicate that the environmental conditions can influence the availability of the pathways and thus the choice of the mechanism to be followed. In addition, the roles of the two active site conserved residues, Asp166 and Lys168, have been analyzed for each reaction mechanism.     

Identification of the smooth muscle-specific protein, sm22, as a novel protein kinase C substrate using two-dimensional gel electrophoresis and mass spectrometry

We report a novel method to identify protein kinase C (PKC) substrates. Tissue lysates were fractionated by ion exchange chromatography and used as substrates in in vitro kinase reactions. The phosphorylated proteins were separated using two-dimensional gel electrophoresis. Spots that contained isolated phosphoproteins were excised and digested with trypsin. The tryptic peptides were analyzed using mass spectrometry. While several of the proteins identified using this technique represent known PKC substrates, we identified a new PKC substrate in the initial screen. This protein, sm22, is expressed in smooth muscle cells and served well as a substrate for PKC in vitro. Sm22 is predominantly associated with the actin cytoskeleton. Upon activation of PKC in vivo, sm22 dissociates from the actin cytoskeleton and is distributed diffusely in the cytoplasm. Our data strongly suggest that phosphorylation by PKC controls the intracellular localization of sm22. This demonstrates that our approach, using a complex mixture of proteins as in vitro kinase substrates and subsequently identifying the newly phosphorylated proteins by mass spectrometry, is a powerful method to identify new kinase substrates.     

Comparative study of the prereactive protein kinase A Michaelis complex with Kemptide substrate

In the present work we have modeled the Michaelis complex of the cyclic-Adenosine Monophosphate Dependent (cAMD) Protein Kinase A (PKA) with Mg(2)ATP and the heptapeptide substrate Kemptide by classical molecular dynamics. The chosen synthetic substrate is relevant for its high efficiency and small size, and it has not been used in previous theoretical studies. The structural analysis of the data generated along the 6 ns simulation indicates that the modeled substrate-enzyme complex mimics the substrate binding pattern known for PKA. The values of the average prereactive distances obtained from the simulation do not exclude any of the two limiting situations proposed as mechanisms in the literature for the phosphorylation reaction (dissociative and associative) because the system oscillates between configurations compatible with each of them. Furthermore, the results obtained for the average interaction distances between active site residues concord in suggesting the plausibility of an alternative third reaction mechanism.