Protein kinase C
The MARCKS protein is a widely distributed cellular substrate for protein kinase C. It is a myristoylprotein that binds calmodulin (see Drosophila calmodulin) and
actin in a manner reversible by protein kinase C-dependent phosphorylation. It is also highly expressed in nervous tissue, particularly
during development. To evaluate a possible developmental role for MARCKS, the gene was disrupted in mice by using the techniques of
homologous recombination. Pups homozygous for the disrupted allele lacked detectable MARCKS mRNA and protein. All
MARCKS-deficient pups died before or within a few hours of birth. Twenty-five percent had exencephaly and 19% had omphalocele
(normal frequencies, < 1%), indicating high frequencies of midline defects, particularly in cranial neurulation. Nonexencephalic
MARCKS-deficient pups had agenesis of the corpus callosum and other forebrain commissures, as well as failure of fusion of the
cerebral hemispheres. All MARCKS-deficient pups also displayed characteristic lamination abnormalities of the cortex and retina.
These studies suggest that MARCKS plays a vital role in the normal developmental processes of neurulation, hemisphere fusion,
forebrain commissure formation, and formation of cortical and retinal laminations. It is concluded that MARCKS is necessary for
normal mouse brain development and postnatal survival (Stumpo, 1995).
MARCKS and the MARCKS-related protein (MRP) are members of a distinct
family of protein kinase C (PKC) substrates that also bind calmodulin regulated by PKC phosphorylation. The kinetics
of PKC-mediated phosphorylation and the calmodulin binding properties of intact, recombinant MARCKS and MRP were investigated
and compared with previous studies of synthetic peptides spanning the PKC phosphorylation site/calmodulin binding domains
(PSCBD) of these proteins. Both MARCKS and MRP were high affinity substrates for the catalytic fragment of PKC, and their
phosphorylation occurs with positive cooperativity.
Affinities are similar to the values determined from studies of their respective PSCBD peptides. Two-dimensional mapping of
MRP and its synthetic PSCBD peptide yield identical patterns of tryptic phosphopeptides; as in the case of
MARCKS, all of the PKC phosphorylation sites in MRP lie within the 24-amino acid PSCBD. Sequence analysis of tryptic
phosphopeptides reveals that the first and third (but not the second) serines in the MRP PSCBD are phosphorylated by PKC. Both
MARCKS and MRP bind dansyl-calmodulin with high affinity, with a Kapp of 4.6 and 9.5 nM, respectively. Phosphorylation of
MARCKS and MRP by PKC disrupt the protein-calmodulin complexes, with half-lives of 4.0 and 3.5 min, respectively. These
studies suggest that intact, recombinant MARCKS and MRP are accurately modeled by their synthetic PSCBD peptides with respect to
PKC phosphorylation kinetics and their phosphorylation-dependent calmodulin binding properties (Verghese, 1994).
A high density of transient A-type K+ channels is located in the distal dendrites of CA1
hippocampal pyramidal neurons and these channels shape EPSPs, limit the back-propagation of action
potentials, and prevent dendritic action potential initiation. Because of the importance of these channels in dendritic signal propagation, their modulation by protein kinases would be of significant interest. The effects of activators of cAMP-dependent protein kinase (PKA) and the Ca2+-dependent phospholipid-sensitive protein kinase (PKC) were investigated on K+ channels in cell-attached patches from the distal dendrites of hippocampal CA1 pyramidal neurons. Inclusion of the membrane-permeant PKA activators 8-bromo-cAMP (8-br-cAMP) or forskolin in the dendritic patch pipette results in a depolarizing shift in the activation curve for the transient channels of approximately 15 mV. Activation of PKC by either of two phorbol esters also results in a 15 mV depolarizing shift of the activation curve. Neither PKA nor PKC activation
affects the sustained or slowly inactivating component of the total outward current. This downregulation of transient K+ channels in the distal dendrites may be responsible for some of the frequently reported increases in cell excitability found after PKA and PKC activation. In support of this hypothesis, it was found that activation of either PKA or PKC significantly increased the amplitude of back-propagating action potentials in distal dendrites (Hoffman, 1998).
One of the most prominent roles of metabotropic glutamate receptors (mGluRs) in the CNS is to serve as presynaptic receptors
that inhibit transmission at glutamatergic synapses. Previous reports suggest that the presynaptic effect of group II mGluRs at
corticostriatal synapses can be inhibited by activators of protein kinase C (PKC). Activation of PKC
inhibits the ability of group II and group III mGluRs to regulate transmission at three major synapses in the hippocampal
formation. Thus, this effect may be a widespread phenomenon that occurs at glutamatergic synapses throughout the CNS. This response is not limited to PKC-activating phorbol esters but activation of A3 adenosine receptors
induces a PKC-dependent inhibition of group III mGluR function at the Schaffer collateral-CA1 synapse. In addition to
inhibiting mGluR modulation of excitatory synaptic transmission, activation of PKC reduces inhibition of
forskolin-stimulated cAMP accumulation by group II and group III mGluRs, suggesting that the effect of PKC on mGluR
signaling is not specific to their effects on neurotransmitter release. This led to the hypothesis that PKC acts upstream
from effector proteins regulated by mGluRs and acts at the level of the receptor or GTP-binding protein. Interestingly, PKC inhibits mGluR-induced increases in [35S]-GTPgammaS binding in cortical synaptosomes. These data
suggest that PKC-induced inhibition of mGluR signaling may be mediated by the inhibition of coupling of mGluRs to
GTP-binding proteins (Macek, 1998).
Syntaxin 1A (see Drosophila Syntaxin) inhibits GABA uptake of an endogenous GABA transporter in neuronal cultures from rat hippocampus and in
reconstitution systems expressing the cloned rat brain GABA transporter GAT1. Evidence of interactions between syntaxin 1A
and GAT1 comes from three experimental approaches: botulinum toxin cleavage of syntaxin 1A, syntaxin 1A antisense
treatments, and coimmunoprecipitation of a complex containing GAT1 and syntaxin 1A. Protein kinase C (PKC), which modulates GABA transporter function, exerts its modulatory effects by regulating the availability of syntaxin 1A
to interact with the transporter, and a transporter mutant that fails to interact with syntaxin 1A is not regulated by PKC. These
results suggest a new target for regulation by syntaxin 1A and a novel mechanism for controlling the machinery involved in
both neurotransmitter release and reuptake (Beckman, 1998).
Protein kinase C (PKC) positively modulates NMDA receptor (NMDAR) currents. In contrast to previous reports, this study determines the
importance of individual exons in the mechanism underlying the potentiation process by examining the complete set of eight naturally
occurring splice variants expressed in Xenopus oocytes, both as homomers and as heteromeric NR1/NR2A or NR1/NR2B complexes. After
PKC stimulation, homomeric currents demonstrate a high level of potentiation (approximately 500% of untreated baseline currents) that
is reduced to a lower level (approximately 300% of baseline) in variants containing the first C-terminal exon (C1). An ANOVA shows that
only C1 and no other exon or interaction of exons determine the degree of NMDAR current modulation by PKC. When recordings are
performed in solutions in which barium replaces calcium, only the lower form of potentiation is observed, regardless of the splice variant
exon composition. This suggests an important role for calcium in the PKC modulation of homomeric NMDA splice variant currents in which
the C1 exon also participates. The effectiveness of the C1 exon to reduce the higher form of potentiation is modulated by heteromeric
assemblies with NR2A heteromers yielding smaller levels of potentiation and a larger C1 exon effect, when compared with NR2B heteromers. The
heteromers demonstrate the higher form of potentiation even in the absence of calcium. Furthermore, calcium has different effects in the
potentiation of the heteromers depending on the NR2 subunit. This study refines the region of the NR1 subunit involved in a modulation
crucial to the function of NMDA receptors and provides evidence that the NR2A and NR2B subunits realize this modulation differentially (Logan, 1999).
Several protein kinases are known to phosphorylate Ser/Thr residues of certain GABAA receptor subunits, yet the effect of phosphorylation
on GABAA receptor function in neurons remains controversial, and the functional consequences of phosphorylating synaptic GABAA
receptors of adult CNS neurons are poorly understood. Whole-cell patch-clamp recordings of GABAA receptor-mediated miniature
IPSCs (mIPSCs) in CA1 pyramidal neurons and dentate gyrus granule cells (GCs) of adult rat hippocampal slices were used to determine the effects of
cAMP-dependent protein kinase (PKA) and Ca2+/phospholipid-dependent protein kinase (PKC) activation on the function of synaptic
GABAA receptors. The mIPSCs recorded in CA1 pyramidal cells and in GCs are differentially affected by PKA and PKC. In pyramidal
cells, PKA reduces mIPSC amplitudes and enhances the fraction of events decaying with a double exponential, whereas PKC has no effect. In contrast, in GCs PKA is ineffective, but PKC increases the peak amplitude of mIPSCs and also favors double exponential
decays. Intracellular perfusion of the phosphatase inhibitor microcystin reveals that synaptic GABAA receptors of pyramidal cells, but not
those of GCs, are continually phosphorylated by PKA and conversely, dephosphorylated, most likely by phosphatase 1 or 2A. This
differential, brain region-specific phosphorylation of GABAA receptors may produce a wide dynamic range of inhibitory synaptic strength in
these two regions of the hippocampal formation (Poisbeau, 1999).
The induction of neurite outgrowth by NGF is a transcription-dependent process in PC12 cells, but the
transcription factors that mediate this process have previously been unknown. The bHLH
transcriptional repressor HES-1 has now been shown to be a mediator of this process. Inactivation of endogenous HES-1 by
forced expression of a dominant-negative protein induces neurite outgrowth in the absence of NGF and
increases response to NGF. In contrast, expression of additional wild-type HES-1 protein represses
and delays response to NGF. Endogenous HES-1 DNA-binding activity is post-translationally inhibited
during NGF signaling in vivo, and phosphorylation of PKC consensus sites in the HES-1 DNA-binding
domain inhibits DNA binding by purified HES-1 in vitro. Mutation of these sites generates a
constitutively active protein that strongly and persistently blocks response to NGF. These results
suggest that post-translational inhibition of HES-1 is both essential for and partially mediates the
induction of neurite outgrowth by NGF signaling. The MASH1 bHLH activator protein is a likely target for direct repression by HES-1. Previous studies have shown that NGF signaling induces the activation or localization of both cytoplasmic and nuclear PKC isoforms in PC12 and other cells. Given that both PKCs and at least some ribosomal S6 kinases are activated during NGF signaling, HES-1 may be a target for multiple kinases activated or functioning during NGF signaling (Strom, 1997).
Expression of specific protein kinase C isoforms correlates with
cell fate in neural chicken embryo cells. Consequently, the effects of PKC activation by
phorbol esters were tested on acquisition of the astrocytic phenotype, using cultured embryonic cortical astrocytes
derived from 15-day-old chick embryos (E15CH). Short term treatment with the phorbol
ester (TPA), which activates PKC-alpha/beta in E15CH, causes
association of PKC with the cytoskeleton. In vitro kinase assays of cytoskeleton-associated PKC
demonstrates phosphorylation of many cytoskeletal proteins. Phosphorylation is blocked by protein
kinase inhibitors (H8), and enhanced by phosphatase inhibitors (calyculin A). Among these PKC
substrates, a most prominent 60-kDa protein was identified as vimentin. Assembly of vimentin into the
cytoskeleton depends on cell type and state of differentiation. 20 min treatment with TPA leads to a 3-fold
increase in the rate of newly synthesized full-length vimentin assembly (posttranslational assembly).
Furthermore, TPA increases cotranslational assembly of vimentin. Long-term TPA treatment, which correlates
with a prolonged phospholipase D (PLD) activation, is mitogenic and causes dramatic changes in the
morphology of astrocytes. In addition these fibrous, polarized astrocytes have decreased activity of the
astrocyte specific enzyme, glutamine synthetase, but have increased abundance of vimentin protein.
These studies provide biochemical evidence on acquisition of a different astrocytic phenotype after
activation of the PKC/PLD pathway in the chick embryo. Therefore PKC and PLD activation is
pivotal for the acquisition and maintenance of phenotypes in chick embryonic astrocytes (Mangoura, 1995).
AMPA receptor subunits interact with a PDZ domain-containing protein called PICK1, which is known to bind protein kinase
C alpha (PKC alpha). PICK1 interacts with sequences within the last ten amino acid residues containing a novel PDZ binding motif (E S V/I K I) of the short C-terminal
alternative splice variants of AMPA receptor subunits. No interaction occurs with the corresponding long splice variants which do not contain the E S V/I K I motif.
The PDZ domain of PICK1 is required for the interaction; the mutation of a single amino acid in this region (Lys-27 to Glu) prevents interaction between PICK1
and GluR2 in the yeast two-hybrid assay. A similar mutation has been reported to prevent the binding of PICK1 to PKC alpha, indicating that the same domain of
PICK1 binds both PKC alpha and GluRs. Flag-tagged PICK1 is retained by a glutathione S-transferase (GST) fusion of the C-terminal of GluR2 (GST-ct-GluR2;
short splice variant) but not by GST-ct-GluR1 (long splice variant). Recombinant full length GluR2 is coimmunoprecipitated with flag-PICK1 using an anti-flag
antibody and flag-PICK1 is coimmunoprecipitated with an N-terminal directed anti-GluR2 antibody. Transient expression of both proteins in COS cells reveals
colocalization and an altered pattern of distribution for each protein, in comparison to the expression patterns when expressed individually. This novel interaction provides a possible regulatory
mechanism to specifically modulate distinct splice variants and may be involved in targeting the phosphorylation of short form GluRs by PKC alpha (Dev, 1999).
Cerebellar LTD requires activation of PKC and is expressed, at least in part, as postsynaptic AMPA receptor internalization. AMPA receptor internalization requires clathrin-mediated endocytosis and depends upon the carboxy-terminal region of GluR2/3. Phosphorylation of Ser-880 in this region by PKC differentially regulates the binding of the PDZ domain-containing proteins GRIP/ABP and PICK1. Peptides, corresponding to the phosphorylated and dephosphorylated GluR2 carboxy-terminal PDZ binding motif, were perfused in cerebellar Purkinje cells grown in culture. Both the dephospho form (which blocks binding of GRIP/ABP and PICK1) and the phospho form (which selectively blocks PICK1) attenuate LTD induction by glutamate/depolarization pairing, as do antibodies directed against the PDZ domain of PICK1. These findings indicate that expression of cerebellar LTD requires PKC-regulated interactions between the carboxy-terminal of GluR2/3 and PDZ domain-containing proteins (Xia, 2000).
Four PDZ domain-containing proteins, syntenin, PICK1, GRIP, and PSD95, have been identified as interactors with the kainate receptor (KAR) subunits GluR52b, GluR52c, and GluR6. Of these, it is shown that both GRIP and PICK1 interactions are required to maintain KAR-mediated synaptic function at mossy fiber-CA3 synapses. In addition, PKCalpha can phosphorylate ct-GluR52b at residues S880 and S886, and PKC activity is required to maintain KAR-mediated synaptic responses. It is proposed that PICK1 targets PKCalpha to phosphorylate KARs, causing their stabilization at the synapse by an interaction with GRIP. Importantly, this mechanism is not involved in the constitutive recycling of AMPA receptors since blockade of PDZ interactions can simultaneously increase AMPAR- and decrease KAR-mediated synaptic transmission at the same population of synapses (Hirbec, 2003).
The finding that KARs and AMPARs can bind to a common pool of PDZ proteins suggests that these proteins may play important general roles in the regulation of glutamatergic synapses. Based on the present findings and previous work on AMPARs, it is possible to speculate on the molecular mechanisms that mediate the differential regulation of AMPARs and KARs by these PDZ proteins. In this scheme, AMPARs are secured in intracellular pools via association of the GluR2 subunit with GRIP and/or ABP. These 'gripped' receptors are immobile over the time course of the electrophysiology experiments. PICK1 exchanges for GRIP and targets PKCalpha, which then phosphorylates S880 of GluR2, thereby preventing the rebinding of GRIP. The S880-phosphorylated AMPARs are mobile and available for surface expression. It is proposed that KARs are also 'gripped' by GRIP, but in this case, PICK1-targetted, PKC-dependent phosphorylation stabilizes the GRIP interaction with GluR5/6 and anchors the receptors at the postsynaptic membrane. These data are entirely consistent with the observations that blockade of either GRIP or PICK1 binding, or inhibition of PKC, results in a rapid decrease in KAR-mediated synaptic currents. It is speculated that, whereas phosphorylation of S880 of GluR2 prevents GRIP binding, phosphorylation of S880 and/or S886 of GluR52b (and/or equivalent residues of GluR6) stabilizes GRIP binding and anchors the receptors at the synapse (Hirbec, 2003).
These differences in the molecular consequences of PKC-mediated phosphorylation of AMPARs and KARs can explain the differential regulation in opposite directions of the functional synaptic responses. The results showing that, at the same population of synapses, disruption of PDZ protein interactions results in an increase in EPSCA
and a simultaneous decrease in EPSCK
suggests that these proteins may act to regulate the relative proportions of AMPARs and KARs at synapses. Physiologically, given the distinct biophysical and functional profiles of AMPARs and KARs, the dynamic regulation of these interactions will play important roles in the modulation of basal glutamatergic synaptic transmission. Furthermore, it has been reported previously that some forms of developmental and activity-dependent synaptic plasticity involve a switch from functionally expressed KARs to AMPARs. The differential effects of PDZ-interacting proteins demonstrated here on these two receptor types provide an attractive molecular mechanism to account for these developmental and activity-dependent changes in the AMPAR and KAR complement at synapses (Hirbec, 2003).
Spine morphology is regulated by intracellular signals, like PKC, that affect
cytoskeletal and membrane dynamics. This study investigated the role of MARCKS
(myristoylated, alanine-rich C-kinase substrate) in dendrites of 3-week-old
hippocampal cultures. MARCKS associates with membranes via the combined action
of myristoylation and a polybasic effector domain, which binds phospholipids
and/or F-actin, unless phosphorylated by PKC. Knockdown of endogenous MARCKS
using RNAi reduces spine density and size. PKC activation induces similar
effects, which are prevented by expression of a nonphosphorylatable mutant.
Moreover, expression of pseudophosphorylated MARCKS is, by itself, sufficient
to induce spine loss and shrinkage, accompanied by reduced F-actin content.
Nonphosphorylatable MARCKS causes spine elongation and increases the mobility of
spine actin clusters. Surprisingly, it also decreases spine density via a novel
mechanism of spine fusion, an effect that requires the myristoylation sequence.
Thus, MARCKS is a key factor in the maintenance of dendritic spines and
contributes to PKC-dependent morphological plasticity (Calabrese, 2005).
At CA1 synapses, activation of NMDA receptors (NMDARs) is required for the induction of both long-term potentiation and depression. The basal level of activity of these receptors is controlled by converging cell signals from G-protein-coupled receptors and receptor tyrosine kinases. Pituitary adenylate cyclase activating peptide (PACAP) is implicated in the regulation of synaptic plasticity because it enhances NMDAR responses by stimulating Gαs-coupled receptors and protein kinase A. However, the major hippocampal PACAP1 receptor (PAC1R) also signals via Gαq subunits and protein kinase C (PKC). In CA1 neurons, PACAP38 enhances synaptic NMDA, and evoked NMDAR, currents in isolated CA1 neurons via activation of the PAC1R, Gαq, and PKC. The signaling was blocked by intracellular applications of the Src inhibitory peptide Src(40-58). Immunoblots confirmed that PACAP38 biochemically activates Src. A Gαq pathway is responsible for this Src-dependent PACAP enhancement because it was attenuated in mice lacking expression of phospholipase C β1, it was blocked by preventing elevations in intracellular Ca2+, and it was eliminated by inhibiting either PKC or cell adhesion kinase β [CAKβ or Pyk2 (proline rich tyrosine kinase 2)]. Peptides that mimic the binding sites for either Fyn or Src on receptor for activated C kinase-1 (RACK1) also enhanced NMDAR in CA1 neurons, but their effects were blocked by Src(40-58), implying that Src is the ultimate regulator of NMDARs. RACK1 serves as a hub for PKC, Fyn, and Src and facilitates the regulation of basal NMDAR activity in CA1 hippocampal neurons (Macdonald, 2005).
Syndecan-4 (Syn4) is a heparan sulphate proteoglycan that is able to bind to some growth factors, including FGF, and can control cell migration. This study describes a new role for Syn4 in neural induction in Xenopus. Syn4 is expressed in dorsal ectoderm and becomes restricted to the neural plate. Knockdown with antisense morpholino oligonucleotides reveals that Syn4 is required for the expression of neural markers in the neural plate and in neuralised animal caps. Injection of Syn4 mRNA induces the cell-autonomous expression of neural, but not mesodermal, markers. Two parallel pathways are involved in the neuralising activity of Syn4: FGF/ERK, which is sensitive to dominant-negative FGF receptor and to the inhibitors SU5402 and U0126, and a PKC pathway, which is dependent on the intracellular domain of Syn4. Neural induction by Syn4 through the PKC pathway requires inhibition of PKCdelta and activation of PKCalpha. PKCalpha inhibits Rac GTPase and c-Jun is a target of Rac. These findings might account for previous reports implicating PKC in neural induction and suggest a link between FGF and PKC signalling pathways during neural induction (Kuriyama, 2009).
Syn4 modulates FGF signalling through its extracellular domain (containing the GAG-binding region, which will present heparin sulphates to which FGF is expected to bind) and by an effect on the transduction of intracellular signals. The data support the idea that FGF is required for neural induction and that Syn4 is a likely modulator, by showing that the inhibition of FGF receptor and of MAPK activity impair neural induction by Syn4. Syn4 could act as a co-receptor of the FGF receptor or as a presenter of the FGF ligand, through binding of FGF to the GAG side-chains, to facilitate the activation of FGF receptor (Kuriyama, 2009).
However, Syn4 also plays a separate role in neural induction involving PKC. It is proposed that this involves inhibition of PKC{delta} and activation of PKC{alpha}, and that PKC{alpha} is an inhibitor of the small GTPase Rac. Since the BMP-inhibiting effects of FGF act through MAPK, this pathway could account for the BMP-inhibition-independent role of FGF signalling in neural induction. Rac is a well-known regulator of cell migration that acts by controlling actin polymerisation, but has not previously been implicated in neural induction. Evidence that Rac can control JNK activity suggested the hypothesis that Syn4/PKC{alpha} might inhibit Rac activity by an increase in AP-1 (c-Fos/c-Jun) activity that is mediated through inhibition of JNK (Kuriyama, 2009).
PKC{alpha} has never been connected with the signalling pathways now known to be involved in neural induction. It was originally shown that PKC{alpha} is activated and translocated to the membrane during neural induction, and it was suggested that this is required to confer neural competence on the ectoderm. This study has confirmed and extended these observations by showing that expression of PKC{alpha} in ventral ectoderm or in animal caps can act as a neuralising signal and that PKC{alpha} activity is regulated by interactions with Syn4 and PKC{delta}. PKC{delta} appears to work as a repressor of PKC{alpha}, whereas Syn4 appears to be required for PKC{alpha} activity; however, it was also shown that PKC{alpha} is required for the neuralising activity of Syn4. Thus, this finding allows proposal of a link between the PKC and FGF pathways, both of which have been identified as being involved in neural induction (Kuriyama, 2009).
These observations have parallels in studies of migrating cells. Syn4 interacts with PIP2, and this stabilises the oligomeric structure of Syn4 and promotes the association of PKC{alpha} and Syn4; the catalytic domain of PKC{alpha} binds to the cytoplasmic domain of Syn4, and PKC{alpha} is 'superactivated'. This interaction between PKC{alpha} and Syn4 provides a satisfactory explanation for the observation that neural induction by Syn4 requires PKC{alpha} and vice versa. In addition, during cell migration, PKC{delta} phosphorylates Syn4, decreases its affinity for PIP2 and abolishes its capacity to activate PKC{alpha}. This study has found a similar negative regulation between PKC{alpha} and PKC{delta} during early neural plate development (Kuriyama, 2009).
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