From the Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney 2010, Australia
Received for publication, February 14, 2001, and in revised form, March 22, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In pancreatic islets the activation
of phospholipase C (PLC) by the muscarinic receptor agonist
carbamyolcholine (carbachol) results in the hydrolysis of both
phosphatidylinositol 4,5-bisphosphate (PtdInsP2) and phosphatidylinositol (PtdIns).
Here we tested the hypothesis that PtdIns hydrolysis is mediated by
PLC Many receptor tyrosine kinases and
GPCRs1 couple to PLC that
catalyzes the cleavage of PtdInsP2 and resultant generation
of the two second messengers Ins(1,4,5)P3, which mobilizes
Ca2+ from intracellular stores, and DAG, which activates
protein kinase C (1-4). These pathways are well described in
the regulation of insulin secretion following occupation of m3 and m1
muscarinic receptors on the surface of pancreatic The PLC family is comprised of four major groups (PLCs We have previously provided limited evidence that hydrolysis of PtdIns
and PtdInsP2 are independently regulated and hence potentially mediated by different PLCs. Thus, in pancreatic Materials--
The sources of all materials were as previously
described (9, 10) except for tissue culture media which was from
ICN Biomedicals (Seven Hills, NSW, Australia), radiochemicals from Amersham Pharmacia Biotech, and scintillant from Canberra
Packard (Gladesville, NSW, Australia). All other biochemicals and
specialized reagents were from Sigma except genistein, daidzein,
LY294002, and wortmannin, which were purchased from Biomol (Plymouth
Meeting, PA). Immunologicals were obtained from the following sources: anti-PLC Islet Isolation and Culture--
Pancreatic islets were isolated
following ductal infusion of collagenase into the exocrine pancreas,
purified on a Histopaque 1077 gradient, and then handpicked under a
binocular microscope (9). They were maintained in tissue culture for
48-72 h in Medium 199 containing 10% fetal calf serum, 14 mM NaHCO3, 11.1 mM glucose, 500 IU/ml penicillin, 100 µg/ml streptomycin, and 50 µg/ml gentamycin.
For the inositol phosphate studies, 10 µCi/ml [2-3H]inositol was present throughout the culture period.
Generation of Adenovirus and Islet Infection--
The two PLCs
were present in slightly different adenoviral vectors necessitating the
use of different control viruses. Rat PLC Inositol Phosphate Studies--
Groups of 50 islets were
preincubated at 37 °C for 15 min in 500 µl of a modified KRB
medium containing 5 mM NaHCO3, 2.8 mM glucose, 1 mM CaCl2, 0.5% BSA,
and 10 mM Hepes, pH 7.4. Prewarmed stimulating solutions
were added as doubly concentrated 500-µl stocks in the same medium.
Reactions were terminated after 1 min by the addition of 110 µl of
100% ice-cold trichloroacetic acid, and the incubation tubes
immediately vortexed and placed at 4 °C for a minimum of 30 min.
Inositol phosphates were extracted and analyzed by HPLC using a
Partisphere PAC column (12.5 cm) and ammonium phosphate gradient
as previously described (10). Radioactive peaks were quantified with a
Radiomatic Flo-1 Carbachol-stimulated Tyrosine Phosphorylation--
Incubations
and stimulations were as for inositol phosphate studies except that
groups of islets were incubated in modified KRB without BSA. Reactions
were terminated by immediate removal of the incubation medium and
addition of 50 µl of ice-cold lysis buffer (50 mM
Tris-HCl, pH 7.6, 5 mM EGTA, 5 mM EDTA, 1%
Triton X-100, 2 mM phenylmethylsulfonylflouride, 5 µg/ml
aprotinin, 2 µM pepstatin A, 2 mM
benzamidine, and 1 mM sodium orthovanadate) and transferred
to ice. Islets were sonicated using a Branson Sonifier 250 (20 pulses
at 10% duty cycle; setting 1) and mixed with 50 µl of 2× Laemmli
sample buffer, boiled for 5 min, and centrifuged for 5 min prior to
carrying out SDS polyacrylamide gel electrophoresis on 10% gels.
Proteins were then transferred to a nitrocellulose membrane at 500 mA
for 2 h and blocked for 1 h in a solution of 20 mM Tris-HCl, 137 mM NaCl, and 0.1% Tween 20, pH 7.5, containing 1% BSA for 1 h. Tyrosine-phosphorylated proteins were detected using the anti-phosphotyrosine antibody RC-20
for 1 h and visualized directly by enhanced chemiluminescence.
PLC Expression--
Islet extracts were prepared from 50 islets
by addition of 100 µl of 1× Laemmli sample buffer, boiled for 5 min,
and centrifuged for 5 min prior to carrying out SDS polyacrylamide gel
electrophoresis on 10% gels. The proteins were then transferred to a
nitrocellulose membrane at 500 mA for 2 h and blocked for 1 h
in a solution of 20 mM Tris-HCl, 137 mM NaCl,
and 0.1% Tween 20, pH 7.5, containing 5% milk powder. The membranes
were then incubated for 2 h with an anti-PLC antibody before
incubating for 1 h with a horseradish peroxidase-conjugated
secondary antibody and detection of immunoreactive proteins using the
enhanced chemiluminescence system.
In the following experiments breakdown of PtdIns and
PtdInsP2 were assessed as increases in Ins(1)P1
and Ins(4)P1 levels, respectively. This approach has been
previously verified on the basis of time course and inhibitor studies,
as well as flux measurements (9). Changes in steady-state levels of
these inositol phosphates at 1 min post-stimulation, as
performed here, are not as pronounced as in end point accumulation
assays, such as following prolonged stimulation in the presence of LiCl
to inhibit inositol phosphate breakdown (30). For technical reasons,
however, the rationale underlying measurement of PtdIns hydrolysis as
an increase in Ins(1)P1 does not necessarily hold at longer
time points nor in the presence of LiCl, which precluded use of these
alternative protocols (9). As a first means of addressing whether
different PLCs might be involved in mediating hydrolysis of PtdIns and
PtdInsP2, we made use of the tyrosine kinase inhibitor,
genistein, which has been widely demonstrated to inhibit activation of
PLC1, which is known to be regulated by activation of tyrosine
kinases and PtdIns 3-kinase. PtdIns breakdown was more sensitive than
that of PtdInsP2 to the tyrosine kinase inhibitor,
genistein. Conversely, the tyrosine phosphatase inhibitor, vanadate,
alone promoted PtdIns hydrolysis and acted non-additively with
carbachol. Vanadate did not stimulate PtdInsP2 breakdown.
Carbachol also stimulated a rapid (maximal at 1-2 min) tyrosine
phosphorylation of several islet proteins, although not of PLC
1
itself. Two structurally unrelated inhibitors of PtdIns 3-kinase,
wortmannin and LY294002, more effectively attenuated the hyrolysis of
PtdIns compared with PtdInsP2. Adenovirally mediated
overexpression of PLC
1 significantly increased carbachol-stimulated
PtdIns hydrolysis without affecting that of PtdInsP2.
Conversely overexpression of PLC
1 up-regulated the
PtdInsP2, but not PtdIns, response. These results indicate that the hydrolysis of PtdIns and PtdInsP2 are
independently regulated in pancreatic islets and that PLC
1
selectively mediates the breakdown of PtdIns. The activation mechanism
of PLC
involves tyrosine phosphorylation (but not of PLC
directly) and PtdIns 3-kinase. Our findings point to a novel
bifurcation of signaling pathways downstream of muscarinic receptors
and suggest that hydrolysis of PtdIns and PtdInsP2 might
serve different physiological ends.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells (5-8).
However we have shown that in addition to the classical pathway of
PtdInsP2 hydrolysis, exposure of pancreatic islets to the
muscarinic receptor agonist carbachol also promotes the hydrolysis of
PtdIns (9, 10). The latter predominates quantitatively over, and is a
precursor of, PtdInsP2. Although hydrolysis of either
phosphoinositide species results in generation of DAG, only
PtdInsP2 gives rise to a Ca2+ signal via
Ins(1,4,5)P3 production. Therefore the two hydrolytic events may have different functional consequences. The inositol phosphate, corresponding to Ins(1,4,5)P3, that is derived
from PtdIns, is Ins(1)P1. This has no biological function
but can be used to quantify PtdIns hydrolysis, at least under the
conditions verified in our previous studies (9).
,
,
,
and
). Members of the PLC
family are activated by G-proteins in
response to occupation of GPCRs (3, 4). The recently discovered protein
kinase C
is a component of ras signaling pathways (11-13). PLC
is the most sensitive of the PLC family to
Ca2+, and a rise in intracellular Ca2+ is
thought to underly its activation in vivo (14, 15). The PLC
family members (PLC
1 and 2) are classically activated
downstream of receptor tyrosine kinases, but they also couple to
muscarinic and other GPCRs in a manner secondary to stimulation of
cytosolic tyrosine kinases (3, 4, 16, 17). Although PLC
is a substrate for many tyrosine kinases there are indications, based both
on activity assays (18-20) and experiments using whole cells (21, 22),
that tyrosine phosphorylation of PLC
itself is not always necessary
for its activation. Indeed there is recent evidence that
phosphatidylinositol 3,4,5-trisphosphate, generated by the enzyme
PtdIns 3-kinase, binds to the pleckstrin homology domain of PLC
and
thereby stimulates its activity (23-26). The precise mechanism
underlying this mode of activation is obscure, but it does not require
tyrosine phosphorylation of PLC
. This PLC is also of interest,
because compared with the PLC
and
family members, which are
relatively selective for PtdInsP2, PLC
is also
capable of efficiently using PtdIns as a substrate, at least in
vitro (27).
-cells, a rise in intracellular Ca2+ levels is sufficient to cause
breakdown of PtdInsP2 but not of PtdIns (10). Our current
aim was to characterize further the mechanism underlying PtdIns
hydrolysis, with particular reference to a potential role for PLC
.
Our results show for the first time that PLC
mediates PtdIns
hydrolysis in vivo and that the underlying mechanism appears
to involve activation of PtdIns 3-kinase and alterations in the
tyrosine phosphorylation of islet cell proteins although not of PLC
itself.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and mixed monoclonal and rabbit antisera, Upstate
Biotechnology, Lake Placid, NY; anti-PLC
1, Santa Cruz Biotechnology,
Santa Cruz, CA; RC20 anti-phosphotyrosine and anti-PLC
1,
Transduction Laboratories, San Diego, CA; goat anti-mouse IgG-Sepharose
4B conjugate and protein A-Sepharose 4B conjugate, Zymed
Laboratories Inc., South San Francisco, CA.
1 (28) was subcloned into
pShuttle-CMV and recombined with pAdEasy-1 as described (29).
Control virus was generated by recombination of pShuttle-CMV and
pAdEasy-1. Virus-encoding rat PLC
1 (30) was a generous gift from
Dr. C. B. Newgard (Dallas, TX). Here the appropriate control virus,
not containing any transgene, was based on the pJM17 adenoviral vector
(31) and was provided by Dr. L. Carpenter. After islets were
prelabeled in tissue culture for 44 h as described above, the
[3H]inositol-containing medium was removed and retained.
Groups of 400-600 islets were then incubated in 140 µl of
inositol-labeled culture media with 5% polyethylene glycol, to which
20 µl of concentrated adenovirus stock was added (2-5 × 107 plaque-forming units/µl
stock).2 Islets were
incubated with virus for 1 h at 37 °C with gentle agitation
every 15 min. Infected islets were washed eight times with fresh media
without labeled inositol before two washes in reserved inositol-labeled
culture media, and recombinant protein was allowed to express during a
further 3-h culture in reserved inositol-labeled culture media.
(Series A-100) on-line detector, using Ultima Flo
AP scintillant. Counts were internally corrected by monitoring
for recovery of the total free inositol in each sample.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(16, 17). As shown in Fig. 1,
genistein reduced carbachol-stimulated PtdIns hydrolysis in a
dose-dependent manner, with 70% inhibition occurring at
100 µM genistein. Most importantly this inhibition was
significantly greater than that seen with an equivalent dose of
daidzein, a genistein analogue that does not affect tyrosine kinase
activity. However the effects of genistein and daidzein were identical
on PtdInsP2 hydrolysis suggesting that, in contrast to
PtdIns hydrolysis, this is not specifically influenced by tyrosine kinase inhibition.
View larger version (41K):
[in a new window]
Fig. 1.
Effect of inhibition of tyrosine kinases on
basal and carbachol-stimulated hydrolysis of PtdIns and
PtdInsP2. Isolated islets were labeled during 48-h
tissue culture with [3H]inositol and, following a 15-min
preincubation, stimulated for 1 min in KRB medium containing 0.5 mM carbachol (Carb) where indicated. In some
instances 10 or 100 µM genistein (Gen) or 100 µM daidzein (Daid) were added 15 min prior to
stimulation. Hydrolysis of PtdIns and PtdInsP2 was
quantified as Ins(1)P1 and Ins(4)P1,
respectively. Inositol monophosphate isomers were separated by HPLC
using an ammonium phosphate gradient and a Partisphere PAC column. For
further details see "Experimental Procedures." Results are
presented as percentages of the carbachol-stimulated increment in
either Ins(1)P1 or Ins(4)P1 and are means ± S.E. of eight to ten individual determinations. As calculated using
Student's t test, * denotes p < 0.05, **
denotes p < 0.001 versus the corresponding
value for carbachol alone, and § denotes p < 0.05 as
indicated.
Although the results above are suggestive of a selective requirement
for tyrosine kinases in the PtdIns response, the high nonspecificity of
genistein complicated further analysis. Similar nonspecificity has been
seen on other parameters when using a range of other tyrosine kinase
inhibitors in islet cells (32). We therefore turned to the converse
approach of using sodium orthovanadate, a tyrosine phosphatase
inhibitor that augments tyrosine phosphorylation of several islet cell
proteins (Ref. 33 and not shown). This potently stimulated PtdIns
hydrolysis to ~75% of the maximal carbachol response (Fig.
2). The combination of carbachol plus
vanadate exerted no greater effects than those because of carbachol
alone. In marked contrast, vanadate alone caused only a very modest
increase in PtdInsP2 hydrolysis and actually potentiated
the response to carbachol. This is probably explained by the ability of
vanadate to stimulate PtdInsP2 hydrolysis in islets
secondary to Ca2+ influx (34) whereas we have previously
shown that Ca2+ influx is insufficient to promote PtdIns
hydrolysis (10). The current experiments therefore suggest that
increased tyrosine phosphorylation is sufficient to activate PtdIns
hydrolysis that occurs via a mechanism that is non-additive (and thus
potentially similar) to that of carbachol. Taken together the findings
of the genistein and vanadate experiments implicate tyrosine
phosphorylation as a component of the mechanism by which carbachol
activates PtdIns hydrolysis. Because there should be no requirement for
activation of tyrosine kinases in activation of PLC and PLC
, in
contrast to PLC
(3, 4), our results also suggest that hydrolysis of
PtdIns and PtdInsP2 are mediated by different PLCs and that PLC
is implicated in the PtdIns response.
|
We next analyzed the ability of carbachol to increase tyrosine
phosphorylation of PLC and other islet cell proteins. Looking first
at islet cell lysates, carbachol increased the tyrosine phosphorylation
of a number of proteins, including prominent species at ~30, 32, 36, 38, 64, and 125 kDa (Fig. 3). These
responses were rapid in onset (maximal at 1-2 min) but remained
elevated over the 30 min of stimulation. Although these experiments
established that carbachol could activate tyrosine kinases, there was
no marked increase in phosphorylation in substrates around the 145-kDa
region, corresponding to PLC
. To address this issue directly, PLC
was immunoprecipitated from lysates of 600 carbachol-treated and
untreated islets. Although PLC
was detectable in the
immunoprecipitates, its phosphotyrosine content was extremely low under
basal conditions and not increased by prior carbachol stimulation
(results not shown).
|
There is now ample evidence that tyrosine phosphorylation of PLC
itself is not always necessary for its activation. One alternative activation mechanism is via stimulation of PtdIns 3-kinase activity and
subsequent generation of phosphatidylinositol 3,4,5-trisphosphate, which binds to the pleckstrin homology domain of PLC
(23-26). This
mechanism was investigated in experiments examining the effects of well
documented, structurally diverse, PtdIns 3-kinase inhibitors (35) on
carbachol-stimulated phosphoinositide hydrolysis. As shown in Fig.
4, 100 nM wortmannin and 50 µM LY294002 inhibited PtdIns hydrolysis by more than 40 and 70%, respectively. Corresponding effects on PtdInsP2
hydrolysis were much less pronounced, with wortmannin exerting no
significant effect and LY294002 inhibiting by less than 30%. Thus
PtdIns hydrolysis exhibits a selective requirement for PtdIns 3-kinase,
consistent with a role for that enzyme as an upstream regulator of
PLC
.
|
To examine the involvement of PLC more directly we used recombinant
adenovirus to overexpress this enzyme in pancreatic islets, as
demonstrated in Fig. 5A. Most
importantly this virus caused a 40% increase in carbachol-stimulated
PtdIns hydrolysis (Fig. 5B). In marked contrast it did not
affect PtdInsP2 hydrolysis at all. The specificity of
these findings were confirmed in control experiments using a PLC
1
adenovirus previously documented to increase PtdInsP2
hydrolysis by ~50% in pancreatic islets treated for 10 min with
carbachol in the presence of LiCl (30). Under the conditions of our
assay (1-min stimulation, no LiCl) the agonist-stimulated PtdInsP2 response was increased to 119.3 ± 4.7%
versus 100 ± 2.3% in carbachol-treated, control
virus-infected islets (n = 12, p < 0.005), whereas PtdIns hydrolysis was not significantly affected; 106.5 ± 10.2 versus 100 ± 4.8 (n = 12). Basal responses were not affected by the PLC
1 virus (not
shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The major finding of the current study is that
carbachol-stimulated PtdIns hydrolysis in pancreatic islets is mediated
by PLC. This conclusion was based partially on the differential sensitivities of the PtdIns and PtdInsP2 responses to
genistein, vanadate, wortmannin, and LY294002, which are consistent
with the known mechanisms of activation of PLC
(3, 4). This was
confirmed directly by the evidence that overexpression of PLC
1 but
not PLC
1 resulted in a selective increase of agonist-stimulated PtdIns breakdown. Although we have not formally assessed the PLC
family members in this context, we consider their involvement highly
unlikely. As is the case in other cell types (14, 15), PLC
is known
to be activated in
-cells as a direct consequence of a rise in
intracellular Ca2+ (36). Although this is sufficient to
promote PtdInsP2 hydrolysis in pancreatic islets (37),
we have previously shown this was not the case for PtdIns breakdown
(10). Our current data are also supported by earlier findings that
PLC
efficiently utilizes both PtdInsP2 and PtdIns as
substrates in activity assays in vitro, whereas the PLC
and
and families show a much higher degree of specificity for
PtdInsP2 (20). However our results are the first to
demonstrate directly that PLC
acts on PtdIns in a more physiological
setting using intact cells. This might seem surprising, but in practice
PtdIns hydrolysis would be difficult to detect unless specifically
addressed. There is one earlier study of epidermal growth
factor-stimulated phosphoinositide turnover in A-431 cells, now known
to involve PLC
, demonstrating a transient rise in inositol trisphosphate but a persistent increase in total inositol monophosphate (38). Although the latter was interpreted as evidence of PtdIns hydrolysis, it is not clear that this increase actually represented Ins(1)P1 derived from PtdIns. Nevertheless it would now be
of interest to re-examine the A-431 cells more closely for direct evidence of PtdIns hydrolysis, using the rigorous analytical criteria we established previously for pancreatic islets (9).
A corollary of our major finding is that PLC appeared not to play a
role in carbachol-stimulated PtdInsP2 hydrolysis in islet cells. This is consistent with studies demonstrating that
PtdInsP2 is targeted in pancreatic islets and
-cell
lines by PLC
1, PLC
3, and PLC
1 (30, 36). However it is clearly
inconsistent with a host of earlier data from other cell types
demonstrating that PLC
does hydrolyze PtdInsP2 (3, 4).
The most obvious explanation for this apparent contradiction is tissue
differences, especially because the great majority of previous studies
were undertaken with transformed cell lines, instead of the terminally
differentiated primary tissue used here. An alternative explanation is
that under conditions following occupation of GPCRs, where both PLCs
are activated, PLC
might compete more effectively than PLC
for
access to PtdInsP2. On the other hand receptor tyrosine
kinases, which would not be expected to couple to PLC
, could
stimulate PtdInsP2 hydrolysis through the activation of
PLC
alone. This proposal would need to be supported by direct
evidence but is attractive in that it might help to explain the
necessity for PLC
activation, which is presumably not to
stimulate PtdInsP2 hydrolysis, because this would be
effectively mediated by PLC
. Another potential explanation is that
the substrate preference of PLC
might be conditioned by its
activation mechanism. For example PtdInsP2 is likely to be
sequestered in vivo by binding proteins such as profilin,
which have much lower affinities for PtdIns (39). In principle,
therefore, PtdIns should be easier for PLC
to access than
PtdInsP2, and indeed there is some evidence that tyrosine phosphorylation of PLC
is necessary for displacement of profilin (40). Consistent with this, our findings would suggest that PLC
does
not need to be tyrosine-phosphorylated to hydrolyze PtdIns,
although there was at least a partial requirement for activation of an
upstream tyrosine kinase. In addition, PtdIns hydrolysis was dependent
on the activation of PtdIns 3-kinase. Although a well documented route
for stimulation of PLC
, this has only been addressed previously in
the context of PtdInsP2 hydrolysis (23-26). Likewise there
is very little information on whether other potential mechanisms for
activation of PLC
, such generation of phosphatidic acid (19) or
arachidonic acid (20), might be also be involved in regulation of
PtdIns hydrolysis.
Increases in protein phosphotyrosine content in response to carbachol or other GPCRs have not been demonstrated previously using pancreatic islets. In one earlier study (43) carbachol increased tyrosine phosphorylation of a single, unidentified protein of 125 kDa in a transformed insulin-secreting cell line, whereas it had been reported previously that 15-min muscarinic stimulation of pancreatic islets was without effect (33). These results are clarified and extended by our findings of an early (maximal at 1-2 min) incorporation of phosphate into tyrosine residues of a number of proteins, including one around 125 kDa. Tyrosine phosphorylation can therefore be added to the other known responses of pancreatic islets to muscarinic receptor occupation. In addition to the hydrolysis of both PtdIns and PtdInsP2 (5-10), these include activation of protein kinase C (44, 45), mobilization of Ca2+ from intracellular stores (8), Na+-dependent membrane depolarization (46), and both gating and subsequent inhibition of voltage-dependent Ca2+ influx (47). Indeed it is possible that tyrosine phosphorylation might lie upstream of some or other of these events.
In conclusion we have mechanistically dissociated the
hydrolysis of PtdIns from that of PtdInsP2 in pancreatic
-cells on the basis of requirements for tyrosine
phosphorylation, PtdIns 3-kinase, and involvement of different PLC
isoforms. The demonstration that PLC
1 targets PtdIns exclusively,
whereas other PLCs acts on PtdInsP2, points to a hitherto
unsuspected diversification of signaling pathways downstream of
muscarinic receptors. This is apparent from the consideration that
although PtdInsP2 breakdown releases the two intracellular
messengers Ins(1,4,5)P3 and DAG, which regulate
Ca2+ mobilization and protein kinase C activation,
respectively, the only product of PtdIns hydrolysis possessing known
biological function is DAG. Thus different protein kinase cascades of
differing Ca2+ dependences could be initiated from the
hydrolysis of each phospholipid species. Alternatively, a DAG/protein
kinase C readout emanating from PtdIns might lend itself to regulation
in a different manner than that of the PtdInsP2 pathway.
There is already some evidence for this in that PtdIns hydrolysis seems
to predominate over longer stimulation and is therefore perhaps less
sensitive to down-regulation than is PtdInsP2 regulation
(10). This might also be consistent with reports of biphasic DAG
generation in response to stimulation of a variety of tissues (41, 42,
48). In addition, hydrolysis of PtdIns, but not of
PtdInsP2, is directly influenced by the prevailing membrane
potential (10) and might therefore serve as a means of integrating
signals arising from different external stimuli. Direct experimental
support for these proposals will await a better understanding of the
mechanisms underlying the two processes, especially of the tyrosine
kinases and their substrates that control PtdIns hydrolysis. However
this would be more conveniently addressed in a model system that is
more amenable to molecular intervention and biochemical analysis than
are isolated pancreatic islets.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Carol Browne, Damien Cordery,
Brooke Carter, and Libby Kerr for islet isolation, Lee Carpenter for
help generating the adenoviruses, and Danielle Lynch, Trevor Lewis,
and Lee Carpenter for critical comments on the manuscript. We also
gratefully acknowledge Sue Gho Rhee for the PLC1 plasmid, Chris
Newgard for the PLC
1 adenovirus, and Tong-Chuan He and
Bert Vogelstein for the components of the pAdEasy system.
![]() |
FOOTNOTES |
---|
* This work was supported by a Block grant from the National Health and Medical Research Council of Australia and a research grant from Aza Pty Ltd.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Cell Signaling Group,
Garvan Inst. of Medical Research, 384 Victoria St., Darlinghurst, Sydney 2010, Australia. Tel.: 61-2-9295-8204; Fax: 61-2-9295-8201; E-mail: t.biden@garvan.unsw.edu.au.
Published, JBC Papers in Press, March 27, 2001, DOI 10.1074/jbc.M101406200
2 L. Carpenter, Z. Z. Xu, P. Poronnik, G. W. Both, and T. J. Biden, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GPCR(s), G-protein-coupled receptor(s); carbachol, carbamylcholine; KRB, Krebs-Ringer bicarbonate; PLC, phospholipase C; DAG, diacylglycerol; PtdIns, phosphatidylinositol; PtdInsP2, phosphatidylinositol 4,5-bisphosphate; BSA, bovine serum albumin; Ins(1, 4,5)P3, inositol 1,4,5-trisphosphate; Ins(1)P1, inositol 1-monophosphate; Ins(4)P1, inositol 4-monophosphate; HPLC, high pressure liquid chromatography.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Berridge, M. J. (1993) Nature 361, 315-325[CrossRef][Medline] [Order article via Infotrieve] |
2. | Irvine, R. F. (1998) Curr. Biol. 8, 557-559 |
3. |
Rhee, S. G.,
and Bae, Y. S.
(1997)
J. Biol. Chem.
272,
15045-15048 |
4. | Sekiya, F., Bae, Y. S., and Rhee, S. G. (1999) Chem. Phys. Lipids 98, 3-11[CrossRef][Medline] [Order article via Infotrieve] |
5. | Zawalich, W. S., and Rasmussen, H. (1990) Mol. Cell. Endocrinol. 70, 119-137[CrossRef][Medline] [Order article via Infotrieve] |
6. | Iismaa, T. P., Kerr, E. A., Wilson, J. R., Carpenter, L., Sims, N., and Biden, T. J. (2000) Diabetes 49, 392-398[Abstract] |
7. | Morgan, N. G., Rumford, G. M., and Montague, W. (1985) Biochem. J. 228, 713-718[Medline] [Order article via Infotrieve] |
8. |
Wollheim, C. B.,
and Biden, T. J.
(1986)
J. Biol. Chem.
261,
8314-8319 |
9. | Biden, T. J., Prugue, M. L., and Davison, A. G. M. (1992) Biochem. J. 285, 541-549[Medline] [Order article via Infotrieve] |
10. |
Biden, T. J.,
Davison, A. G. M.,
and Prugue, M. L.
(1993)
J. Biol. Chem.
268,
11065-11072 |
11. |
Song, C.,
Hu, C.-D.,
Masago, M.,
Kariya, K.,
Yamawaki-Kataoka, Y.,
Shibatohge, M.,
Wu, D.,
Satoh, T.,
and Kataoka, T.
(2001)
J. Biol. Chem.
276,
2752-2757 |
12. |
Lopez, I.,
Mak, E. C.,
Ding, J.,
Hamm, H. E.,
and Lomasney, J. W.
(2001)
J. Biol. Chem.
276,
2758-2765 |
13. |
Kelley, G. G.,
Reks, S. E.,
Ondrako, J. M.,
and Smrcka, A. V.
(2001)
EMBO J.
20,
743-754 |
14. |
Banno, Y.,
Okano, Y.,
and Nozawa, Y.
(1994)
J. Biol. Chem.
269,
15846-15852 |
15. |
Kim, Y.-H.,
Park, T.-J.,
Lee, Y. H.,
Baek, K. J.,
Suh, P.-G.,
Ryu, S. H.,
and Kim, K.-T.
(1999)
J. Biol. Chem.
274,
26127-26134 |
16. |
Gusovsky, F.,
Leuders, J. E.,
Kohn, E. C.,
and Felder, C. C.
(1993)
J. Biol. Chem.
268,
7768-7772 |
17. |
Marrero, M. B.,
Paxton, W. G.,
Duff, J. L.,
Berk, B. C.,
and Bernstein, K. E.
(1994)
J. Biol. Chem.
269,
10935-10939 |
18. | Hernandez-Sotomayor, S. M. T., and Carpenter, G. (1993) Biochem. J. 293, 507-511[Medline] [Order article via Infotrieve] |
19. |
Jones, G. A.,
and Carpenter, G.
(1993)
J. Biol. Chem.
268,
20845-20850 |
20. |
Hwang, S. C.,
Jhon, D.-K.,
Bae, Y. S.,
Kim, J. H.,
and Rhee, S. G.
(1996)
J. Biol. Chem.
271,
18342-18349 |
21. | Yeo, E.-J., Provost, J. J., and Exton, J. H. (1997) Biochim. Biophys. Acta 1256, 308-320 |
22. | Baldassare, J. J., Henderson, P. A., Tarver, A., and Fisher, G. J. (1997) Biochem. J. 324, 283-287[Medline] [Order article via Infotrieve] |
23. |
Falasca, M.,
Logan, S. K.,
Lehto, V. P.,
Baccante, G.,
Lemmon, M. A.,
and Schlessenger, J.
(1998)
EMBO J.
17,
414-422 |
24. |
Bae, Y. S.,
Cantley, L. G.,
Chen, C.-S.,
Kim, S.-R.,
Kwon, K.-S.,
and Rhee, S. G.
(1998)
J. Biol. Chem.
273,
4465-4469 |
25. |
Rameh, L. E.,
Rhee, S. G.,
Spokes, K.,
Kazlauskas, A.,
Cantley, L. C.,
and Cantley, L. G.
(1998)
J. Biol. Chem.
273,
23750-23757 |
26. | Gratacap, M.-P., Payrastre, B., Viala, C., Mauco, G., Plantavid, M., and Chap, H. (1998) J. Biol. Chem. 38, 24314-24321[CrossRef] |
27. | Ryu, S. H., Suh, P. G., Cho, K. S., Lee, K. Y., and Rhee, S. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6649-6653[Abstract] |
28. | Suh, P. G., Ryu, S. H., Moon, K. H., Suh, H. W., and Rhee, S. G. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5419-5423[Abstract] |
29. |
He, T.-C.,
Zhou, S.,
da Costa, L. T., Yu, J.,
Kinzler, K. W.,
and Vogelstein, B.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
2509-2514 |
30. | Gasa, R., Trinh, K. Y., Yu, K., Wilkie, T. M., and Newgard, C. B. (1999) Diabetes 48, 1035-1044[Abstract] |
31. | Becker, T. C., Noel, R. J., Coats, W. S., Gomez-Foix, A. M., Alam, T., Gerard, R. D., and Newgard, C. B. (1994) Methods Cell Biol. 43, 161-189[Medline] [Order article via Infotrieve] |
32. | Jonas, J.-C., Plant, T. D., Gilon, P., Detimary, P., Nenquin, M., and Henquin, J.-C. (1995) Br. J. Pharmacol. 114, 872-880[Abstract] |
33. | Jonas, J.-C., and Henquin, J.-C. (1996) Biochem. J. 315, 49-55[Medline] [Order article via Infotrieve] |
34. |
Zhang, A.,
Gao, Z.-Y.,
Gilon, P.,
Nenquin, M.,
Drews, G.,
and Henquin, J.-C.
(1991)
J. Biol. Chem.
266,
21649-21656 |
35. | Davies, S. P., Reddy, H., Caivano, M., and Cohen, P. (2000) Biochem. J. 351, 95-105[CrossRef][Medline] [Order article via Infotrieve] |
36. | Ishihara, H., Wada, T., Kizuki, N., Asano, T., Yazaki, Y., Kikuchi, M., and Oka, Y. (1999) Biochem. Biophys. Res. Commun. 254, 77-82[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Biden, T. J.,
Peter-Riesch, B.,
Schlegel, W.,
and Wollheim, C. B.
(1987)
J. Biol. Chem.
262,
3567-3571 |
38. | Tilly, B. C., Van Paridon, P. A., Verlaan, I., De Laat, S. W., and Moolenaar, W. H. (1988) Biochem. J. 252, 857-863[Medline] [Order article via Infotrieve] |
39. | Goldschmidt-Clermont, P. J., Machesky, L. M., Baldassare, J. J., and Pollard, T. D. (1990) Science 247, 1575-1578[Medline] [Order article via Infotrieve] |
40. | Goldschmidt-Clermont, P. J., Kim, J. W., Machesky, L. M., Rhee, S. G., and Pollard, T. D. (1991) Science 251, 1231-1233[Medline] [Order article via Infotrieve] |
41. | Nakashima, S., Suganuma, A., Matsui, A., and Nozawa, Y. (1991) Biochem. J. 275, 355-361[Medline] [Order article via Infotrieve] |
42. |
Tokuwa, Y.,
Takuwa, N.,
and Rasmussen, H.
(1986)
J. Biol. Chem.
261,
14670-14675 |
43. |
Konrad, R. J.,
Dean, R. M.,
Young, R. A.,
Billings, P. C.,
and Wolf, B. A.
(1996)
J. Biol. Chem.
271,
24179-24186 |
44. | Persaud, S. J., Jones, P. M., Sugden, D., and Howell, S. J. (1989) Biochem. J. 264, 753-758[Medline] [Order article via Infotrieve] |
45. |
Easom, R. A.,
Landt, M.,
Colca, J. R.,
Hughes, J. H.,
Turk, J.,
and McDaniel, M.
(1990)
J. Biol. Chem.
265,
14938-14946 |
46. | Henquin, J.-C., Garcia, M.-C., Bozem, M., Hermans, M. P., and Nenquin, M. (1988) Endocrinology 122, 2134-2142[Abstract] |
47. | Gilon, P., Nenquin, M., and Henquin, J.-C. (1995) Biochem. J. 311, 259-267[Medline] [Order article via Infotrieve] |
48. |
Griendling, K. K.,
Rittenhouse, S. E.,
Brock, T. A.,
Ekstein, L. S.,
Gimbrone, M. A.,
and Alexander, W. A.
(1986)
J. Biol. Chem.
261,
5901-5906 |