Biomedical subjects

A J Czernik

Publications and source records attributed to A J Czernik.

At least 19 recordsLinked to original sources

PubMed2001

Opposing changes in phosphorylation of specific sites in synapsin I during Ca2+-dependent glutamate release in isolated nerve terminals.

Synapsins are major neuronal phosphoproteins involved in regulation of neurotransmitter release. Synapsins are well established targets for multiple protein kinases within the nerve terminal, yet little is known about dephosphorylation processes involved in regulation of synapsin function. Here, we observed a reciprocal relationship in the phosphorylation-dephosphorylation of the established phosphorylation sites on synapsin I. We demonstrate that, in vitro, phosphorylation sites 1, 2, and 3 of synapsin I (P-site 1 phosphorylated by cAMP-dependent protein kinase; P-sites 2 and 3 phosphorylated by Ca(2+)-calmodulin-dependent protein kinase II) were excellent substrates for protein phosphatase 2A, whereas P-sites 4, 5, and 6 (phosphorylated by mitogen-activated protein kinase) were efficiently dephosphorylated only by Ca(2+)-calmodulin-dependent protein phosphatase 2B-calcineurin. In isolated nerve terminals, rapid changes in synapsin I phosphorylation were observed after Ca(2+) entry, namely, a Ca(2+)-dependent phosphorylation of P-sites 1, 2, and 3 and a Ca(2+)-dependent dephosphorylation of P-sites 4, 5, and 6. Inhibition of calcineurin activity by cyclosporin A resulted in a complete block of Ca(2+)-dependent dephosphorylation of P-sites 4, 5, and 6 and correlated with a prominent increase in ionomycin-evoked glutamate release. These two opposing, rapid, Ca(2+)-dependent processes may play a crucial role in the modulation of synaptic vesicle trafficking within the presynaptic terminal.

4-Aminopyridine↗

PubMed2001

Identification of synapsin I peptides that insert into lipid membranes.

The synapsins constitute a family of synaptic vesicle-associated phosphoproteins essential for regulating neurotransmitter release and synaptogenesis. The molecular mechanisms underlying the selective targeting of synapsin I to synaptic vesicles are thought to involve specific protein-protein interactions, while the high-affinity binding to the synaptic vesicle membrane may involve both protein-protein and protein-lipid interactions. The highly hydrophobic N-terminal region of the protein has been shown to bind with high affinity to the acidic phospholipids phosphatidylserine and phosphatidylinositol and to penetrate the hydrophobic core of the lipid bilayer. To precisely identify the domains of synapsin I which mediate the interaction with lipids, synapsin I was bound to liposomes containing the membrane-directed carbene-generating reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine and subjected to photolysis. Isolation and N-terminal amino acid sequencing of 125I-labelled synapsin I peptides derived from CNBr cleavage indicated that three distinct regions in the highly conserved domain C of synapsin I insert into the hydrophobic core of the phospholipid bilayer. The boundaries of the regions encompass residues 166-192, 233-258 and 278-327 of bovine synapsin I. These regions are surface-exposed in the crystal structure of domain C of bovine synapsin I and are evolutionarily conserved among isoforms across species. The present data offer a molecular explanation for the high-affinity binding of synapsin I to phospholipid bilayers and synaptic vesicles.

Amino Acid Sequence↗

PubMed2001

Tonically active protein kinase A regulates neurotransmitter release at the squid giant synapse.

1. Electrophysiological and microinjection methods were used to examine the role of cyclic AMP-dependent protein kinase A (PKA) in regulating transmitter release at the squid giant synapse. 2. Excitatory postsynaptic potentials (EPSPs) evoked by presynaptic action potentials were not affected by presynaptic injection of an exogenous active catalytic subunit of mammalian PKA. 3. In contrast, presynaptic injection of PKI-amide, a peptide that inhibits PKA with high potency and specificity, led to a reversible inhibition of EPSPs. 4. Injection of several other peptides that serve as substrates for PKA also reversibly inhibited neurotransmitter release. The ability of these peptides to inhibit release was correlated with their ability to serve as PKA substrates, suggesting that these peptides act by competing with endogenous substrates for phosphorylation by active endogenous PKA. 5. We suggest that the phosphorylation of PKA substrates is maintained at a relatively high state under basal conditions and that this tonic activity of PKA is to a large degree required for evoked neurotransmitter release at the squid giant presynaptic terminal.

Action Potentials↗

PubMed2001

Phosphorylation of protein phosphatase inhibitor-1 by Cdk5.

Protein phosphatase inhibitor-1 is a prototypical mediator of cross-talk between protein kinases and protein phosphatases. Activation of cAMP-dependent protein kinase results in phosphorylation of inhibitor-1 at Thr-35, converting it into a potent inhibitor of protein phosphatase-1. Here we report that inhibitor-1 is phosphorylated in vitro at Ser-67 by the proline-directed kinases, Cdk1, Cdk5, and mitogen-activated protein kinase. By using phosphorylation state-specific antibodies and selective protein kinase inhibitors, Cdk5 was found to be the only kinase that phosphorylates inhibitor-1 at Ser-67 in intact striatal brain tissue. In vitro and in vivo studies indicated that phospho-Ser-67 inhibitor-1 was dephosphorylated by protein phosphatases-2A and -2B. The state of phosphorylation of inhibitor-1 at Ser-67 was dynamically regulated in striatal tissue by glutamate-dependent regulation of N-methyl-d-aspartic acid-type channels. Phosphorylation of Ser-67 did not convert inhibitor-1 into an inhibitor of protein phosphatase-1. However, inhibitor-1 phosphorylated at Ser-67 was a less efficient substrate for cAMP-dependent protein kinase. These results demonstrate regulation of a Cdk5-dependent phosphorylation site in inhibitor-1 and suggest a role for this site in modulating the amplitude of signal transduction events that involve cAMP-dependent protein kinase activation.

Animals↗

PubMed2001

ARPP-16/ARPP-19: a highly conserved family of cAMP-regulated phosphoproteins.

ARPP-16 and ARPP-19 are closely related cAMP-regulated phosphoproteins that were initially discovered in mammalian brain as in vitro substrates for protein kinase A (PKA). ARPP-16 is enriched in dopamine-responsive medium spiny neurons in the striatum, while ARPP-19 is ubiquitously expressed. ARPP-19 is highly homologous to alpha-endosulfine and database searches allowed the identification of novel related proteins in D. melanogaster, C. elegans, S. mansoni and yeast genomes. Using isoform-specific antibodies, we now show that ARPP-19 is composed of at least two differentially expressed isoforms (termed ARPP-19 and ARPP-19e/endosulfine). All ARPP-16/19 family members contain a conserved consensus site for phosphorylation by PKA (RKPSLVA in mammalian ARPP-16 and ARPP-19), and this site was shown to be efficiently phosphorylated in vitro by PKA. An antibody that specifically recognized the phosphorylated form of ARPP-16/19/19e was used to examine the phosphorylation of ARPP-16/19 family members in intact cells. In striatal slices, the phosphorylation of ARPP-16 was increased in response to activation of D(1)-type dopamine receptors, and decreased in response to activation of D(2)-type dopamine receptors. In non-neuronal cells, ARPP-19 was highly phosphorylated in response to activation of PKA. These results establish that ARPP-16/19 proteins constitute a family of PKA-dependent intracellular messengers that function in all cells. The high levels of ARPP-16 in striatal neurons and its bi-directional regulation by dopamine suggest a specific role in dopamine-dependent signal transduction. The conservation of this protein family through evolution suggests that it subserves an important cellular function that is regulated by PKA.

Amino Acid Sequence↗

PubMed2000

Synapsins as mediators of BDNF-enhanced neurotransmitter release.

We examined enhancement of synaptic transmission by neurotrophins at the presynaptic level. In a synaptosomal preparation, brain-derived neurotrophic factor (BDNF) increased mitogen-activated protein (MAP) kinase-dependent synapsin I phosphorylation and acutely facilitated evoked glutamate release. PD98059, used to inhibit MAP kinase activity, markedly decreased synapsin I phosphorylation and concomitantly reduced neurotransmitter release. The stimulation of glutamate release by BDNF was strongly attenuated in mice lacking synapsin I and/or synapsin II. These results indicate a causal link of synapsin phosphorylation via BDNF, TrkB receptors and MAP kinase with downstream facilitation of neurotransmitter release.

Animals↗

PubMed2000

Use of phosphosynapsin I-specific antibodies for image analysis of signal transduction in single nerve terminals.

We have developed a semi-quantitative method for indirectly revealing variations in the concentration of second messengers (Ca(2+), cyclic AMP) in single presynaptic boutons by detecting the phosphorylation of the synapsins, excellent nerve terminal substrates for cyclic AMP- and Ca(2+)/calmodulin-dependent protein kinases. For this purpose, we employed polyclonal, antipeptide antibodies recognising exclusively synapsin I phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (at site 3) or synapsins I/II phosphorylated by either cAMP-dependent protein kinase or Ca(2+)/calmodulin-dependent protein kinase I (at site 1). Cerebellar granular neurones in culture were double-labelled with a monoclonal antibody to synapsins I/II and either of the polyclonal antibodies. Digitised images were analysed to determine the relative phosphorylation stoichiometry at each individual nerve terminal. We have found that: (i) under basal conditions, phosphorylation of site 3 was undetectable, whereas site 1 exhibited some degree of constitutive phosphorylation; (ii) depolarisation in the presence of extracellular Ca(2+) was followed by a selective and widespread increase in site 3 phosphorylation, although the relative phosphorylation stoichiometry varied among individual terminals; and (iii) phosphorylation of site 1 was increased by stimulation of cyclic AMP-dependent protein kinase but not by depolarisation and often occurred in specific nerve terminal sub-populations aligned along axon branches. In addition to shedding light on the regulation of synapsin phosphorylation in living nerve terminals, this approach permits the spatially-resolved analysis of the activation of signal transduction pathways in the presynaptic compartment, which is usually too small to be studied with other currently available techniques.

Animals↗

PubMed1999

Phosphorylation of DARPP-32 by Cdk5 modulates dopamine signalling in neurons.

The physiological state of the cell is controlled by signal transduction mechanisms which regulate the balance between protein kinase and protein phosphatase activities. Here we report that a single protein can, depending on which particular amino-acid residue is phosphorylated, function either as a kinase or phosphatase inhibitor. DARPP-32 (dopamine and cyclic AMP-regulated phospho-protein, relative molecular mass 32,000) is converted into an inhibitor of protein phosphatase 1 when it is phosphorylated by protein kinase A (PKA) at threonine 34. We find that DARPP-32 is converted into an inhibitor of PKA when phosphorylated at threonine 75 by cyclin-dependent kinase 5 (Cdk5). Cdk5 phosphorylates DARPP-32 in vitro and in intact brain cells. Phospho-Thr 75 DARPP-32 inhibits PKA in vitro by a competitive mechanism. Decreasing phospho-Thr 75 DARPP-32 in striatal slices, either by a Cdk5-specific inhibitor or by using genetically altered mice, results in increased dopamine-induced phosphorylation of PKA substrates and augmented peak voltage-gated calcium currents. Thus DARPP-32 is a bifunctional signal transduction molecule which, by distinct mechanisms, controls a serine/threonine kinase and a serine/threonine phosphatase.

Animals↗

PubMed1999

Synapsins as major neuronal Ca2+/S100A1-interacting proteins.

The mammalian S100A1 protein can activate the invertebrate myosin-associated giant protein kinase twitchin in a Ca(2+)-dependent manner by more than 1000-fold in vitro; however, no mammalian S100-dependent protein kinases are known. In an attempt to identify novel mammalian Ca(2+)/S100A1-regulated protein kinases, brain extracts were subjected to combined Ca(2+)-dependent affinity chromatography with S100A1 and an ATP analogue. This resulted in the purification to near-homogeneity of the four major synapsin isoforms Ia, Ib, IIa and IIb. All four synapsins were specifically affinity-labelled with the ATP analogue 5'-p-fluorosulphonylbenzoyladenosine. S100A1 bound to immobilized synapsin IIa in BIAcore experiments in a Ca(2+)-dependent and Zn(2+)-enhanced manner with submicromolar affinity; this interaction could be competed for with synthetic peptides of the proposed S100A1-binding sites of synapsin. Double-labelling confocal immunofluorescence microscopy demonstrated that synapsins and S100A1 are both present in the soma and neurites of PC12 cells, indicating their potential to interact in neurons in vivo.

Adenosine Triphosphate↗

PubMed1999

Phosphorylation of the cytoplasmic domain of Alzheimer's beta-amyloid precursor protein at Ser655 by a novel protein kinase.

The cytoplasmic domain of Alzheimer's beta-amyloid precursor protein (APP) can be phosphorylated at Thr654, Ser655, and Thr668 (APP695 isoform numbering). Previous studies demonstrated that Ser655 of APP was phosphorylated by protein kinase C (PKC) and calmodulin-dependent protein kinase II (CaMKII) in vitro and by unidentified protein kinase(s) in vivo. We report here the characterization of a novel protein kinase (designated APP kinase I) which phosphorylates Ser655 of APP. APP kinase I was partially purified over 7,000-fold from rat brain and identified as a approximately 43 kDa protein that is distinct from a number of known protein kinases, including PKC and extracellular signal-regulated kinases (ERKs). The identification of a novel protein kinase that phosphorylates Ser655 will hopefully contribute to our understanding of the metabolism and/or function of APP in the pathogenesis of Alzheimer's disease (AD).

Alzheimer Disease↗

PubMed1999

Phylogenetically conserved CK-II phosphorylation site of the murine homeodomain protein Hoxb-6.

In an effort to characterize the signal transduction mechanisms that operate to regulate homeodomain protein function, we have analyzed the phosphorylation state of two homeodomain proteins, Hoxb-6 and Hoxc-8, in vitro and in vivo. The baculovirus expression system was employed to demonstrate that Hoxb-6 is phosphorylated in Sf9 cells while Hoxc-8 is not. Using two-dimensional tryptic phosphopeptide mapping and purified protein kinases, we demonstrate that Hoxb-6 is phosphorylated in vitro by casein kinase II and cAMP-dependent protein kinase. The casein kinase II phosphorylation site was mapped to serine-214. Two-dimensional tryptic phosphopeptide mapping of immunoprecipitated Hoxb-6 from mouse embryonic spinal cords demonstrates that the same peptide phosphorylated in vitro and in Sf9 cells by casein kinase II is also phosphorylated in vivo. The conservation of this site in several homeodomain proteins from various species is discussed.

Amino Acid Sequence↗

PubMed1999

Synapsins as regulators of neurotransmitter release.

One of the crucial issues in understanding neuronal transmission is to define the role(s) of the numerous proteins that are localized within presynaptic terminals and are thought to participate in the regulation of the synaptic vesicle life cycle. Synapsins are a multigene family of neuron-specific phosphoproteins and are the most abundant proteins on synaptic vesicles. Synapsins are able to interact in vitro with lipid and protein components of synaptic vesicles and with various cytoskeletal proteins, including actin. These and other studies have led to a model in which synapsins, by tethering synaptic vesicles to each other and to an actin-based cytoskeletal meshwork, maintain a reserve pool of vesicles in the vicinity of the active zone. Perturbation of synapsin function in a variety of preparations led to a selective disruption of this reserve pool and to an increase in synaptic depression, suggesting that the synapsin-dependent cluster of vesicles is required to sustain release of neurotransmitter in response to high levels of neuronal activity. In a recent study performed at the squid giant synapse, perturbation of synapsin function resulted in a selective disruption of the reserve pool of vesicles and in addition, led to an inhibition and slowing of the kinetics of neurotransmitter release, indicating a second role for synapsins downstream from vesicle docking. These data suggest that synapsins are involved in two distinct reactions which are crucial for exocytosis in presynaptic nerve terminals. This review describes our current understanding of the molecular mechanisms by which synapsins modulate synaptic transmission, while the increasingly well-documented role of the synapsins in synapse formation and stabilization lies beyond the scope of this review.

Animals↗

PubMed1999

Regulation of synaptotagmin I phosphorylation by multiple protein kinases.

Synaptotagmin I has been suggested to function as a low-affinity calcium sensor for calcium-triggered exocytosis from neurons and neuroendocrine cells. We have studied the phosphorylation of synaptotagmin I by a variety of protein kinases in vitro and in intact preparations. SyntagI, the purified, recombinant, cytoplasmic domain of rat synaptotagmin I, was an effective substrate in vitro for Ca2+/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC), and casein kinase II (caskII). Sequencing of tryptic phosphopeptides from syntagI revealed that CaMKII and PKC phosphorylated the same residue, corresponding to Thr112, whereas caskII phosphorylated two residues, corresponding to Thr125 and Thr128. Endogenous synaptotagmin I was phosphorylated on purified synaptic vesicles by all three kinases. In contrast, no phosphorylation was observed on clathrin-coated vesicles, suggesting that phosphorylation of synaptotagmin I in vivo occurs only at specific stage(s) of the synaptic vesicle life cycle. In rat brain synaptosomes and PC12 cells, K+-evoked depolarization or treatment with phorbol ester caused an increase in the phosphorylation state of synaptotagmin I at Thr112. The results suggest the possibility that the phosphorylation of synaptotagmin I by CaMKII and PKC contributes to the mechanism(s) by which these two kinases regulate neurotransmitter release.

Amino Acid Sequence↗

PubMed1998

Ca2+/calmodulin-dependent kinase II mediates simultaneous enhancement of gap-junctional conductance and glutamatergic transmission.

While chemical synapses are very plastic and modifiable by defined activity patterns, gap junctions, which mediate electrical transmission, have been classically perceived as passive intercellular channels. Excitatory transmission between auditory afferents and the goldfish Mauthner cell is mediated by coexisting gap junctions and glutamatergic synapses. Although an increased intracellular Ca2+ concentration is expected to reduce gap junctional conductance, both components of the synaptic response were instead enhanced by postsynaptic increases in Ca2+ concentration, produced by patterned synaptic activity or intradendritic Ca2+ injections. The synaptically induced potentiations were blocked by intradendritic injection of KN-93, a Ca2+/calmodulin-dependent kinase (CaM-K) inhibitor, or CaM-KIINtide, a potent and specific peptide inhibitor of CaM-KII, whereas the responses were potentiated by injection of an activated form of CaM-KII. The striking similarities of the mechanisms reported here with those proposed for long-term potentiation of mammalian glutamatergic synapses suggest that gap junctions are also similarly regulated and indicate a primary role for CaM-KII in shaping and regulating interneuronal communication, regardless of its modality.

Animals↗

PubMed1998

A third member of the synapsin gene family.

Synapsins are a family of neuron-specific synaptic vesicle-associated phosphoproteins that have been implicated in synaptogenesis and in the modulation of neurotransmitter release. In mammals, distinct genes for synapsins I and II have been identified, each of which gives rise to two alternatively spliced isoforms. We have now cloned and characterized a third member of the synapsin gene family, synapsin III, from human DNA. Synapsin III gives rise to at least one protein isoform, designated synapsin IIIa, in several mammalian species. Synapsin IIIa is associated with synaptic vesicles, and its expression appears to be neuron-specific. The primary structure of synapsin IIIa conforms to the domain model previously described for the synapsin family, with domains A, C, and E exhibiting the highest degree of conservation. Synapsin IIIa contains a novel domain, termed domain J, located between domains C and E. The similarities among synapsins I, II, and III in domain organization, neuron-specific expression, and subcellular localization suggest a possible role for synapsin III in the regulation of neurotransmitter release and synaptogenesis. The human synapsin III gene is located on chromosome 22q12-13, which has been identified as a possible schizophrenia susceptibility locus. On the basis of this localization and the well established neurobiological roles of the synapsins, synapsin III represents a candidate gene for schizophrenia.

Alternative Splicing↗

PubMed1998

Two sites of action for synapsin domain E in regulating neurotransmitter release.

Synapsins, a family of synaptic vesicle proteins, have been shown to regulate neurotransmitter release; the mechanism(s) by which they act are not fully understood. Here we have studied the role of domain E of synapsins in neurotransmitter release at the squid giant synapse. Two squid synapsin isoforms were cloned and found to contain a carboxy (C)-terminal domain homologous to domain E of the vertebrate a-type synapsin isoforms. Presynaptic injection of a peptide fragment of domain E greatly reduced the number of synaptic vesicles in the periphery of the active zone, and increased the rate and extent of synaptic depression, suggesting that domain E is essential for synapsins to regulate a reserve pool of synaptic vesicles. Domain E peptide had no effect on the number of docked synaptic vesicles, yet reversibly inhibited and slowed the kinetics of neurotransmitter release, indicating a second role for synapsins that is more intimately associated with the release process itself. Thus, synapsin domain E is involved in at least two distinct reactions that are crucial for exocytosis in presynaptic terminals.

Amino Acid Sequence↗

PubMed1998

Regulation of secretion of Alzheimer amyloid precursor protein by the mitogen-activated protein kinase cascade.

Activation of protein kinase C (PKC) regulates the processing of Alzheimer amyloid precursor protein (APP) into its soluble form (sAPP) and amyloid beta-peptide (A beta). However, little is known about the intermediate steps between PKC activation and modulation of APP metabolism. Using a specific inhibitor of mitogen-activated protein (MAP) kinase kinase activation (PD 98059), as well as a dominant negative mutant of MAP kinase kinase, we show in various cell lines that stimulation of PKC by phorbol ester rapidly induces sAPP secretion through a mechanism involving activation of the MAP kinase cascade. In PC12-M1 cells, activation of MAP kinase by nerve growth factor was associated with stimulation of sAPP release. Conversely, M1 muscarinic receptor stimulation, which is known to act in part through a PKC-independent pathway, increased sAPP secretion mainly through a MAP kinase-independent pathway. A beta secretion and its regulation by PKC were not affected by PD 98059, supporting the concept of distinct secretory pathways for A beta and sAPP formation.

Amyloid beta-Peptides↗

PubMed1997

Synapsin I interacts with c-Src and stimulates its tyrosine kinase activity.

Synapsin I is a synaptic vesicle-associated phosphoprotein that has been implicated in the formation of presynaptic specializations and in the regulation of neurotransmitter release. The nonreceptor tyrosine kinase c-Src is enriched on synaptic vesicles, where it accounts for most of the vesicle-associated tyrosine kinase activity. Using overlay, affinity chromatography, and coprecipitation assays, we have now shown that synapsin I is the major binding protein for the Src homology 3 (SH3) domain of c-Src in highly purified synaptic vesicle preparations. The interaction was mediated by the proline-rich domain D of synapsin I and was not significantly affected by stoichiometric phosphorylation of synapsin I at any of the known regulatory sites. The interaction of purified c-Src and synapsin I resulted in a severalfold stimulation of tyrosine kinase activity and was antagonized by the purified c-Src-SH3 domain. Depletion of synapsin I from purified synaptic vesicles resulted in a decrease of endogenous tyrosine kinase activity. Portions of the total cellular pools of synapsin I and Src were coprecipitated from detergent extracts of rat brain synaptosomal fractions using antibodies to either protein species. The interaction between synapsin I and c-Src, as well as the synapsin I-induced stimulation of tyrosine kinase activity, may be physiologically important in signal transduction and in the modulation of the function of axon terminals, both during synaptogenesis and at mature synapses.

Animals↗