Identification of a novel nuclear-localized adenylate kinase from Drosophila melanogaster

As a step to further understand the role of adenylate kinase (AK) in the energy metabolism network, we identified, purified, and characterized a previously undescribed adenylate kinase in Drosophila melanogaster. The cDNA encodes a 175-amino acid protein, which shows 47.85% identity in 163 amino acids to human AK6. The recombinant protein was successfully expressed in Escherichia coli BL21(DE3) strain. Characterization of this protein by enzyme activity assay showed adenylate kinase activity. AMP and CMP were the preferred substrates, and UMP can also be phosphorylated to some extent, with ATP as the best phosphate donor. Subcellular localization study showed a predominantly nuclear localization. Therefore, based on the substrate specificity, the specific nuclear localization in the cell, and the sequence similarity with human AK6, we named this novel adenylate kinase identified from the fly DAK6.     

Study of global motions in proteins by weighted masses molecular dynamics: Adenylate kinase as a test case

The weighted masses molecular dynamics (WMMD) technique is applied to the protein adenylate kinase. A novel set of restraints has been developed to allow the use of this technique with proteins. The WMMD simulation is successful in predicting the flexibility of the two mobile domains of the protein. The end product of the simulation is similar to the known open and AMP bound forms of the enzyme. The biological relevance of the restraints used and potential methods of improving the technique are discussed. © 1996 Wiley-Liss, Inc.     

Heterologous sensitization of adenylate cyclase is protein kinase A-dependent in Cath.a differentiated (CAD)-D2L cells

Persistent activation of Galphai/o-coupled receptors results in a paradoxical enhancement of subsequent drug-stimulated adenylate cyclase activity. The exact mechanism of this up-regulation in the cyclic AMP signaling pathway, known as heterologous sensitization, remains undefined. The present study was designed to investigate the involvement of cyclic AMP-dependent protein kinase in D2L receptor-mediated sensitization in a neuronal cellular environment. The current studies were conducted in the Cath.a differentiated (CAD) cell line transfected stably with the D2L dopamine receptor (CAD-D2L). Long-term 18 h treatment with the D2 receptor agonist, quinpirole, resulted in a two-fold enhancement of forskolin-stimulated cyclic AMP accumulation. Similarly, long-term treatment with the PKA inhibitors, H89 or Rp-8Br-cAMP, also enhanced adenylate cyclase activity. In contrast, long-term activation of protein kinase A (PKA) by forskolin, isobutylmethylxanthine (IBMX), or dibutyryl cyclic AMP caused a significant reduction in subsequent forskolin-stimulated cyclic AMP accumulation and reduced both quinpirole- and H89-induced heterologous sensitization. The effects of PKA inhibitors and activators did not involve changes in PKA subunit expression. RT-PCR analysis of adenylate cyclase isoform expression patterns revealed the expression of mRNA for ACVI and ACIX in CAD-D2L cells. The ability of ACVI to be negatively regulated by PKA is consistent with the observation that inhibition of PKA results in heterologous sensitization of adenylate cyclase activity in CAD-D2L cells.     

A computational analysis of substrate binding strength by phosphorylase kinase and protein kinase A

Protein kinases exhibit various degrees of substrate specificity. The large number of different protein kinases in the eukaryotic proteomes makes it impractical to determine the specificity of each enzyme experimentally. To test if it were possible to discriminate potential substrates from non-substrates by simple computational techniques, we analysed the binding enthalpies of modelled enzyme-substrate complexes and attempted to correlate it with experimental enzyme kinetics measurements. The crystal structures of phosphorylase kinase and cAMP-dependent protein kinase were used to generate models of the enzyme with a series of known peptide substrates and non-substrates, and the approximate enthalpy of binding assessed following energy minimization. We show that the computed enthalpies do not correlate closely with kinetic measurements, but the method can distinguish good substrates from weak substrates and non-substrates.     

Comparative surface geometry of the protein kinase family

Identifying conserved pockets on the surfaces of a family of proteins can provide insight into conserved geometric features and sites of protein–protein interaction. Here we describe mapping and comparison of the surfaces of aligned crystallographic structures, using the protein kinase family as a model. Pockets are rapidly computed using two computer programs, FADE and Crevasse. FADE uses gradients of atomic density to locate grooves and pockets on the molecular surface. Crevasse, a new piece of software, splits the FADE output into distinct pockets. The computation was run on 10 kinase catalytic cores aligned on the αF‐helix, and the resulting pockets spatially clustered. The active site cleft appears as a large, contiguous site that can be subdivided into nucleotide and substrate docking sites. Substrate specificity determinants in the active site cleft between serine/threonine and tyrosine kinases are visible and distinct. The active site clefts cluster tightly, showing a conserved spatial relationship between the active site and αF‐helix in the C‐lobe. When the αC‐helix is examined, there are multiple mechanisms for anchoring the helix using spatially conserved docking sites. A novel site at the top of the N‐lobe is present in all the kinases, and there is a large conserved pocket over the hinge and the αC‐β4 loop. Other pockets on the kinase core are strongly conserved but have not yet been mapped to a protein–protein interaction. Sites identified by this algorithm have revealed structural and spatially conserved features of the kinase family and potential conserved intermolecular and intramolecular binding sites.     

Photokinetic microassay of adenylate kinase using the firefly luciferase reaction.

A new rapid photokinetic method is described for determining the activity of adenylate kinase (ATP:AMP phosphotranspherase, EC 2.7.4.3) in 0.1--5.0 micrograms of freeze-dried tissue. This represents a weight range far below that obtainable by fine-needle biopsy. The reaction 2 ADP in equilibrium with AMP + ATP was employed and the ATP formed assayed with firefly luciferase as light yielder. The light emission was recorded on a multi-channel scaler. The adenylate kinase activities found in tissues of mice were in the same range as previously described in a study using fluorometric microassay.     

Identification of protein kinase substrates by proteomic approaches

This review describes the current status of proteomic approaches to identify kinase substrates, which may lead to valuable medical applications. It guides the reader towards various methods using 2DE and liquid chromatography-tandem mass spectrometry. Dynamic changes of phosphorylation during extracellular stimuli can be quantitatively monitored by both technologies. Among appropriate prefractionation procedures, the purification of phosphoproteins and phosphopeptides is an absolute step for success. The temporal change and stoichiometry of phosphorylation are the important criteria to evaluate the physiological meaning of the reaction. Kinase substrates can also be identified by in vitro phosphorylation systems employing protein arrays, fractionated lysates, genetically engineered kinases and phage libraries. The final section contains an expert opinion on the current strategies and the issues we are going to challenge in the next 5 years.     

A novel view of domain flexibility in E. coli adenylate kinase based on structural mode-coupling (15)N NMR relaxation.

Adenylate kinase from Escherichia coli (AKeco), consisting of a single 23.6 kDa polypeptide chain folded into domains CORE, AMPbd and LID, catalyzes the reaction AMP+ATP-->2ADP. In the ligand-free enzyme the domains AMPbd and LID execute large-amplitude movements controlling substrate binding and product release during catalysis. Domain flexibility is investigated herein with the slowly relaxing local structure (SRLS) model for (15)N relaxation. SRLS accounts rigorously for coupling between the global and local N-H motions through a local ordering potential exerted by the protein structure at the N-H bond. The latter reorients with respect to its protein surroundings, which reorient on the slower time scale associated with the global protein tumbling. AKeco diffuses globally with correlation time tau(m)=15.1 ns, while locally two different dynamic cases prevail. The domain CORE features ordering about the equilibrium N-H bond orientation with order parameters, S(2), of 0.8-0.9 and local motional correlation times, tau, mainly between 5-130 ps. This represents a conventional rigid protein structure with rapid small-amplitude N-H fluctuations. The domains AMPbd and LID feature small parallel (Z(M)) ordering of S(2)=0.2-0.5 which can be reinterpreted as high perpendicular (Y(M)) ordering. M denotes the local ordering/local diffusion frame. Local motion about Z(M) is given by tau( parallel) approximately 5 ps and local motion of the effective Z(M) axis about Y(M) by tau( perpendicular)=6-11 ns. Z(M) is tilted at approximately 20 degrees from the N-H bond. The orientation of the Y(M) axis may be considered parallel to the C(alpha)(i-1)-C(alpha)(i) axis. The tau( perpendicular) mode reflects collective nanosecond peptide-plane motions, interpretable as domain motion. A powerful new model of protein flexibility/domain motion has been established. Conformational exchange (R(ex)) processes accompany the tau( perpendicular) mode. The SRLS analysis is compared with the conventional model-free analysis.     

Cyclooxygenase-2 and adenylate cyclase/protein kinase A signaling pathway enhances angiogenesis through induction of vascular endothelial growth factor in rat sponge implants

Angiogenesis is reportedly enhanced by prostaglandins (PGs). In the present experiment, we tested whether or not COX-2 and adenylate cyclase/protein kinase A (AC/PKA)-dependent VEGF induction enhanced angiogenesis in this model. Angiogenesis was enhanced by topical injection of human recombinant basic fibroblast growth factor (bFGF). The enhanced angiogenesis by bFGF was inhibited by indomethacin or selective COX-2 inhibitors, NS398, nimesulide, and JTE-522. Topical daily injections of 8-bromo-cAMP enhanced angiogenesis in a dose-dependent manner. Forskolin, an activator of AC, also facilitated angiogenesis in a dose-dependent manner, as did amrinone, an inhibitor of phosphodiesterase. VEGF induction was confirmed by the increased levels in the fluids in the sponge matrix after topical injection of 8-bromo-cAMP. Immunohistochemical investigation further revealed the VEGF-expressed cells in the sponge granulation tissues to be fibroblasts, and the intensity of positive reactions was enhanced by bFGF, 8-bromo-cAMP, forskolin, and amrinone. Angiogenesis was inhibited by indometacin or selective COX-2 inhibitors, NS-398, nimesulide, and JTE-522. In addition, angiogenesis without topical injections of the above compounds was also suppressed by SQ22,536, an inhibitor for AC. or H-89, an inhibitor for PKA, with concomitant reductions in VEGF levels. Daily topical injections of neutralizing antibody or anti-sense oligonucleotide against VEGF significantly suppressed angiogenesis. These results suggested that COX-2 and AC/PKA-dependent induction of VEGF certainly enhanced angiogenesis, and that pharmacological tools for controlling this signaling pathway may be able to facilitate the management of conditions involving angiogenesis.     

Geographic variation of muscle adenylate kinase in the loach Misgurnus anguillicaudatus

The electrophoretic pattern of the loach muscle adenylate kinase was composed of one or two major bands. Each major band was preceded by two minor bands. Three codominant alleles were postulated to segregate in loach. Each allele coded for one major band with different mobility.
Adenylate kinase (AK. E.C. 2.7.4.3.) catalyses the reaction 2ADP ATP + AMP. and is known as a heat stable protein constituent of skeletal muscle. Electrophoretic variation of AK has been reported in the pika Ochotona r. rufescens (Vergnes et al. 1974). the teleostean fish Zoarces viviparus (Frydenberg & Si-monsen, 1973), the mussel Mvtilus edulis (Ahmad et al. 1977). and the tunicate Ciona intestinalis (Schmidtke & Engel. 1980). In this note, individual variation of AK in muscle extracts of the fresh-water fish Misgurnus anguillicaudatus is described.
Loach were collected in ponds or purchased from fish shops (Table 1 & Fig. 1). Three populations (OS. AS and KN) were purchased from fish shops in Osaka. Akashi and Kanazawa cities, respectively. Their exact sampling locations were not known. For reference, the locations of these cities are indicated by an open circle in Fig. i. Fish were collected and stored frozen at – 20 ^oC at the sampling time given in Table 1. Muscle extracts were prepared and examined in the period February-April 1981.The method for preparing muscle extract and-the starch gel electrophoretic procedures were the same as those reported previously (Kimura, 1976). The amine-citrate buffer system as described by Clayton & Tretiak (1972) was used. AK was stained by the method of Allendorf et al. (1977). After electrophoresis, an inhibition test was also performed by immersing the gel in 10^-3 M 5.5'-dithiobis-(2-nitrobenzoate) solution for 30 minutes at room temperature.
Under the electrophoretic condition used in the present study, all of the AK     

Modulation of adenylate cyclase activity by Ca2+, phospholipid-dependent protein kinase in rat brain striatum

Summary The effects of purified Ca²⁺, phospholipid-dependent protein kinase (C-kinase) were studied on adenylate cyclase activity from rat brain striatum. C-kinase treatment of the membranes stimulated adenylate cyclase activity, the maximal stimulation between 50–80% was observed at 3.5 U/ml, whereas the catalytic subunit of cAMP dependent protein kinase did not show any effect on enzyme activity. The inclusion of Ca²⁺ and phosphatidyl serine did not augment the percent stimulation of adenylate cyclase by C-kinase, however EGTA inhibited the stimulatory effect of C-kinase on enzyme activity. Furthermore, the known inhibitors of C-kinase such as polymyxin-B and 1-(5-Isoquinoline sulfonyl)-2-methylpiperazine dihydrochloride (H-7) also inhibited the stimulatory effect of C-kinase on adenylate cyclase activity. In addition, in the presence of GTP the stimulatory effects of C-kinase on basal and N-Ethylcarboxamide adenosine- (NECA-), dopamine-(DA) and forskolin- (FSK) sensitive adenylate cyclase activities were augmented. On the other hand, the inhibitory effect of high concentrations of GTP on enzyme activity was attenuated by C-kinase treatment. In addition, oxotremorine inhibited adenylate cyclase activity in a concentration dependent manner, with an apparent Ki of about 10 µM and C-kinase treatment almost completely abolished this inhibition. These data suggest that C-kinase may play an important role in the regulation of adenylate cyclase activity possibly by interacting with a guanine nucleotide regulatory protein.Abbreviations C-kinase Ca^2– phospholipid-dependent protein kinase - NECA N-Ethylcarboxamide adenosine - DA Dopamine - FSK Forskolin - PMA Phorbol 12-(-Myristate), 13-Acetate, H-7, 1-(5-isoquinoline sulfonyl)-2-methylpiperazine dihydrochloride Presented in part at the VIth International Conference on Cyclic nucleotides, calcium and protein phosphorylation signal transduction in biological systems. September 2-6, 1986, Bethesda, MD (USA).M.B.A.-S. was Canadian Heart Foundation Scholar during the course of these studies.     

Phosphorylation of microtubule-associated protein tau by isoforms of c-Jun N-terminal kinase (JNK)

Microtubule-associated protein tau in a hyperphosphorylated state is the major component of the filamentous lesions that define a number of neurodegenerative diseases commonly referred to as tauopathies. Hyperphosphorylation of tau at most sites appears to precede filament assembly. Many of the hyperphosphorylated sites are serine/threonine-proline sequences. Here we show that c-Jun N-terminal kinases JNK1, JNK2 and JNK3 phosphorylate tau at many serine/threonine-prolines, as assessed by the generation of the epitopes of phosphorylation-dependent anti-tau antibodies. Of the three protein kinases, JNK2 phosphorylated the most sites in tau, followed by JNK3 and JNK1. Phosphorylation by JNK isoforms resulted in a greatly reduced ability of tau to promote microtubule assembly. These findings extend the number of candidate protein kinases for the hyperphosphorylation of tau in Alzheimer's disease and other neurodegenerative disorders.     

Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis

The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser²⁰⁵⁶, Thr²⁶⁴⁷ and Thr²⁶⁰⁹ in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser³²⁰⁵ and Thr³⁹⁵⁰ in mitosis. Phosphorylation of Thr³⁹⁵⁰ is DNA-PK-dependent, whereas phosphorylation of Ser³²⁰⁵ requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser³²⁰⁵ in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser³²⁰⁵ in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr⁶⁸ in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs’ role in mitosis may be mechanistically distinct from its well-established role in NHEJ.     

Fast collapse but slow formation of secondary structure elements in the refolding transition of E. coli adenylate kinase

The various models proposed for protein folding transition differ in their order of appearance of the basic steps during this process. In this study, steady state and time-resolved dynamic non-radiative excitation energy transfer (FRET and trFRET) combined with site specific labeling experiments were applied in order to characterize the initial transient ensemble of Escherichia coli adenylate kinase (AK) molecules upon shifting conditions from those favoring denaturation to refolding and from folding to denaturing. Three sets of labeled AK mutants were prepared, which were designed to probe the equilibrium and transient distributions of intramolecular segmental end-to-end distances. A 176 residue section (residues 28-203), which spans most of the 214 residue molecule, and two short secondary structure chain segments including an alpha-helix (residues 169-188) and a predominantly beta-strand region (residues 188-203), were labeled. Upon fast change of conditions from denaturing to folding, the end-to-end distance of the 176 residue chain section showed an immediate collapse to a mean value of 26 A. Under the same conditions, the two short secondary structure elements did not respond to this shift within the first ten milliseconds, and retained the characteristics of a fully unfolded state. Within the first 10 ms after changes of the solvent from folding to denaturing, only minor changes were observed at the local environments of residues 203 and 169. The response of these same local environments to the shift of conditions from denaturing to folding occurred within the dead time of the mixing device. Thus, the response of the CORE domain of AK to fast transfer from folding to unfolding conditions is slow at all three conformational levels that were probed, and for at least a few milliseconds the ensemble of folded molecules is maintained under unfolding conditions. A different order of the changes was observed upon initiation of refolding. The AK molecules undergo fast collapse to an ensemble of compact structures where the local environment of surface probes seems to be native-like but the two labeled secondary structure elements remain unfolded.     

The natively helical chain segment 169-188 of Escherichia coli adenylate kinase is formed in the latest phase of the refolding transition

The refolding transition of Escherichia coli adenylate kinase (AK) was investigated by monitoring the refolding kinetics of a selected 20 residue helical segment in the CORE domain of the protein. Residues 169 and 188 were labeled by 1-acetamido-methyl-pyrene, and by bimane, respectively. The experiment combines double-jump stopped-flow fast mixing initiation of refolding and time-resolved F?rster energy transfer spectroscopy for monitoring the conformational transitions (double-kinetics experiment). Two kinetic phases were found in the denaturant-induced unfolding of AK. In the first phase, the fluorescence quantum yields of both probes decreased. The distribution of the distances between them transformed from the native state's narrow distribution with the mean distance corresponding to the distance in the crystal structure, to a distribution compatible with an unordered structure. In the second, slow step of denaturation, neither the fluorescence parameters of the probes nor the distance distribution between them changed. This step appeared to be a transformation of the fast-folding species formed in the first phase, to the slow-folding species. Refolding of the fast-folding species of the denatured state of AK was also a two-phase process. During the first fast phase, within less than 5ms, the fluorescence emission of both probes increased, but the distance distribution between the labeled sites was unchanged. Only during the second slow refolding step did the intramolecular distance distribution change from the characteristic of the denatured state to the narrow distribution of the native state. This experiment shows that for the case of the CORE domain of AK, the large helical segment of residues 169-188 was not formed in the first compaction step of refolding. The helical conformation of this segment is established only in the second, much slower, refolding phase, simultaneously with the completion of the native structure.     

In situ regulation of cell-cell communication by the cAMP-dependent protein kinase and protein kinase C

The effects of cAMP-dependent protein kinase A and protein kinase C on cell-cell communication have been examined in primary ovarian granulosa cells microinjected with purified components of these two regulatory cascades. These cells possess connexin43 ( 1)-type gap junctions, and are well-coupled electrotonically and as judged by the cell-to-cell transfer of fluorescent dye. Within 2–3 min after injection of the protein kinase A inhibitor (PKI) communication was sharply reduced or ceased, but resumed in about 3 min with the injection of the protein kinase A catalytic subunit. A similar resumption also occurred in PKI-injected cells after exposure to follicle stimulating hormone. Microinjection of the protein kinase C inhibitor protein caused a transient cessation of communication that spontaneously returned within 15–20 min. Treatment of cells with activators of protein kinase C, TPA or OAG for 60 min caused a significant reduction in communication that could be restored within 2–5 min by the subsequent injection of either the protein kinase C inhibitor or the protein kinase A catalytic subunit. With a longer exposure to either protein kinase C activator communication could not be restored and this appeared to be related to the absence of aggregates of connexin43 in membrane as detected immunologically. In cells injected with alkaline phosphatase communication stopped but returned either spontaneously within 20 min or within 2–3 min of injecting the cell with either the protein kinase A catalytic subunit or with protein kinase C. When untreated cells were injected with protein kinase C communication diminished or ceased within 5 min. Collectively these results demonstrate that cell-cell communication is regulated by both protein kinase A and C, but in a complex interrelated manner, quite likely by multiple phosphorylation of proteins within or regulating connexin-43 containing gap junctions.Abbreviations C catalytic subunit of protein kinase A - CKI protein kinase C inhibitor protein - Cx connexin protein - dbcAMP N⁶,2-O-dibutyryladenosine 3:5-cyclic monophosphate - OAG 1-oleoyl-2-acetyl-sn-glycerol - protein kinase A cAMP-dependent protein kinase - protein kinase C Ca²⁺-sensitive phospholipid-dependent protein kinase - PKI protein kinase A inhibitor protein - R regulatory subunit of protein kinase A - TRA 12-O-tetradecanoylphorbol-13-acetate - 8Br-cAMP 8-bromoadenosine 3:5 cyclic monophosphate     

Electrostatic interactions determine entrance/release order of substrates in the catalytic cycle of adenylate kinase

Adenylate kinase is a monomeric phosphotransferase with important biological function in regulating concentration of adenosine triphosphate (ATP) in cells, by transferring the terminal phosphate group from ATP to adenosine monophosphate (AMP) and forming two adenosine diphosphate (ADP) molecules. During this reaction, the kinase may undergo a large conformational transition, forming different states with its substrates. Although many structures of the protein are available, atomic details of the whole process remain unclear. In this article, we use both conventional molecular dynamics (MD) simulation and an enhanced sampling technique called parallel cascade selection MD simulation to explore different conformational states of the Escherichia coli adenylate kinase. Based on the simulation results, we propose a possible entrance/release order of substrates during the catalytic cycle. The substrate-free protein prefers an open conformation, but changes to a closed state once ATP·Mg enters into its binding pocket first and then AMP does. After the reaction of ATP transferring the terminal phosphate group to AMP, ADP·Mg and ADP are released sequentially, and finally the whole catalyze cycle is completed. Detailed contact and distance analysis reveals that the entrance/release order of substrates may be largely controlled by electrostatic interactions between the protein and the substrates.     

An expanded adenylate kinase gene family in the protozoan parasite Trypanosoma cruzi

Adenylate kinases supply energy routes in cellular energetic homeostasis. In this work, we identified and characterized the adenylate kinase activity in extracts from the flagellated parasite Trypanosoma cruzi, the causative agent of Chagas' disease. Adenylate kinase activity was detected in different subcellular fractions and the cytosolic isoform was biochemically characterized. Cytosolic adenylate kinase specific activity increases continuously during the epimastigote growth and is down-regulated when other soluble phosphotransferase, arginine kinase, is overexpressed. Six different genes of adenylate kinase isoforms were identified and the mRNA expression was confirmed by RT-PCR and Northern Blot. Three open reading frames coding for different enzyme isoforms named TzADK1, TzADK2 and TzADK5 were cloned and functionally expressed in E. coli. This work reports an unusually large number of genes of adenylate kinases and suggests a coordinated regulation of phosphotransferase-mediated ATP regenerating pathways in the unicellular parasite Trypanosoma cruzi.     

Identification of PSF as a protein kinase Calpha-binding protein in the cell nucleus

Protein kinase C (PKC) isoforms are present in the cell nucleus in diverse cell lines and tissues. Since little is known about proteins interacting with PKC inside the cell nucleus, we used Neuro-2a neuroblastoma cells, in which PKCalpha is present in the nucleus, to screen for nuclear binding partners for PKC. Applying overlay assays, we detected several nuclear proteins which bind to PKCalpha. Specificity of binding was shown by its dependence on PKC activation by phorbol ester, calcium, and phosphatidylserine. The PKC-binding proteins were partially purified and analyzed by microsequencing and mass spectrometry. Four proteins could be identified: PTB-associated splicing factor (PSF), p68 RNA helicase, and the heterogeneous nuclear ribonucleoprotein (hnRNP) proteins A3 and L. In the case of PSF, binding to PKC could also be demonstrated in a GST-pull-down assay using GST-PKCalpha, expressed in insect cells. Phosphorylation experiments revealed that PSF is a weak in vitro substrate for PKCalpha.     

Phosphorylation of the carboxyl terminal region of dystrophin by mitogen-activated protein (MAP) kinase

Dystrophin is the 427-kDa protein product of the Duchenne muscular dystrophy gene (DMD). The function of this protein remains to be elucidated. We have recently reported that dystrophin is phosphorylated,in vivo, in rat skeletal muscle primary cell culture (RE Milner, JL Busaan, CFB Holmes, JH Wang, M Michalak (1993) J Biol Chem 268: 21901–21905). This observation suggests that protein phosphorylation may have some role in modulating the function of dystrophin or its interaction with membrane associate dystroglycan. We report here that the carboxyl-terminal of dystrophin is phosphorylated by the MAP kinase p44^mpk (mitogen-activated protein kinase), from the sea star oocytes and by soluble extracts of rabbit skeletal muscle. Importantly we showed that native dystrophin in isolated sarcolemmal vesicles is phosphorylated by sea star p44^mpk. Partial purification and immunological analysis show that a mammalian kinase related to p44^mpk is present in the skeletal muscle extracts and that it contributes to phosphorylation of the carboxyl-terminal of dystrophin. This kinase phosphorylates dystrophin on a threonine residue(s). We conclude that phosphorylation of dystrophin may play an important role in the function of this cytoskeletal protein.Abbreviations MAP kinase mitogen-activated protein kinase - DMD Duchenne muscular dystrophy - GST Glutathione S-transferase - PAGE polyacrylamide gel electrophoresis - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - MOPS 4-morpholinepropanesulfonic acid