Characterization of a calmodulin kinase II inhibitor protein in brain

Ca2+/calmodulin-dependent protein kinase II (CaM-KII) regulates numerous physiological functions, including neuronal synaptic plasticity through the phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors. To identify proteins that may interact with and modulate CaM-KII function, a yeast two-hybrid screen was performed by using a rat brain cDNA library. This screen identified a unique clone of 1.4 kb, which encoded a 79-aa brain-specific protein that bound the catalytic domain of CaM-KII α and β and potently inhibited kinase activity with an IC50 of 50 nM. The inhibitory protein (CaM-KIIN), and a 28-residue peptide derived from it (CaM-KIINtide), was highly selective for inhibition of CaM-KII with little effect on CaM-KI, CaM-KIV, CaM-KK, protein kinase A, or protein kinase C. CaM-KIIN interacted only with activated CaM-KII (i.e., in the presence of Ca2+/CaM or after autophosphorylation) by using glutathione S-transferase/CaM-KIIN precipitations as well as coimmunoprecipitations from rat brain extracts or from HEK293 cells cotransfected with both constructs. Colocalization of CaM-KIIN with activated CaM-KII was demonstrated in COS-7 cells transfected with green fluorescent protein fused to CaM-KIIN. In COS-7 cells phosphorylation of transfected α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors by CaM-KII, but not by protein kinase C, was blocked upon cotransfection with CaM-KIIN. These results characterize a potent and specific cellular inhibitor of CaM-KII that may have an important role in the physiological regulation of this key protein kinase.

Protein kinase C chimeras: catalytic domains of alpha and beta II protein kinase C contain determinants for isotype-specific function.

Protein kinase C (PKC) is involved in the proliferation and differentiation of many cell types. In human erythroleukemia (K-562) cells, the PKC isoforms alpha and beta II play distinct functional roles. alpha PKC is involved in phorbol 12-myristate 13-acetate-induced cytostasis and megakaryocytic differentiation, whereas beta II PKC is required for proliferation. To identify regions within alpha and beta II PKC that allow participation in these divergent pathways, we constructed chimeras in which the regulatory and catalytic domains of alpha and beta II PKC were exchanged. These PKC chimeras can be stably expressed, exhibit enzymatic properties similar to native alpha and beta II PKC in vitro, and participate in alpha and beta II PKC isotype-specific pathways in K-562 cells. Expression of the beta/alpha PKC chimera induces cytostasis in the same manner as overexpression of wild-type alpha PKC. In contrast, the alpha/beta II PKC chimera, like wild-type beta II PKC, selectively translocates to the nucleus and leads to increased phosphorylation of the nuclear envelope polypeptide lamin B in response to bryostatin-1. Therefore, the catalytic domains of alpha and beta II PKC contain determinants important for alpha and beta II PKC isotype function. These results suggest that the catalytic domain represents a potential target for modulating PKC isotype activity in vivo.

A peptide export–import control circuit modulating bacterial development regulates protein phosphatases of the phosphorelay

The phosphorelay signal transduction system activates developmental transcription in sporulation of Bacillus subtilis by phosphorylation of aspartyl residues of the Spo0F and Spo0A response regulators. The phosphorylation level of these response regulators is determined by the opposing activities of protein kinases and protein aspartate phosphatases that interpret positive and negative signals for development in a signal integration circuit. The RapA protein aspartate phosphatase of the phosphorelay is regulated by a peptide that directly inhibits its activity. This peptide is proteolytically processed from an inactive pre-inhibitor protein encoded in the phrA gene. The pre-inhibitor is cleaved by the protein export apparatus to a putative pro-inhibitor that is further processed to the active inhibitor peptide and internalized by the oligopeptide permease. This export–import circuit is postulated to be a mechanism for timing phosphatase activity where the processing enzymes regulate the rate of formation of the active inhibitor. The processing events may, in turn, be controlled by a regulatory hierarchy. Chromosome sequencing has revealed several other phosphatase–prepeptide gene pairs in B. subtilis, suggesting that the use of this mechanism may be widespread in signal transduction.

Ca2+/calmodulin-dependent protein kinase II phosphorylation of the presynaptic protein synapsin I is persistently increased during long-term potentiation

Long-term potentiation (LTP) is an increase in synaptic responsiveness thought to be involved in mammalian learning and memory. The localization (presynaptic and/or postsynaptic) of changes underlying LTP has been difficult to resolve with current electrophysiological techniques. Using a biochemical approach, we have addressed this issue and attempted to identify specific molecular mechanisms that may underlie LTP. We utilized a novel multiple-electrode stimulator to produce LTP in a substantial portion of the synapses in a hippocampal CA1 minislice and tested the effects of such stimulation on the presynaptic protein synapsin I. LTP-inducing stimulation produced a long-lasting 6-fold increase in the phosphorylation of synapsin I at its Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) sites without affecting synapsin I levels. This effect was fully blocked by either the N-methyl-d-aspartate receptor antagonist d(−)-2-amino-5-phosphonopentanoic acid (APV) or the CaM kinase II inhibitor KN-62. Our results indicate that LTP expression is accompanied by persistent changes in presynaptic phosphorylation, and specifically that presynaptic CaM kinase II activity and synapsin I phosphorylation may be involved in LTP expression.

Modulation of a calcium-sensitive nonspecific cation channel by closely associated protein kinase and phosphatase activities

Regulation of nonspecific cation channels often underlies neuronal bursting and other prolonged changes in neuronal activity. In bag cell neurons of Aplysia, it recently has been suggested that an intracellular messenger-induced increase in the activity of a nonspecific cation channel may underlie the onset of a 30-min period of spontaneous action potentials referred to as the “afterdischarge.” In patch clamp studies of the channel, we show that the open probability of the channel can be increased by an average of 10.7-fold by application of ATP to the cytoplasmic side of patches. Duration histograms indicate that the increase is primarily a result of a reduction in the duration and percentage of channel closures described by the slowest time constant. The increase in open probability was not observed using 5′-adenylylimidodiphosphate, a nonhydrolyzable ATP analog, and was blocked in the presence of H7 or the more specific calcium/phospholipid-dependent protein kinase C (PKC) inhibitor peptide(19–36). Because the increase in activity observed in response to ATP occurred without application of protein kinase, our results indicate that a kinase endogenous to excised patches mediates the effect. The effect of ATP could be reversed by exogenously applied protein phosphatase 1 or by a microcystin-sensitive phosphatase also endogenous to excised patches. These results, together with work demonstrating the presence of a protein tyrosine phosphatase in these patches, suggest that the cation channel is part of a regulatory complex including at least three enzymes. This complex may act as a molecular switch to activate the cation channel and, thereby, trigger the afterdischarge.

Adenosine activates ATP-sensitive potassium channels in arterial myocytes via A2 receptors and cAMP-dependent protein kinase.

The mechanism by which the endogenous vasodilator adenosine causes ATP-sensitive potassium (KATP) channels in arterial smooth muscle to open was investigated by the whole-cell patch-clamp technique. Adenosine induced voltage-independent, potassium-selective currents, which were inhibited by glibenclamide, a blocker of KATP currents. Glibenclamide-sensitive currents were also activated by the selective adenosine A2-receptor agonist 2-p-(2-carboxethyl)-phenethylamino-5'-N- ethylcarboxamidoadenosine hydrochloride (CGS-21680), whereas 2-chloro-N6-cyclopentyladenosine (CCPA), a selective adenosine A1-receptor agonist, failed to induce potassium currents. Glibenclamide-sensitive currents induced by adenosine and CGS-21680 were largely reduced by blockers of the cAMP-dependent protein kinase (Rp-cAMP[S], H-89, protein kinase A inhibitor peptide). Therefore, we conclude that adenosine can activate KATP currents in arterial smooth muscle through the following pathway: (i) Adenosine stimulates A2 receptors, which activates adenylyl cyclase; (ii) the resulting increase intracellular cAMP stimulates protein kinase A, which, probably through a phosphorylation step, opens KATP channels.

Cellular protein kinase C isoform zeta regulates human parainfluenza virus type 3 replication.

Phosphorylation of the P proteins of nonsegmented negative-strand RNA viruses is critical for their function as transactivators of the viral RNA polymerases. Using unphosphorylated P protein of human parainfluenza virus type 3 (HPIV3) expressed in Escherichia coli, we have shown that the cellular protein kinase that phosphorylates P in vitro is biochemically and immunologically indistinguishable from cellular protein kinase C isoform zeta (PKC-zeta). Further, PKC-zeta is specifically packaged within the progeny HPIV3 virions and remains tightly associated with the ribonucleoprotein complex. The P protein seems also to be phosphorylated intracellularly by PKC-zeta, as shown by the similar protease digestion pattern of the in vitro and in vivo phosphorylated P proteins. The growth of HPIV3 in CV-1 cells is completely abrogated when a PKC-zeta-specific inhibitor pseudosubstrate peptide was delivered into cells. These data indicate that PKC-zeta plays an important role in HPIV3 gene expression by phosphorylating P protein, thus providing an opportunity to develop antiviral agents against an important human pathogen.

Mutant mice lacking the gamma isoform of protein kinase C show decreased behavioral actions of ethanol and altered function of gamma-aminobutyrate type A receptors.

Calcium/phospholipid-dependent protein kinase (protein kinase C, PKC) has been suggested to play a role in the sensitivity of gamma-aminobutyrate type A (GABAA) receptors to ethanol. We tested a line of null mutant mice that lacks the gamma isoform of PKC (PKC gamma) to determine the role of this brain-specific isoenzyme in ethanol sensitivity. We found that the mutation reduced the amount of PKC gamma immunoreactivity in cerebellum to undetectable levels without altering the levels of the alpha, beta I, or beta II isoforms of PKC. The mutant mice display reduced sensitivity to the effects of ethanol on loss of righting reflex and hypothermia but show normal responses to flunitrazepam or pentobarbital. Likewise, GABAA receptor function of isolated brain membranes showed that the mutation abolished the action of ethanol but did not alter actions of flunitrazepam or pentobarbital. These studies show the unique interactions of ethanol with GABAA receptors and suggest protein kinase isoenzymes as possible determinants of genetic differences in response to ethanol.

Degradation of the cleaved leader peptide of thiolase by a peroxisomal proteinase.

A peroxisomal location for insulin-degrading enzyme (IDE) has been defined by confocal immunofluorescence microscopy of stably transfected CHO cells overexpressing IDE and digitonin-permeabilization studies in normal nontransfected fibroblasts. The functional significance of IDE in degrading cleaved leader peptides of peroxisomal proteins targeted by the type II motif was evaluated with a synthetic peptide corresponding to the type II leader peptide of prethiolase. The peptide effectively competed for degradation and cross-linking of the high-affinity substrate 125I-labeled insulin to IDE. Direct proteolysis of the leader peptide of prethiolase was confirmed by HPLC; degradation was inhibited by immunodepletion with an antibody to IDE. Phylogenetic analysis of proteinases related to IDE revealed sequence similarity to mitochondrial processing peptidases.

A release mechanism for stored ATP in ocular ciliary epithelial cells

Purines can modify ciliary epithelial secretion of aqueous humor into the eye. The source of the purinergic agonists acting in the ciliary epithelium, as in many epithelial tissues, is unknown. We found that the fluorescent ATP marker quinacrine stained rabbit and bovine ciliary epithelia but not the nerve fibers in the ciliary bodies. Cultured bovine pigmented and nonpigmented ciliary epithelial cells also stained intensely when incubated with quinacrine. Hypotonic stimulation of cultured epithelial cells increased the extracellular ATP concentration by 3-fold; this measurement underestimates actual release as the cells also displayed ecto-ATPase activity. The hypotonically triggered increase in ATP was inhibited by the Cl−-channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) in both cell types. In contrast, the P-glycoprotein inhibitors tamoxifen and verapamil and the cystic fibrosis transmembrane conductance regulator (CFTR) blockers glybenclamide and diphenylamine-2-carboxylate did not affect ATP release from either cell type. This pharmacological profile suggests that ATP release is not restricted to P-glycoprotein or the cystic fibrosis transmembrane conductance regulator, but can proceed through a route sensitive to NPPB. ATP release also was triggered by ionomycin through a different NPPB-insensitive mechanism, inhibitable by the calcium/calmodulin-activated kinase II inhibitor KN-62. Thus, both layers of the ciliary epithelium store and release ATP, and purines likely modulate aqueous humor flow by paracrine and/or autocrine mechanisms within the two cell layers of this epithelium.

Efficient loading of HLA-DR with a T helper epitope by genetic exchange of CLIP

The HLA class II-associated invariant chain (Ii)-derived peptide (CLIP) occupies the peptide binding groove during assembly in the endoplasmic reticulum, travels with HLA class II to endosomal compartments, and is subsequently released to allow binding of antigenic peptides. We investigated whether the exchange of CLIP with a known T helper epitope at the DNA level would lead to efficient loading of this helper epitope onto HLA class II. For this purpose, a versatile Ii-encoding expression vector was created in which CLIP can be replaced with a helper epitope of choice. Upon supertransfection of HLA-DR1-transfected 293 cells with an Ii vector encoding a known T helper epitope (HA307–319), predominantly length variants of this epitope were detected in association with the HLA-DR1 molecules of these cells. Moreover, this transfectant was efficiently recognized by a peptide-specific T helper clone (HA1.7). The results suggest that this type of Ii vector can be used to create potent class II+ cellular vaccines in which defined T cell epitopes are continuously synthesized.

Trypanosoma cruzi Calreticulin Is a Lectin That Binds Monoglucosylated Oligosaccharides but Not Protein Moieties of Glycoproteins

Trypanosoma cruzi is a protozoan parasite that belongs to an early branch in evolution. Although it lacks several features of the pathway of protein N-glycosylation and oligosaccharide processing present in the endoplasmic reticulum of higher eukaryotes, it displays UDP-Glc:glycoprotein glucosyltransferase and glucosidase II activities. It is herewith reported that this protozoan also expresses a calreticulin-like molecule, the third component of the quality control of glycoprotein folding. No calnexin-encoding gene was detected. Recombinant T. cruzi calreticulin specifically recognized free monoglucosylated high-mannose-type oligosaccharides. Addition of anti-calreticulin serum to extracts obtained from cells pulse–chased with [35S]Met plus [35S]Cys immunoprecipitated two proteins that were identified as calreticulin and the lysosomal proteinase cruzipain (a major soluble glycoprotein). The latter but not the former protein disappeared from immunoprecipitates upon chasing cells. Contrary to what happens in mammalian cells, addition of the glucosidase II inhibitor 1-deoxynojirimycin promoted calreticulin–cruzipain interaction. This result is consistent with the known pathway of protein N-glycosylation and oligosaccharide processing occurring in T. cruzi. A treatment of the calreticulin-cruzipain complexes with endo-β-N-acetylglucosaminidase H either before or after addition of anti-calreticulin serum completely disrupted calreticulin–cruzipain interaction. In addition, mature monoglucosylated but not unglucosylated cruzipain isolated from lysosomes was found to interact with recombinant calreticulin. It was concluded that the quality control of glycoprotein folding appeared early in evolution, and that T. cruzi calreticulin binds monoglucosylated oligosaccharides but not the protein moiety of cruzipain. Furthermore, evidence is presented indicating that glucosyltransferase glucosylated cruzipain at its last folding stages.

Posttranscriptional regulation of urokinase plasminogen activator receptor messenger RNA levels by leukocyte integrin engagement

As an adhesion receptor, the β2 integrin lymphocyte function-associated antigen-1 (LFA-1) contributes a strong adhesive force to promote T lymphocyte recirculation and interaction with antigen-presenting cells. As a signaling molecule, LFA-1-mediates transmembrane signaling, which leads to the generation of second messengers and costimulation resulting in T cell activation. We recently have demonstrated that, in costimulatory fashion, LFA-1 activation promotes the induction of T cell membrane urokinase plasminogen activator receptor (uPAR) and that this induced uPAR is functional. To investigate the mechanism(s) of this induction, we used the RNA polymerase II inhibitor 5,6-dichloro-1-β-d-ribobenzimidazole and determined that uPAR mRNA degradation is delayed by LFA-1 activation. Cloning of the wild-type, deleted and mutated 3′-untranslated region of the uPAR cDNA into a serum-inducible rabbit β-globin cDNA reporter construct revealed that the AU-rich elements and, in particular the nonameric UUAUUUAUU sequence, are crucial cis-acting elements in uPAR mRNA degradation. Experiments in which Jurkat T cells were transfected with reporter constructs demonstrated that LFA-1 engagement was able to stabilize the unstable reporter mRNA containing the uPAR 3′-untranslated region. Our study reveals a consequence of adhesion receptor-mediated signaling in T cells, which is potentially important in the regulation of T cell activation, including production of cytokines and expression of proto-oncogenes, many of which are controlled through 3′ AU-rich elements.

Etoposide induces heritable chromosomal aberrations and aneuploidy during male meiosis in the mouse

Etoposide, a topoisomerase II inhibitor widely used in cancer therapy, is suspected of inducing secondary tumors and affecting the genetic constitution of germ cells. A better understanding of the potential heritable risk of etoposide is needed to provide sound genetic counseling to cancer patients treated with this drug in their reproductive years. We used a mouse model to investigate the effects of clinical doses of etoposide on the induction of chromosomal abnormalities in spermatocytes and their transmission to zygotes by using a combination of chromosome painting and 4′,6-diamidino-2-phenylindole staining. High frequencies of chromosomal aberrations were detected in spermatocytes within 64 h after treatment when over 30% of the metaphases analyzed had structural aberrations (P < 0.01). Significant increases in the percentages of zygotic metaphases with structural aberrations were found only for matings that sampled treated pachytene (28-fold, P < 0.0001) and preleptotene spermatocytes (13-fold, P < 0.001). Etoposide induced mostly acentric fragments and deletions, types of aberrations expected to result in embryonic lethality, because they represent loss of genetic material. Chromosomal exchanges were rare. Etoposide treatment of pachytene cells induced aneuploidy in both spermatocytes (18-fold, P < 0.01) and zygotes (8-fold, P < 0.05). We know of no other report of an agent for which paternal exposure leads to an increased incidence of aneuploidy in the offspring. Thus, we found that therapeutic doses of etoposide affect primarily meiotic germ cells, producing unstable structural aberrations and aneuploidy, effects that are transmitted to the progeny. This finding suggests that individuals who undergo chemotherapy with etoposide may be at a higher risk for abnormal reproductive outcomes especially within the 2 months after chemotherapy.

Full-length myotonin protein kinase (72 kDa) displays serine kinase activity.

We describe the full-length (72 kDa) myotonin protein kinase (Mt-PK) and demonstrate its kinase activity. The 72-kDa protein corresponds to the translation product from the first in-frame AUG codon. This protein was found in the cytoplasmic fraction, whereas the previously reported 55-kDa protein was observed in nuclear extracts. Only the 72-kDa protein was phosphorylated by [32P]phosphate in normal human fibroblasts. To investigate the putative kinase activity of Mt-PK, a construct containing the full-length open reading frame of Mt-PK was expressed in bacterial cells. The recombinant Mt-PK autophosphorylates a Ser residue and phosphorylates the synthetic peptide Gly-Arg-Gly-Leu-Ser-Leu-Ser-Arg, which contains a Ser residue in the phosphorylation site. We examined phosphorylation of the voltage-dependent Ca(2+)-release channel, or dihydropyridine receptor (DHPR), by recombinant Mt-PK. We observed that the beta subunit of DHPR was phosphorylated in vitro by Mt-PK. A beta-subunit DHPR peptide containing some of the Ser residues predicted to be phosphorylated was synthesized and found to be a substrate for Mt-PK in vitro. We conclude that the 72-kDa Mt-PK has a protein kinase activity specific for Ser residues.