Funding, Deals & Partnerships: BIOLOGICS & MEDICAL DEVICES; BioMed e-Series; Medicine and Life Sciences Scientific Journal – http://PharmaceuticalIntelligence.com
Pancreatic cancer survival is determined by ratio of two enzymes, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 1: Next Generation Sequencing (NGS)
Reporter and Curator: Dr. Sudipta Saha, Ph.D.
Protein kinase C (PKC) isozymes function as tumor suppressors in increasing contexts. These enzymes are crucial for a number of cellular activities, including cell survival, proliferation and migration — functions that must be carefully controlled if cells get out of control and form a tumor. In contrast to oncogenic kinases, whose function is acutely regulated by transient phosphorylation, PKC is constitutively phosphorylated following biosynthesis to yield a stable, autoinhibited enzyme that is reversibly activated by second messengers. Researchers at University of California San Diego School of Medicine found that another enzyme, called PHLPP1, acts as a “proofreader” to keep careful tabs on PKC.
The researchers discovered that in pancreatic cancer high PHLPP1 levels lead to low PKC levels, which is associated with poor patient survival. They reported that the phosphatase PHLPP1 opposes PKC phosphorylation during maturation, leading to the degradation of aberrantly active species that do not become autoinhibited. They discovered that any time an over-active PKC is inadvertently produced, the PHLPP1 “proofreader” tags it for destruction. That means the amount of PHLPP1 in patient’s cells determines his amount of PKC and it turns out those enzyme levels are especially important in pancreatic cancer.
This team of researchers reversed a 30-year paradigm when they reported evidence that PKC actually suppresses, rather than promotes, tumors. For decades before this revelation, many researchers had attempted to develop drugs that inhibit PKC as a means to treat cancer. Their study implied that anti-cancer drugs would actually need to do the opposite — boost PKC activity. This study sets the stage for clinicians to one day use a pancreatic cancer patient’s PHLPP1/PKC levels as a predictor for prognosis, and for researchers to develop new therapeutic drugs that inhibit PHLPP1 and boost PKC as a means to treat the disease.
The ratio — high PHLPP1/low PKC — correlated with poor prognoses: no pancreatic patient with low PKC in the database survived longer than five-and-a-half years. On the flip side, 50 percent of the patients with low PHLPP1/high PKC survived longer than that. While still in the earliest stages, the researchers hope that this information might one day aid pancreatic diagnostics and treatment. The researchers are next planning to screen chemical compounds to find those that inhibit PHLPP1 and restore PKC levels in low-PKC-pancreatic cancer cells in the lab. These might form the basis of a new therapeutic drug for pancreatic cancer.
neurohumoral activity and vesicles vital and essential for all functions related to
cell movement,
migration, and
contraction.
Calmodulin and Protein Kinase C Increase Ca–stimulated Secretion by Modulating
Membrane-attached Exocytic Machinery
YA Chen, V Duvvuri, H Schulmani, and RH.Scheller‡
From the ‡Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology,
and the Department of Neurobiology, Stanford University School of Medicine, Stanford, CA
The molecular mechanisms underlying the Ca2+ regulation of hormone and neurotransmitter release
are largely unknown.
Using a reconstituted [3H]norepinephrine release assay in permeabilized PC12 cells, we found
essential proteins that support the triggering stage of Ca2+-stimulated exocytosis
are enriched in an EGTA extract of brain membranes.
Fractionation of this extract allowed purification of two factors that stimulate secretion
in the absence of any other cytosolic proteins.
These are calmodulin and protein kinase Ca (PKCa). Their effects on secretion were
confirmed using commercial and recombinant proteins.
Calmodulin enhances secretion
in the absence of ATP, whereas
PKC requires ATP to increase secretion, suggesting that
phosphorylationis involved in PKC-mediated stimulation
but not calmodulin mediated stimulation.
Both proteins modulate
The half-maximal increase was elicited by 3 nM PKC and 75 nM calmodulin.
These results suggest that calmodulin and PKC increase Ca2+-activated exocytosis by
directly modulating the membrane- or cytoskeleton-attached exocytic machinery downstream of Ca2+ elevation.
The abbreviations used are:
NE, norepinephrine; PKC, protein kinase C; CaM, calmodulin; SNAP-25, synaptosome-associated protein of 25 kDa; CAPS, calcium-dependent activator protein for secretion; SNARE, SNAP (soluble N-ethylmaleimide-sensitive factor attachment proteins) receptor; CaMK, Ca2+/calmodulin-dependent protein kinase; PAGE, polyacrylamide gel electrophoresis; AMP-PNP, adenosine 59-(b,g- imido) triphosphate; HA, hydroxyapatite
*This work was supported in part by Conte Center Grant MH48108. The costs of publication of
this article were defrayed in part by the payment of page charges. This article has been marked
“advertisement” in accordance with 18 U.S.C. Section 1734.
The molecular mechanisms of presynaptic vesicle release have been extensively examined
by a combination of
biochemical,
genetic, and
electrophysiological techniques.
A series of protein-protein interaction cascadeshave been proposed to lead to vesicle
docking and fusion (1–3). The SNARE protein family, including
syntaxin, SNAP-25, and vesicle-associated membrane protein
(VAMP, also called synaptobrevin),
plays an essential role in promoting membrane fusion, and
is thought to comprise the basic fusion machinery (4, 5).
In Ca2+-stimulated exocytosis, many additional proteins are important in the Ca2+ regulation
of the basic membrane trafficking apparatus.Calcium
not only triggers rapid fusion of release-competent vesicles, but is also involved in
earlier processes which replenish the pool of readily releasable vesicles (6).
Furthermore, it appears to be critical in initiating several forms of synaptic plasticity including
post-tetanic potentiation (7).
The molecular mechanisms by which Ca2+ regulates these processes is not well understood.
PC12 cells have often been utilized to study Ca2+-activated exocytosis, as
they offer a homogeneous cell population that possesses the same basic exocytic machinery as neurons (8).
In this study, we used an established cracked cell assay, in which
[3H]norepinephrine (NE)1 labeled PC12 cells are
permeabilized by mechanical “cracking” and
then reconstituted for secretion of NE in the presence of test proteins (9).
Transmitter-filled vesicles and intracellular cytoskeletal structures
remain intact in these cells,
while cytosolic proteins leak out (10).
These cracked cells readily release NE upon addition of
ATP,
brain cytosol, and
1 mM free Ca2+
at an elevated temperature.
We term this a “composite assay,” as
all essential components are added into one reaction mixture.
Alternatively, cracked cells can be
first primed with cytosol and ATP, washed, then
reconstituted for NE release with cytosol and Ca2+ (11).
This sequential priming-triggering protocol is useful
for determining whether a protein acts early or late in the exocytic pathway, and
whether its effect is dependent on Ca2+ or ATP.
This semi-intact cell system serves as
a bridge between an in vitro system comprised of purified components, and
electro-physiological systems that monitor release in vivo.
It provides information on protein functions in a cell with an intact membrane infrastructure while being easily manipulatable.
Ca2+ regulation by membrane depolarization is no longer a concern, as intra-cellular Ca2+ concentrationcan be controlled by a buffered solution.
Indirect readout of neurotransmitter release using a postsynaptic cell is replaced by
direct readout of [3H]NEreleased into the buffer.
Complications associated with interpreting overlapping
exo- and endocytotic signals are also eliminated as only one round of exocytosis is measured.
Finally, concentration estimates are likely to be accurate, since
added compounds do not need to diffuse long distances along axons and dendrites to their sites of action.
Using this assay, several proteins required for NE release have been purified from rat brain cytosol, including
phosphatidyl-inositol transfer protein (12),
phosphatidylinositol-4-phosphate 5-kinase (13), and
calcium-dependent activator protein for secretion (CAPS) (9).
The validity of the cracked cell system is confirmed by the finding that
phosphatidylinositol transfer protein and CAPS are mammalian homologues of
yeast SEC14p (12) and
nematode UNC31p, respectively (14),
both proteins involved in membrane trafficking (15, 16).
Calmodulin is the most ubiquitous calcium mediator in eukaryotic cells, yet its involvement in membrane trafficking has not been well established. Some early studies showed
that calmodulin inhibitors (17–19), anti-calmodulin antibodies (20,21),
However, in other studies, calmodulin-binding peptides and an anti-calmodulin antibody led to the conclusionthat
calmodulin is only involved in endocytosis,
not exocytosis (23).
More recently, it was reported that
Ca2+/ calmodulin signals the completion of docking and
triggers a late step of homotypic vacuole fusion in yeast,
thus suggesting an essential role for Ca2+/calmodulin in constitutive intracellular membrane fusion (24).
If calmodulin indeed plays an important role in exocytosis,
a likely target of calmodulin is
Ca2+/calmodulin-dependent protein kinase II (CaMKII),
a multifunctional kinase that is found on synaptic vesicles (25) and
has been shown to potentiate neurotransmitter release (26, 27).
Another Ca2+ signaling molecule, PKC, has also been implicated in regulated exocytosis.
In various cell systems, it has been shown that
the phorbol esters stimulate secretion (28, 29).
It is usually assumed that phorbol esters effect on exocytosis is
through activation of PKC,
but Munc13-1 was recently shown to be a presynaptic phorbol ester receptor that enhances neurotransmitter release (30, 31),
which complicates the interpretation of some earlier reports. The mode of action of PKC remains controversial. There is evidence
that PKC increases the intracellular Ca2+ levels by modulating plasma membrane Ca2+ channels (32, 33),
that it increases the size of the release competent vesicle pool (34, 35), or
that it increases the Ca2+ sensitivity of the membrane trafficking apparatus (36).
no consensus on these issues has been reached.
PKC substrates that have been implicated in exocytosis include
SNAP-25 (37),
synaptotagmin (28),
CAPS (38), and
nsec1 (39).
It is believed that upon phosphorylation, these PKC substrates might
interact differently with their binding partners, which, in turn,
leads to the enhancement of exocytosis.
In addition, evidence is accumulating that PKC and calmodulin interfere with each others actions, as
PKC phosphorylation sites are embedded in the calmodulin-binding domains of substrates such as
neuromodulin and
neurogranin (40).
It is therefore possible that PKC could modulate exocytosis via
a calmodulin-dependent pathway by synchronously releasing calmodulin from storage proteins.
In this study, we fractionated an EGTA extract of brain membranes in order to identify active components that could reconstitute release in the cracked cell assay system. We identified calmodulin and PKCas two active factors. Thus, we demonstrate that
calmodulin and PKC play a role in the Ca2+ regulation of exocytosis, and provide further insight into the mechanisms of their action.
DISCUSSION
In this study, we first identified an EGTA extract of brain membranes as a protein source
capable of reconstituting Ca2+- activated exocytosis in cracked PC12 cells.
EGTA only extracts a small pool of Ca2+-dependent membrane-associating proteins,
it served as an efficient initial purification step.
Further protein chromatography led to the identification of two active factors in the starting extract,
calmodulin and PKC,
which together accounted for about half of the starting activity.
Upon confirmation with commercially obtained proteins, this result unambiguously demonstrated
that calmodulin and PKC mediate aspects of Ca2+-dependent processes in exocytosis.
The finding that brain membrane EGTA extract alone is able
to replace cytosol in supporting Ca2+-triggered NE secretion
in PC12 cells is somewhat surprising. We suggest that the likely explanation is 2-fold.
some cytosolic proteins essential for exocytosis have a membrane-bound pool within permeabilized cells, whose activity might be sufficient for a normal level of exocytosis.
although the 100,000 3 g membrane pellet was washed to remove as many cytosolic proteins as possible,
some cytosolic proteins that associate with membranes in a
Ca2+-independent manner are probably present in the membrane EGTA extract.
these proteins likely constitute only a small percentage of the proteins in the extract, as
the characteristics of the activity triggered by the membrane extract
are quite different to that of cytosol (Fig. 2).
Using an unbiased biochemical purification method, we demonstrated that
calmodulin and PKC directly modulate the exocytotic machinery downstream of Ca2+ entry
they signal through membrane-attached molecules to increase exocytosis.
These targets include integral and peripheral membrane proteins, and cytosolic proteins that have a significant
membrane-bound pool. The modest stimulation by calmodulin and PKC on secretion might suggest a regulatory
role. However, it is also possible that some intermediates in their signaling pathways are in limiting amounts in the cell ghosts, so that their full effects were not observed. Half-maximal stimulation was obtained at
about 3 nM for PKC and
at about 75 nM for calmodulin.
This is consistent with an enzymatic role for PKC, and predicts a high-affinity interaction between
calmodulin and its substrate protein.
Ca2+ regulates exocytosis at many different levels. Prior studies indicated that Ca2+ signaling occurs in
the priming steps as well
as in triggering steps (49, 50).
Our priming triggering protocol
does not allow Ca2+-dependent priming events to be assayed, as EGTA is present in the priming reaction.
a different approach revealed the existence of both high and low Ca2+-dependent processes (Fig. 2).
this analysis indicated that late triggering events require high [Ca2+], whereas
early priming events require low [Ca2+]. If, as proposed, there is
a pronounced intracellular spatial and temporal [Ca2+] gradient from
the point of Ca2+ entry during depolarization (51),
perhaps triggered events occur closer to the point of Ca2+ entry,
while Ca2+-dependent priming events occur further away from the point of Ca2+ entry.
Distinct Ca2+ sensors at these stages might be appropriately tuned to different [Ca2+] to handle different tasks.
By analyzing the Ca2+ sensitivity of calmodulin-and PKC-stimulated release, we addressed the question of
whether calmodulin and PKC plays an early or a late role in vesicle release.
they both require relatively high [Ca2+] (Fig. 8B),
implying that calmodulin and PKC both mediate late triggering events, consistent with some earlier reports
(34, 52, 53).
In addition, it is interesting to note that PKC does not alter the calcium sensitivity of release in cracked cells, in contrast
to observations from the chick ciliary ganglion (36). Therefore, in contrast to previous electrophysiological studies (28),
we are able to limit the possible modes of PKC action in our system to an increase in the readily releasable vesicle pool or
release sites, or an enhancement of the probability of release of individual vesicles upon Ca2+ influx.
The experiments assaying the calcium sensitivity of release (Figs. 2, 5, and 8) demonstrated
a drop in release at very high [Ca2+].
This decline in release at high [Ca2+] has been previously reported (49, 51), and may represent
the true Ca2+ sensitivity of the Ca2+-sensing mechanism inside cells.
However, in our system, it could also be due to the activation of a variety of Ca2+ -activated proteases, as experiments are usually performed in the presence of crude extracts, which include unsequestered proteases.
What might the molecular targets of PKC and calmodulin be? An obvious calmodulin target molecule is CaMKII.
but calmodulin’s effect on exocytosis is ATP-independent, rendering the involvement of a kinase unlikely.
Calmodulin has also been shown to associate with
synaptic vesicles in a Ca2+-dependent fashion through synaptotagmin (54),
probably by binding to its C-terminal tail (55), and to promote Rab3A dissociation from synaptic vesicles (56).
However, there was little calcium-dependent binding of calmodulin to synaptotagmin
either on synaptic vesicles, in a bead binding assay with recombinant proteins,
or in a calmodulin overlay (data not shown).
In addition, using immobilized calmodulin, we did not see
significant Ca2+-dependent pull-down of synaptotagmin or Rab3A from rat brain extract (data not shown).
Recent work has suggested three other candidate targets for calmodulin, Munc13, Pollux, and CRAG (57).
Pollux has similarity to a portion of a yeast Rab GTPase-activating protein, while
CRAG is related to Rab3 GTPase exchange proteins.
Further work is required to investigate the role of their interactions with calmodulin in vivo.
The recent report that calmodulin mediates yeast vacuole fusion (24) is intriguing, as it raises the possibility that
calmodulin, a highly conserved ubiquitous molecule,
may mediate many membrane trafficking events.
It is not yet known if
the effector molecule of calmodulin is conserved or variable across species and different trafficking steps.
It is enticing to propose a model for Ca2+ sensing whereby
calmodulin is a high affinity Ca2+ sensor for both constitutive and regulated membrane fusion.
In the case of constitutive fusion, calmodulin may be the predominant Ca2+ sensor.
In the case of slow, non-local exocytosis of large dense core granules, an additional requirement for
the concerted actions of other molecule(s) that are better tuned to intermediate rises in [Ca2+] might exist.
At the highly localized sites of fast exocytosis of small clear vesicles where high [Ca2+] is reached,
specialized low affinity sensor(s) are likely required
in addition to calmodulin to achieve membrane fusion.
Therefore, although calmodulin participates in multiple types of vesicle fusion,
the impact of Ca2+ sensing by calmodulin on vesicle release likely varies.
Due to the fact that calmodulin binding to some proteins can be modulated by PKC phosphorylation, one might suspect
PKC action on exocytosis proceeds through a calmodulin-dependent pathway.
but the effects of calmodulin and PKC are additive within our system,
suggesting that PKC does not act by releasing calmodulin from a substrate
that functions as a calmodulin storage protein.
How Ca2+ regulates presynaptic vesicle release has been an open question for many years. By
identifying calmodulin and PKC as modulators of Ca21-regulated exocytosis and clarifying their functions,
we have extended our knowledge of the release process.
While the basic machinery of membrane fusion is becoming better understood,
the multiple effects of Ca2+ on exocytosis remain to be elucidated at the molecular level.
In addition, the ways that Ca2+ regulation may be important to
the mechanisms of synaptic plasticity in the central nervous system
EXPERIMENTAL PROCEDURES
Materials Rat Brain Cytosol Preparation Membrane EGTA Extract Preparation
Cracked Cell Assay
PC12 cells were maintained and [3H]NE labeled as described previously (11). Labeled cells were harvested by pipetting with ice-cold potassium glutamate buffer (50 mM Hepes, pH 7.2, 105 mM potassium glutamate, 20 mM potassium acetate, 2 mM EGTA) containing 0.1% bovine serum albumin. Subsequent manipulations were carried out at 0–4 °C. Labeled cells (1–1.5 ml/dish) were mechanically permeabilized passage through a stainless steel homogenizer. The cracked cells were adjusted to 11 mM EGTA and
incubated on ice for 0.5–3 h, followed by three washes in which
the cells were centrifuged at 800 3 g for 5 min and
resuspended in potassium glutamate buffer containing 0.1% bovine serum albumin.
Composite Assay
Each release reaction contains 0.5–1 million cracked cells, 1.5 mM free Ca2+, 2 mM MgATP,
and the protein solution to be tested in potassium glutamate buffer. Release reactions were initiated
by incubation at 30 °C and terminated by returning to ice. The supernatant of each reaction was
isolated by centrifugation at 2,500 3 g for 30 min at 4 °C, and the
released [3H]NE was quantified by scintillation counting (Beckman LS6000IC).
Cell pellets were dissolved in 1% Triton X-100, 0.02% azide and similarly counted. NE release
was calculated as a percentage of total [3H] in the supernatant.
Priming Assay
A priming reaction contains about
1–2 million cracked cells,
2 mM MgATP, and
the protein solution to be tested.
Ca2+ is omitted.
The primed cells were spun down, washed once with fresh potassium glutamate buffer, and
distributed into two triggering reactions, each containing
rat brain cytosol and free Ca2+
The triggering reaction was performed at 30 °C for 3 min, and
the NE release was measured
as in a composite assay.
Triggering Assay
Cracked cells were primed …, centrifuged, washed …, and
distributed into triggering reactions containing
1.5 mM free Ca2+ and the protein solution
To inhibit any ATP dependent activity in the triggering reaction, an
ATP depletion system of
hexokinase
MgCl2,
glucose or
a non-hydrolyzable ATP analogue AMPPNP
was added into the triggering reaction. NE release was measured as above.
Free Ca2+ Concentration Determination
The range of Ca2+free in the release reaction (Fig. 2B) was achieved
by adding Ca2+ into potassium glutamate buffer to reach final [Ca2+] total values of
0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 1.9, and 2.0 mM.
The pH of the reaction was 7.24 when no Ca2+ was added and
7.04 when 2.0 mM Ca2+ was added,
in the absence ofprotein extracts or cracked cells.
Fig. 2B. The range of [Ca21]free in the release reaction (Fig. 2B)
Free Ca2+ concentrations were determined using video microscopic
measurements of fura-2 fluorescence (41). [Ca2+]free was calculated from the equation
[Ca2+]free 5 Kd*3 (R 2 Rmin)/(Rmax 2 R)(42).
The values of Rmin, Rmax, and Kd* were determined in the following solutions:
potassium glutamate buffer (PGB) containing
8 x 3 10^6 cracked cells/ml, 2 mM MgATP (PGB+CC)
1) Rmin: PGB+CC and 10 mM additional EGTA;
2) Rmax: PBG+CC, and 10 mM total Ca2+;
3) Kd*: PGB+CC, 28 mM additional EGTA, and 18 mM total Ca2+, pH 7.2
([Ca2+]free 5 = 169 nM, determined in the absence of cells and MgATP
based on fura-2 calibration in cell-free solutions).
These solutions were
incubated at 37 °C ,
mixed with fura-2 pentapotassium salt
(100 mM; Molecular Probes, Eugene, OR), and
imaged.
This procedure allowed us to take into account
changes in fura-2 properties
caused by the presence of
permeabilized cells.
Duplicate measurements of the above range of [Ca2+] total gave
the following average [Ca2+] free values:
106, 146, 277, 462, 971, 1468, 1847, and 2484 nM.
Purification of Active Proteins
All procedures were carried out at 4 °C or on ice. Membrane EGTA extract of one or two bovine brain(s) was
filtered through cheesecloth and
loaded overnight onto a column packed with DEAE-Sepharose
CL-6B beads (Amersham Pharmacia Biotech).
The column was then
washed with (20 mM Hepes, pH 7.5, 0.25 mM sucrose, 2 mM EGTA, 1 mM dithiothreitol) and
step eluted with 10 column volumes of elution buffer
(20 mM Hepes, pH 7.5, 2 mM EGTA, 400 mM KCl, 1 mM dithiothreitol). 100 ml of every other fraction was
dialyzed overnight into PGB, and
tested in a composite release assay for activity.
The active fractions were pooled and dialyzed into zero salt buffer (20 mM Hepes, pH 7.5, 2 mM EGTA) and
batch bound to 10 ml of Affi-Gel Blue beads (Bio-Rad) or DyeMatrex-Green A beads (Amicon)
Blue beads were used in earlier experiments, and Green beads were used later to
specifically deplete CAPS, which was known to bind to Green beads (9).
The unbound material was
collected,
concentrated to about 2 ml using a Centriprep-10 (Amicon), and
loaded onto a 120-ml HiPrep Sephacryl S-200 gel filtration column
(Amersham Pharmacia Biotech).
Samples were run on the S-200 column in PGB at a flow rate of 7 ml/h.
10–50 ml of every other fraction was tested for
activity in the cracked cell composite assay, and
two peaks of activity were observed (Fig. 3).
The first peak of activity had a predicted molecular mass of 85 kDa.
The corresponding material was
adjusted to 10 mM potassium phosphate concentration (pH 7.2) and
loaded onto a 1-ml column packed with hydroxyapatite Bio-Gel HT (Bio-Rad).
The bound material was
eluted with a linear K-PO4 gradient from 10 to 500 mM (pH 7.2)
at a flow rate of about 0.1 ml/min, and
0.4–0.5-ml fractions were collected.
each fraction was dialyzed into PGB and
tested for activity.
The fractions were also analyzed by
SDS-PAGE and silver staining (Sigma silver stain kit).
The active material was concentrated and resolved
on an 8% poly-acrylamide gel.
Two Coomassie-stained protein bands that matched the activity profile (Fig. 6)
were excised from the gel,
sequenced by the Stanford PAN facility.
The two polypeptide sequences obtained from the upper band were:
LLNQEEGEYYNVPIXEGD
IRSTLNPRWDESFT.
The only bovine protein that contains both polypeptides is PKCa.
The four polypeptide sequences obtained from the lower band were:
YELTGKFERLIVGLMRPPAY,
LIEILASRTNEQIHQLVAA,
MLVVLLQGTREEDDVVSEDL, and
EMSGDVRDVFVAIVQSVK.
Based on these sequences, the protein band was
unambiguously identified to be bovine annexin VI.
The second S-200 peak has a predicted molecular mass of 25 kDa.
The corresponding material was
dialyzed into zero salt buffer
(20 mM Tris, pH 7.5, 1 mM EGTA) and
injected onto a Mono-Q HR 5/5 FPLC column
(Pharmacia).
The FPLC runwas performed at 18 °C at 1 ml/min and
1-ml fractions were collected
with a linear salt gradient from 0 to 1 M KCl over 71 ml.
The fractions containing proteins (determined by A280) were
dialyzed into PGB and
tested in the cracked cell assay.
Western Blot
Anti-calmodulin antibody and anti-PKC antibody were used, and
ECL (Amersham) was used for detection.
RESULTS
A Membrane EGTA Extract Supports NE Release
Brain cytosol, prepared as the supernatant of the brain homogenate,
effectively stimulates NE release
in the cracked cell assay (Fig. 1) as previously shown (9).
We wondered whether crude extracts other than cytosol
could support NE release, and we focused on
extractable peripheral membrane proteins.
We found that a salt or EGTA extract of brain membranes,
membranes defined as the
100,000 3 g pellet of the crude homogenate,
reconstituted secretion in the absence of cytosol.
the salt extract only slightly enhanced NE release
above background (data not shown), the
EGTA extract not only stimulated NE release to a high level,
similar to that supported by cytosol, but also
had a higher specific activity than cytosol (Fig. 1).
FIG. 1. The EGTA extract of brain membranes can support NE release in the absence of cytosol. Rat brain membrane EGTA extract (closed triangles) and rat brain cytosol (closed squares) were prepared as described under “Experimental Procedures.” NE release was measured in a composite reaction mixture of cracked cells, MgATP, Ca2+, and the indicated amount of crude extracts.
The ability of the membrane EGTA extract to support secretion is consistent with the fact that
following cracking, the cells are immediately extracted with EGTA, and are presumably
devoid of most membrane EGTA-extractable factors.
This also suggests that these factors, some of which are probably
Ca2+-dependent membrane-associating proteins,
participate in Ca2+- triggered exocytosis.
The Membrane EGTA Extract Is Enriched in Triggering Fators
NE release in cracked cells can be resolved into two sequential stages,
an ATP-dependent priming stage and
an ATP-independent Ca21-dependent triggering stage (11), and
proteins can be tested for activity in either stage.
An effect in priming indicates
an early role for the protein, and
an effect in triggering a late ATP-independent role.
Since the protein composition of the
membrane EGTA extract and cytosol are different,
we tested whether they had different activities
in the priming stage versus the triggering stage.
We found that the membrane EGTA extract is enrichedin factors that
act during triggering stage of NE release, as
the same amount of protein from the membrane EGTA extract as cytosol
gave a higher stimulation in the triggering assay, but
not in the priming assay (Fig. 2A).
Regular cytosol is prepared in a buffer containing 2 mM EGTA, and thus
presumably contains some of the proteins present in the membrane EGTA extract.
Cytosol prepared in the absence of EGTA showed an even lower specific activity
in the triggering assay compared with regular cytosol (Fig. 2A).
Identification of Calmodulin as an Active Triggering Factor in the EGTA Extract
Biochemical fractionation of the bovine brain membrane EGTA extract was carried out
to identify the active components capable of reconstituting NE release.
Activity was assayed in a composite reaction mixture containing
cracked cells,
ATP,
Ca2+, and
the test protein(s).
Except for the presence of bovine serum albumin in the basal buffer,
no other proteins were added to the cell ghosts except for the test protein(s).
Initial tests indicated that at least
part of the activity in the membrane EGTA extract binds to and
can be efficiently eluted from an anion exchanger and hydroxyapatite resin,
but does not bind to Amicon color resins.
The starting material was, therefore, sequentially purified using
DEAE, Affi-Gel Blue (or Matrex Green-A), and gel filtration chromotography.
Gel filtration fractionation indicated the presence of two peaks of activity with
predicted molecular masses of 25 and 85 kDa, respectively (Fig. 3).
FIG. 3. Gel filtration chromatography reveals two stimulatory factors in the membrane EGTA extract.
In order to purify the active component(s) in the membrane EGTA extract, the crude extract from one bovine brain was fractionated chromatographically (see Experimental Procedures” for details). Fractions from a Sephacryl S-200 gel filtration column were tested for their activity in stimulating NE release in the composite assay. The two activity peaks have predicted molecular masses of 85 and 25 kDa, respectively. The arrows indicate the retention volume of standard proteins run on the same column.
The low molecular weight active factor was purified to homogeneity, as judged by a
Coomassie-stained SDS-PAGE gel, after a subsequent Mono-Q fractionation (Fig. 4).
FIG. 4. The low molecular wen.ight active factor is calmodulin
A, the membrane EGTA extract from one bovine brain (Start) was subjected to sequential fractionation on DEAE, Blue A, and
Sephacryl S-200 columns. The pooled material containing the activity after each chromotographic step was analyzed by SDS-
PAGE and Coomassie staining. The arrowheads indicate the presence of calmodulin in all the lanes. Calmodulin shows a
mobility shift depending on whether or not Ca2+ is present during electrophoresis (see panel C). B, the active material pooled from Sephacryl S-200 was fractionated on a Mono-Q FPLC column and the fractions
(5 ml/fraction) were tested for activity in a composite assay. The activity peak is shown. C,the active Mono-Q fractions (5 ml/fraction) were subjected to SDS-PAGE in the presence of 1 mM EGTA or 0.1 mM Ca2+,
and the gels stained with Coomassie Blue. D, fraction 47 (1 ml) was probed by Western blotting with a monoclonal anti-calmodulin antibody. No Ca2+ or EGTA was
added during SDS-PAGE.
We reasoned that the protein might be calmodulin (43) based on the following:
1) It is a relatively small protein (14–18 kDa) that is abundant in the
starting extract (Fig. 4A).
2) It elutes at a very high salt concentration (0.41 M KCl) on the
Mono-Q column.
3) It stains negatively in silver stain (data not shown).
4) Its electrophoretic mobility shifts depending on the presence or
absence of Ca21 (Fig. 4C).
A Western blot with an anti-calmodulin monoclonal antibody gave a
positive signal (Fig. 4D), confirming our prediction.
Properties of Calmodulin-stimulated Exocytosis
We used commercial calmodulin or bacterially expressed recombinant calmodulin to confirm our purification result; both sources of authentic calmodulin stimulated NE release as expected. Moreover, we found that calmodulin stimulates secretion in a triggering assay as well as in a composite assay (Fig. 5A).
The half-maximal increase was at 75 nM (250 ng/200 ml) final calmodulin concentration. This is within the broad
range of affinities between calmodulin and its various targets and suggests that the interaction between
calmodulin and its target molecule in exocytosis is in the physiological range. When the triggering reaction was
performed at different Ca2+ concentrations, calmodulin increased NE release only at high [Ca2+] (0.4 – 2 mM)
similar to the crude EGTA extract (Fig. 5B),
suggesting that calmodulin contributes to the triggering activity of the membrane EGTA extract. Calmodulin’s affinity for Ca2+ has
been reported to be around 1 mM (25),
consistent with the Ca2+ requirementfor
calmodulin-stimulated secretion that we observed.
FIG. 5. Calmodulin stimulates NE release in the triggering stage. A, calmodulin (obtained from Sigma) increased NE release in the triggering assay in a dose-dependent fashion, in the absence of ATP
or any other cytosolic proteins. In this particular experiment, the
maximal release achieved by addition of rat brain cytosol was 46.5%.
B,the triggering assay was performed with different concentrations
of free Ca2+. Calmodulin (3 mg bacterially expressed recombinant
protein; closed squares) increased NE release with a similar Ca2+
sensitivity to rat brain membrane EGTA extract (10 mg; closed
triangles), as compared with conditions in which no protein was
added (open squares).
Western analysis with commercial protein as standards indicated that calmodulin
constitutesabout 5% of total proteins in the rat brain membrane EGTA extract
and about 2% of total proteins in the rat brain cytosol(data not shown).
In addition, a significant amount of calmodulin appears to be left
in the washed cell ghosts (data not shown).
Based on the activity of saturating levels of
pure calmodulin (releasing 6–10% of total [3H]NE)
and crude EGTA extract (releasing ;45% of total [3H]NE),
we estimated that
calmodulin accounts for 13–22% of total activity of the extract.
Consistent with this,
a high affinity calmodulin-binding peptide (CaMKIIa(291–312) (44), used at 5 mM) and
an anti-calmodulin antibody (2 mg/200 ml)
inhibited about 20% of the membrane EGTA extract-stimulated release (6.7 mg of extract added; data not shown).
We showed that calmodulin increased NE release
in the triggering stage.
Since regular triggering reactions were performed
in the absence of any added ATP,
this suggests that
calmodulin enhanced secretion in an ATP-independent fashion.
Furthermore, residual ATP in the cell ghosts did not play a role, since
addition of a hexokinase ATP depletion system that
can deplete millimolar concentrations of ATP
within a few minutes (11) had little effect, as did
addition of 5 mM AMPPNP,
which blocks ATP-dependent enzymatic activity (Fig.8A).
Therefore, we ruled out the possibility that a kinase mediates calmodulin’s effect.
FIG. 8. PKC and calmodulin stimulate the late triggering reaction in
an ATP-dependent and ATP-independent manner respectively. A, triggering assays were performed to test the activity of calmodulin
(recombinant; black bars) and PKC (purified rat brain PKC from
Calbiochem; shaded bars) in the absence of ATP. A regular triggering
assay is done in the absence of ATP (2ATP). To deplete residual ATP
in the cells, hexokinase-based ATP depletion was employed (1Hexo).
Alternatively, 5 mM AMP-PNP (1AMP-PNP) was added in the triggering
reaction. Under all three conditions, calmodulin increased release
as compared with the background (buffer only; white bars), whereas
PKC did not. B, NE release in a composite assay was measured with varying
concentrations of free Ca2+ in the presence of 10 mg of calmodulin
(recombinant; closed triangles), 70 ng of PKC (purified rat brain PKC
from Calbiochem; closed squares), or buffer only (open squares).
A series of calmodulin mutants from Paramecium and chicken were tested
for their ability to enhance Ca2+-stimulated secretion, and
none of the mutations abolished the calmodulin effect (data not shown).
These mutations include
S101F, M145V, E54K, G40E/D50N, V35I/D50N within Paramecium
calmodulin (45), and M124Q, M51A/V55A, and M51A/V55A/L32A
within chicken calmodulin (46, 47).
The Paramecium calmodulin mutants are the result of
naturally occurring mutations that result in aberrations in their behavior.
These mutants can be grouped into two categories according to their
behavior, reflecting their loss of either
a Ca2+-dependent Na1 current (calmodulin N-terminal lobe mutants: E54K, G40E/D50N, and V35I/D50N) or
a Ca21-dependent K1 current (calmodulin C-terminal lobe mutants: S101F and M145V) (45).
The chicken calmodulin mutants have been shown to
differentially activate myosin light chain kinase (M124Q, M51A/V55A, and M51A/V55A/L32A),
CaMKII (M124Q), and CaMKIV (M124Q),
and the mutated residues are thought to be important in
Other related articles published in this Open Access Online Scientific Journal include the following:
The role of ion channels in Na(+)-K(+)-ATPase: regulation of ion transport across the plasma membrane has been studies by our Team in 2012 and 2013. Chiefly, our sources of inspiration were the following:
1. 2013 Nobel work on vesicles and calcium flux at the neuromuscular junction Machinery Regulating Vesicle Traffic, A Major Transport System in our Cells The 2013 Nobel Prize in Physiology or Medicine is awarded to Dr. James E. Rothman, Dr. Randy W. Schekman and Dr. Thomas C. Südhof
for their discoveries of machinery regulating vesicle traffic,
a major transport system in our cells.
This represents a paradigm shift in our understanding of how the eukaryotic cell, with its complex internal compartmentalization, organizes
the routing of molecules packaged in vesicles
to various intracellular destinations,
as well as to the outside of the cell
Specificity in the delivery of molecular cargo is essential for cell function and survival.
3. Professor David Lichtstein, Hebrew University of Jerusalem, Dean, School of Medicine
Lichtstein’s main research focus is the regulation of ion transport across the plasma membrane of eukaryotic cells.
His work led to the discovery that specific steroids that have crucial roles, as
the regulation of cell viability,
heart contractility,
blood pressure and
brain function.
His research has implications for the fundamental understanding of body functions,
as well as for several pathological states such as
heart failure, hypertension
and neurological and psychiatric diseases.
Physiologist, Professor Lichtstein, Chair in Heart Studies at The Hebrew University elected
Dean of the Faculty of Medicine at The Hebrew University of Jerusalem
4. Professor Roger J. Hajjar, MD at Mount Sinai School of Medicine
Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension
and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
6. Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
This study presents the possible correlation between Myocardial Ischemia (Coronary Artery Disease (CAD)) aka Ischemic Heart Disease (IHD) and single-nucleotide polymorphisms (SNPs) genes encoding several regulators involved in Coronary Blood Flow Regulation (CBFR), including
ion channels acting in vascular smooth muscle and/or
endothelial cells of coronary arteries.
They completely analyzed exon 3 of both KCNJ8 and KCNJ11 genes (Kir6.1 and Kir6.2 subunit, respectively) as well as
the whole coding region of KCN5A gene (Kv1.5 channel).
The work suggests certain genetic polymorphisms may represent a non-modifiable protective factor that could be
used to identify individuals at relatively low-risk for cardiovascular disease
an independent protective role of the
rs5215_GG against developing CAD and
a trend for rs5219_AA to be associated with protection against coronary microvascular dysfunction
Other related articles published on this Open Access Online Scientific Journal include the following:
Part XII: Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN