| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Division of Endocrinology (L.B.L., J.B.N.), Oregon Health Sciences University, Portland, Oregon 97201; Ludwig Institute (M.C.F.), Melbourne, Australia; Howard Hughes Medical Institute (J.D.S.), Vollum Institute, Portland, Oregon 97201
Address all correspondence and requests for reprints to: Dr. Linda Lester, Division of Endocrinology L-607, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97201. E-mail: lesterl{at}ohsu.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
cells
is regulated by the intracellular metabolism of nutrients, primarily
glucose (1). However, nonnutrient signals can augment this
process. For example, the hormone glucagon-like peptide 1
(GLP-1) is a potent insulin secretagogue but only in the
presence of elevated glucose levels (2, 3, 4, 5, 6, 7). These
observations suggest that nutrient and nonnutrient signaling pathways
are integrated in the regulation of insulin secretion. One mechanism
integrating these signals is the activation of specific kinases and
phosphatases resulting in the reversible phosphorylation of cell
proteins (8, 9, 10, 11). This concept is supported by a recent
study where activation of kinases, including protein kinase C (PKC),
protein kinase A (PKA), and the calcium, calmodulin-dependent kinase
(CaM K II) and concomitant inhibition of protein phosphatases lead to
enhanced insulin secretion in cells (12). Furthermore,
transient inhibition of the calcium, calmodulin-dependent phosphatase,
PP-2B, by the immunosuppressant drug cyclosporin (CsA) increased
insulin secretion (13). This finding contrasts a body of
evidence suggesting that long-term treatment with the
immunosuppressants CsA or FK506 impairs insulin secretion
(14) and results in the development of diabetes
(15). Both observations highlight a role for phosphatases,
particularly PP-2B, in regulating insulin secretion. Furthermore, these
studies show contrasting effects for transient vs. sustained
inhibition of PP-2B on insulin secretion. This suggests that PP-2B
activity must be tightly regulated to ensure that reversible
phosphorylation events can occur repeatedly. This idea is supported by
parallel studies in neurons where proteins involved in
exocytosis/endocytosis are targets for reversible protein
phosphorylation by PKA, PKC, and PP-2B. The dephosphorylation of these
proteins by PP-2B allows for repeated exocytotic events stimulated by
activation of the kinase (16).
In this study, we demonstrate that changes in insulin secretion are
associated with phosphorylation of cell substrates by PKA followed
by dephosphorylation by PP-2B. These findings extend our previous
observations that subcellular targeting of PKA through an A-Kinase
Anchoring Protein (AKAP) facilitates hormone mediated insulin
secretion (2). We now show that the coordinate action of
PKA and PP-2B may be due in part to their recruitment into a signaling
complex resulting in the activation of PP-2B by PKA, thus directly
linking the activities of these two enzymes. These observations are
consistent with our previous findings that demonstrate a role for the
subcellular targeting of PKA through associations with AKAPs in
GLP-1-mediated insulin secretion (2). We go
on to show that the anchoring proteins, AKAP79/150 (17, 18) may coordinate the action of PKA and PP-2B by maintaining a
signaling complex that regulates reversible phosphorylation events.
Subcellular targeting of protein kinases and phosphatases has emerged
as a prominent mechanism for regulating reversible phosphorylation
events in neurons, T-Lymphocytes and cardiac myocytes
(19, 20, 21). In this paper, we provide evidence that
subcellular targeting of PKA and PP-2B regulates insulin secretion
through reversible phosphorylation events in cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Preparation of primary islets and transfected RINm5F cells
Pancreatic islets were isolated by collagenase digestion, hand
picked, and plated in Falcon tissue culture dishes as previously
described (22). Islets were maintained in culture for up
to 5 days in RPMI 1640 containing 5 mM glucose and
supplemented with streptomycin (100 µg/ml) and penicillin (100
IU/ml). RINm5F cells at passage 20–25 were transfected with the
mammalian expression vectors pcDNA3, pcAKAP79, or pcHt31 using
Lipofectamine (Life Technologies, Inc., Gaithersburg, MD).
Transfected cell lines were selected by growth in media supplemented
with G418. Cells were maintained in low glucose DMEM (1000 mg/liter
D-glucose), with 10% FCS and 0.8 mg/ml G418. Expression of
AKAP79 and Ht31 was monitored by immunoblotting.
Insulin secretion assay
Insulin secretion from rat islets and RINm5F cells was measured
in static culture using Krebs Ringer HEPES Buffer, KRBH (10
mM HEPES, pH 7.4, 0.1% BSA, 130 mM NaCl, 5.2
mM KCl, 1.3 mM
KH2PO4, 1.6 mM
MgCl, 2.8 mM CaCl2, 20 mM
NaHCO3 and 2.8 mM glucose). Islets or
RINm5F cells were pretreated for 30 min at 37 C with FK506,
cyclosporin, rapamycin or DMSO as indicated. Cells were stimulated with
16.8 mM glucose, ± forskolin and ± FK506. Media was
collected and centrifuged at 15,000 RPM x 10 min. The supernatant
was stored at -20 C before determining insulin content by RIA using
rat insulin as a standard (Linco Research, Inc.).
Total insulin content was determined by incubating RINm5F cells and
RINm5F cells expressing AKAP79 with 0.22M HCL and 95% ETOH at -20 C
for 60 min. The protein content of the supernatant was determined using
a colorimetric assay kit (Bio-Rad Laboratories, Inc.).
PP-2B and PKA assays
RINm5F and rat islets were incubated with FK506 or DMSO for 30
min at 37 C in KRBH. The cells were stimulated with 16.8 mM
glucose ± 10 µM forskolin for 30 min at 37 C.
Following the stimulation, the cells were rinsed with PBS and incubated
with 200 µl of lysis buffer (50 mM Tris, pH 8.0, 150
mM NaCl, 1% NP-40, 2 µg/ml Pepstatin/leupeptin, 1
mM 4-(2-Aminoethyl)benzenesulfonyl Fluoride (AEBSF) and 10
µM IBMX) on ice for 60 min. Cells were scraped, pelleted,
and sonicated. PKA activity was measured by the filter paper method of
Corbin and Rieman (23) using Kemptide (LRRASLG) as a
peptide substrate. PP-2B activity was measured in triplicate
(24) in a 20 µl reaction mixture containing 40
mM Tris-HCL pH 7.5, 0.1 M KCl, 0.1
mM CaCl2, 6 mM magnesium
acetate, 0.5 mM DTT, 0.1 mg/ml BSA, 1.5 µM
calmodulin and 32P-RII peptide (20
µM) as the substrate at 30 C.
Subcellular fractionation
RINm5F cells were grown to near confluency (5 x
105 cells per dish) and rinsed with KRBH. The
cells were treated for 30 min with KRBH, 10 µM forskolin,
or 10 µM forskolin + 10 µM FK506. The media
was removed and the cells washed 3 times with PBS. The cells were
scraped and lysed with lysis buffer [20 mM HEPES, pH 7.4,
0.2% Triton X-100, 20 mM NaCl, 5 mM EDTA, 1
mM DTT, 2 µg/ml leupeptin, 2 µg/ml of pepstatin, 1
mM benzamidine and 0.1 mM phenylmethylsulfonyl
fluoride (PMSF)] and dounce homogenized. A supernatant and particulate
fraction were obtained by centrifugation at 40,000 x g
for 30 min. Protein concentration performed by a colorimetric assay
(Bio-Rad Laboratories, Inc.). Twenty micrograms of protein
from each fraction were separated by SDS-PAGE.
Affinity purification
Cell lysates were used for immunoprecipitations, calmodulin
purification, and cAMP purification. The cell lysates used for these
experiments were prepared by plating RINm5F cells in 150-cm plates and
culturing until approximately 80% confluency. The cells were washed
twice with KRBH and incubated in either KRBH + DMSO or 10
µM FK506 for 30 min at 37 C. The cells were stimulated
for the stated period of time. Cells were washed 3 times in PBS and
incubated in lysis buffer (20 mM Tris-HCl, pH 7.9, 250
mM NaCl, 50 mM NaF, 5 mM EGTA, 0.1
mM PMSF, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 1
mM Benzamidine and 1% vol/vol NP-40) for 15 min on ice.
The lysate was cleared by centrifugation at 15,000 rpm for 10 min.
Lysates were incubated with either 2 µg of antibody (for the
immunoprecipitations), calmodulin Sepharose (for the calmodulin
purification) or cAMP agarose (for the cAMP affinity purification) as
previously described (2). Affinity columns were washed 7
times and eluted by boiling with SDS-PAGE buffer (unless stated
otherwise). The C-subunit was eluted from the protein A Sepharose
column by incubating the column with pH 7.4, 20 mM HEPES
buffer containing 75 mM cAMP for 15 min at room
temperature. PKA assays were performed as described above. All other
protein elutions were subjected to SDS-PAGE electrophoresis,
transferred to PVDF membranes and probed for the specified protein.
Phosphoprotein determination
RINm5F cells were grown to near confluency in 150-mm dishes. The
cells were rinsed in prewarmed KRBH without bovine-serum albumin and
treated with DMSO or FK506 for 30 min at 37 C. The media was replaced
with KRBH containing 10 µM IBMX and 16.8 mM
glucose plus 10 µM forskolin or 100 µM
FK506 if indicated for 30 min. At the end of the incubation period,
cells were rinsed with PBS, scraped and pelleted by centrifugation. The
cells were lysed by adding 100 µl of RIPA buffer containing 2 µg/ml
pepstatin/leupeptin, 1 mM AEBSF and 10 µM
IBMX on ice for 60 min followed by 15 sec sonication (x3). A 30 µl
aliquot of cell lysate was denatured in Laemmli sample buffer and
subjected to SDS-PAGE. Proteins were transferred to PVDF membranes and
blocked for a minimum of 1 h in Tris-buffered saline containing 50
mM NaCl, 0.1% (vol/vol) Tween 20 and 5% (wt/vol) BSA.
Blots were incubated with primary antibody at the recommended
concentration of 1 µg/ml (Zymed Laboratories, Inc., San
Francisco, CA). Blots were incubated with secondary antibody for 2
h at room temperature. Proteins were visualized by chemiluminescence.
In vitro phosphorylation of synapsin 1 immunoprecipitates
was performed in a 30 µl reaction volume containing 5 µCi
32P 
-ATP and 0.07 µg PKA catalytic subunit
for 60 min at 30 C in the presence or absence of 15
µM PKI. The reaction was stopped by adding 5
µl of 0.5 M EDTA and boiling in SDS-PAGE sample
buffer. Phosphoproteins were identified by autoradiography.
Confocal microscopy
RINm5F cells were grown on no. 1 coverslips until 30%
confluent. The cells were rinsed in PBS three times and fixed with
3.7% formaldehyde for 5 min at 20 C. After washing, the cells were
permeablized with ice-cold acetone for 1 min. The cells were washed and
blocked with PBS containing 0.1% BSA for 10 min. AKAP 79 was
visualized using a rabbit polyclonal antibody (J. D. Scott) and a
Texas-Red conjugated antirabbit secondary antibody. The regulatory
subunit of PKA was visualized using a goat-anti-RII antibody (J.
D. Scott) and a FITC conjugated antigoat secondary antibody. The cells
were incubated with the primary and secondary antibodies for 60 min at
20 C. The cover-slips were mounted with Prolong (Molecular Probes, Inc.) and confocal sections were taken using a
laser-scanning confocal microscope (Carl Zeiss).
Statistical analysis
Data are represented as means ± SE. The data
were evaluated for significance by a two-sided nonpaired Student’s
t test using Prism software (GraphPad Software, Inc., San Diego, CA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
cells (RINm5F). Rat islets were
preincubated with FK506 for 30 min at 37 C and static insulin secretion
was measured by RIA following 30 min of stimulation with either glucose
or glucose + forskolin. FK506 treatment increased forskolin stimulated
insulin secretion by 2.5-fold ± 0.5 (n = 6) over controls
(Fig. 1A
). Likewise, transformed
cells, RINm5F cells, treated with FK506 and forskolin had a
2.11-fold ± 0.3 (n = 6) increase in insulin secretion over
RINm5F cells treated with forskolin (Fig. 1B). Insulin secretion was
increased to a similar magnitude [(2.48-fold ± 0.7 (n =
3)] using a more physiologic agonist, GLP-1 (1
nM) (Fig. 1C). Importantly, insulin secretion from rat
islets was increased to a similar extent [2.6-fold ± 0.5 (n
= 4)] upon application of another PP-2B inhibitor, CsA (Fig. 1D).
Further control experiments were performed using a structurally related
macrolide, rapamycin, that binds the immunophilin FKBP12 but does not
inhibit PP-2B (26, 27). As expected, pretreatment of rat
islets with excess rapamycin (10 µM) blocked the FK506
affect on cAMP-mediated insulin secretion but rapamycin did not
affect glucose-mediated insulin secretion independent of FK506
(Fig. 1E). This observation is consistent with a role for rapamycin as
a competitor with FK506 for binding to FKBP12 (28, 29).
Collectively, these experiments confirm that short-term inhibition of
PP-2B activity by FK506 or CsA is responsible for an increase in
cAMP-mediated insulin secretion from rat islets or transformed
cells.
|
). There was no significant difference
in basal secretion between the islet groups (Fig. 2, zero time point).
However, treatment of islets with FK506 for 1 h increased
forskolin and glucose mediated insulin secretion [2.7-fold ± 0.4
(n = 4)] over the non-FK506-treated islets (Fig. 2 and 1
h
time point). However, three hours of FK506 treatment inhibited cAMP
mediated insulin release and this inhibition was maintained during the
remaining 24 h of FK506 treatment (Fig. 2). Therefore, we
confirmed that prolonged inhibition of PP-2B with FK506 results in a
loss of insulin secretion (14, 15). However, we now
demonstrate that the FK506-mediated inhibition of insulin secretion
occurs with nonnutrient stimuli (cAMP) as well as with nutrient stimuli
(glucose).
|
cell proteins would be
phosphorylated in response to forskolin activation of PKA and
dephosphorylated by PP-2B. Inhibiting PP-2B would temporarily increase
the phosphorylation state of these proteins resulting in increased
insulin secretion. To evaluate this possibility, we identified proteins
reversibly phosphorylated by PKA and PP-2B using antiphosphoserine and
antiphosphothreonine antibodies as previously described
(31). Forskolin stimulation of RINm5F cells decreased the
serine phosphorylation of an 84-kDa protein whereas forskolin and FK506
treatment increased the serine phosphorylation (Fig. 3, A and B). No change in phosphorylation
was identified in phosphothreonine containing proteins following
treatment with either forskolin or FK506 (data not shown). Therefore,
the 84-kDa phosphoprotein fulfilled the criterion for a cell PKA
and PP-2B substrate. The mobility of this protein was similar to
synapsin 1, a recently identified 84-kDa cell phosphoprotein
(32) that is a substrate for PKA (33) and
PP-2B (13).
|
, lower panel). Serine phosphorylation of the
immunoprecipitated band was decreased following forskolin treatment,
whereas forskolin and FK506 treatment increased the serine
phosphorylation (Fig. 3C, lanes 1 and 3). Differences in the serine
phosphorylation of the synapsin 1 protein were determined by
densitometry (Fig. 3D), and these changes paralleled those found in
whole RINm5F lysates (Fig. 3A). Collectively, these results suggest
that synapsin 1 underwent reversible phosphorylation following
forskolin treatment of cells.
To confirm that phosphorylation of synapsin 1 was mediated through the
activation of PKA, experiments were repeated in the presence of the PKA
inhibitor, PKI (34). Synapsin 1 immunoprecipitates were
incubated with the catalytic subunit of PKA in the presence or absence
of FK506 and PKI. Synapsin 1 phosphorylation was greater in the
presence of FK506 and PKA than when PKI was also present (Fig. 3E).
These findings suggest that activation of PKA is necessary for the
forskolin-mediated phosphorylation and dephosphorylation of cell
synapsin 1.
Activation of PKA increases PP-2B activity
Given the observation that the activities of PKA and PP-2B were
coordinated resulting in the reversible phosphorylation of synapsin 1,
we hypothesized that activation of PKA may result in increased PP-2B
activity. To understand the interaction between PP-2B and PKA
activities in cells we measured kinase and phosphatase activities
in FK506 and forskolin treated RINm5F whole cell lysates. FK506
treatment decreased both basal and forskolin treated cellular PP-2B
activity (Fig. 4A). However, application
of forskolin and activation of PKA was associated with a 2-fold
(P < 0.001) increase in cellular PP-2B activity
compared with basal levels (Fig. 4A). As expected, forskolin treatment
also increased PKA activity (Fig. 4B). However, inhibition of PP-2B
activity did not significantly change PKA activity (Fig. 4B). These
data support a role for PKA activation in the activation of PP-2B;
however, the cellular mechanism that coordinates the activities of PKA
and PP-2B is currently unknown. Previous studies have suggested that
colocalization of PKA and PP-2B can regulate their enzymatic activities
(35).
|
, lane 3) and inhibiting the catalytic
activity of PKA (Fig. 4C; lane 4) blocked the dephosphorylation of the
84-kDa band seen following forskolin application (Fig. 4C, lane 2).
These data suggest that targeted PKA is needed for the activation of
PP-2B. To confirm this finding, we measured forskolin stimulated PP-2B
activity in RINm5F cells constitutively expressing the anchoring
inhibitor protein, Ht31 (Fig. 4D). Forskolin stimulated PP-2B activity
(Fig. 4D) was significantly lower in the Ht31 cells compared with
RINm5F cells transfected with vector alone (pcDNA). Together, these
data support a role for targeted PKA in PP-2B activation and reversible
phosphorylation of synapsin 1 in cells.
Association of PP-2B and PKA in pancreatic cells
In neurons, both PKA and PP-2B are colocalized by the PKA
targeting protein AKAP79/150 (18, 20). Given the
association we found between PKA and PP-2B in cells, we
hypothesized that AKAP79/150 may target these two enzymes in pancreatic
cells. To investigate this possibility, we looked for an
association between these two enzymes. Initially, we identified
AKAP150, the rat homologue of AKAP79, in pancreatic cells by
calmodulin affinity chromatography. AKAP150 was identified in the
eluate by both a radiolabeled RII binding assay (RII overlay) and by
immunoblotting (Fig. 5A). AKAP150 is the
upper band seen in both the immunoblots and radiolabeled overlays
whereas the lower band is a presumed breakdown product. Subsequently,
we identified an association between PP-2B and PKA in cells using
two complementary biochemical methods. First, we isolated PKA binding
proteins from RINm5F lysate using a cAMP affinity purification method
as previously described (36). PP-2B was identified in the
lysate, flow-through (FT) and eluate by immunoblotting with a specific
PP-2B antibody (Fig. 5B). Secondly, we immunoprecipitated PP-2B from
RINm5F lysates and measured PKA activity. PKA specific activity was
approximately 30-fold ± 2 (n = 4) greater in the PP-2B
immunoprecipitate than in a preimmune immunoprecipitate (Fig. 5C).
Finally, AKAP150 was identified in the eluate of a PP-2B
immunoprecipitation from RINm5F cells but not in the preimmune elution
(Fig. 5D). Together, these data strongly support an association between
a subset of PKA and PP-2B in cells probably via AKAP79/150.
|
cells cell function. To test this hypothesis,
we expressed AKAP79, the human homologue of AKAP150, in RINm5F cells.
RINm5F cells expressing AKAP79 (79OE) or vector alone (pcDNA) were
treated with forskolin and FK506 as described in Fig. 1. The RINm5F
cells expressing AKAP79 had lower forskolin and forskolin + FK506 (Fig. 6A) stimulated insulin secretion than did
wild-type RINm5F or pcDNA controls. Because the 79OE cells had lower
forskolin mediated insulin secretion, we calculated the fold increase
in insulin secretion of FK506 + forskolin over forskolin alone (Fig. 6B). 79OE cells had significantly lower FK506 fold insulin secretion
than did either RIN wild-type (wt) or vector controls (pcDNA)
(P < 0.05) suggesting that at least part of the 79OE
phenotype was related to changes in PP-2B activity. Other investigators
have shown that AKAP79 binds to PP-2B via residues 108–427 resulting
in an inhibition of PP-2B activity (20, 35). Because
constitutive inhibition of PP-2B by FK506 treatment can also decrease
forskolin stimulated insulin secretion (as shown in Fig. 2) we
hypothesized that constitutive expression of AKAP79 would inhibit PP-2B
activity. To test this hypothesis, we measured PP-2B activity in the
lysates of cells constitutively expressing AKAP79. Expression of AKAP79
decreased PP-2B activity compared with RINm5F or pcDNA lysates (Fig. 6C). This effect was not secondary to differences in the amount of
PP-2B found in the cell lysates as determined by immunoblotting with
specific PP-2B antibody (Fig. 6D). Changes in insulin messenger RNA and
insulin content have also been associated with immunosuppressant
treatment of islets or cells (30, 37). Therefore, to
determine if the diminished insulin secretion observed in the 79OE
cells was associated with changes in insulin content, we measured total
insulin content in the wild-type RINm5F, pcDNA transfected RIN, and 79
transfected RIN cells. We found no statistical difference in insulin
content of 79OE cells compared with either the RIN wild-type or RIN
pcDNA cells (Fig. 6E). Therefore, the decrease in forskolin and FK506
mediated insulin secretion in AKAP79 expressing cells was not secondary
to significant decreases in insulin content. Of interest, the RIN
wild-type cells have slightly diminished insulin secretion, lower PP-2B
activity and insulin content compared with the pcDNA transfected cells.
The reason for this difference is not known, however, we suspect that
differences in the tissue culture conditions (i.e. the
presence or absence of the selecting agent, G418) or passage number
could have produced these changes (38). Importantly, the
79OE and pcDNA transfected cells were treated identically therefore,
the pcDNA cell line is the appropriate control for the 79OE cells.
Despite the differences in cell culturing, the 79OE cells had
diminished FK506 and forskolin-mediated insulin secretion and PP-2B
activity compared with both the RIN Wt and pcDNA cells. Taken together
this data supports a role for AKAP150/79 as an endogenous inhibitor of
PP-2B activity and suggests that targeting of PP-2B may regulate its
activation.
|
). In contrast, the
regulatory subunit of PKA is found at the cell periphery in the 79OE
cells. The altered subcellular localization of PKA is associated with
increased AKAP79 targeting at the cell periphery. Minimal signal for
AKAP79 is seen in the wild-type RIN cells consistent with the low
levels of expression of AKAP150 in these cells and the diminished
affinity of the AKAP79 antibody for AKAP150. Together, these data show
that AKAP79 expression alters the distribution of PKA. However, this
does not completely explain the change in insulin secretion because
expression of AKAP 18 also targets PKA to the periphery of cells
but results in an increase in PKA mediated insulin secretion
(39). Therefore, we believe that AKAP79 targeting may also
affect the subcellular location of PP-2B, thus producing the decrease
in insulin secretion. To evaluate this possibility, we measured AKAP150
and PP-2B in soluble and particulate fractions of both basal and
forskolin stimulated RINm5F cells (Fig. 7, B and C, upper
panels). PP-2B was found predominately in the soluble fraction.
However, forskolin treatment resulted in a greater portion of PP-2B in
the particulate fraction (Fig. 7B, upper panel). AKAP150 is
predominately found in the particulate fraction with a portion moving
into the soluble fraction following forskolin treatment (Fig. 7C, upper panel). Expression of AKAP79 appears to diminish the
movement of PP-2B to the particulate fraction (Fig. 7B, lower
panel), whereas AKAP79 was found in both the soluble and
particulate fractions in the AKAP79 OE cells (Fig. 7C, lower
panel). The increased amount of AKAP79 in the soluble fraction may
be associated with the decrease in PP-2B activity and the decreased
movement of PP-2B to the particulate fraction in the 79 OE cells. This
suggests that AKAP targeting of PKA, depending upon cellular
conditions, could modify the subcellular movement of PP-2B and alter
the interaction between the two signaling enzymes.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
cells
(12). To understand the role of reversible phosphorylation
in cells, it is important to identify both the enzymes involved and
the mechanisms regulating the activities of the enzymes. In this paper,
we show that PKA activation of PP-2B results in the reversible
phosphorylation of cell proteins including synapsin 1 and regulates
insulin release. Furthermore, our data supports a role for AKAP
targeting of these enzymes in the regulation of reversible
phosphorylation events involved in insulin secretion.
We initially observed a synergistic effect of forskolin and FK506 on
insulin secretion suggesting that an increase in PKA and a decrease in
PP-2B activities regulate insulin release. This finding is consistent
with the previous observations that acute activation of kinases or
inhibition of phosphatases increased insulin secretion (12, 40). In addition, we found that prolonged (>3 h) PP-2B
inhibition results in suppressed PKA mediated insulin secretion
reflecting a possible role for PP-2B in resensitizing the cell for
repeated stimulation. Previous studies have demonstrated that
dephosphorylation of cell proteins involved in insulin exocytosis
results in desensitization of the cell to additional stimulations
(41). Chronic inhibition of PP-2B could desensitize
cells resulting in decreased insulin secretion. This would explain the
long-term effects of immunosuppressant drugs (CsA and FK506) on
decreased insulin release and development of type 2 DM
(15). Furthermore, we showed that the effect of FK506 on
insulin secretion could be blocked by cotreatment with another
immunosuppressant, rapamycin (Sirolimus). Although the effect of
rapamycin in cells is not well known, both rapamycin and FK506 must
bind to FKBP12 before they can activate their target proteins, mTOR and
PP-2B, respectively (42). Therefore, rapamycin may compete
with FK506 for binding to FKBP12 preventing inhibition of PP-2B by
FK506 (29). The interaction between these
immunosuppressant drugs on cell function is quite important because
recent studies have shown that mixtures of immunosuppressants may
affect cell function following islet cell transplantation
(43). Understanding the targets for these drugs in
cell will provide important insight into the use of immunosuppressants
for future islet transplantation.
Our data clearly demonstrates that FK506 treatment inhibits PP-2B
activity leading to the persistent phosphorylation of cell proteins
including synapsin 1. Synapsin 1 has recently been identified as a
cell protein (32) and is phosphorylated by PKA and
dephosphorylated by PP-2B (13, 33, 44). Therefore, it is
not surprising to find changes in the phosphorylation state of synapsin
1 resulting from PKA activation and PP-2B inhibition. However, because
we did not perform peptide mapping of synapsin 1, we cannot distinguish
between direct PKA phosphorylation of synapsin 1 vs. an
indirect effect of PKA activation on other kinases, such as CaM K II.
Our data does indicate that reversible phosphorylation of synapsin 1
was dependent upon PKA activation of PP-2B, suggesting that the
activities of these enzymes were closely linked. Although the specific
mechanism linking the activities of PKA and PP-2B is currently unknown,
there are two models that could explain this. First, PKA activation
could increase intracellular calcium and calmodulin concentrations and
subsequently activate PP-2B. Although such a mechanism is plausible
because PKA has documented effects on intracellular calcium levels
(5), we controlled the concentration of calcium and
calmodulin in our evaluation of PP-2B activity thereby minimizing the
effect of PKA mediated increases in calcium. Secondly, PKA could
directly phosphorylate PP-2B thus decreasing its phosphatase activity.
PP-2B is a substrate for PKA phosphorylation, but this is readily
blocked by calmodulin, which was present in our assay mixture
(45). Therefore, neither model adequately explains the
forskolin stimulated PP-2B activity we found in our cells
suggesting the presence of an alternative regulatory process.
Because targeting of PP-2B by AKAP79 results in an inhibition of PP-2B
catalytic activity (18, 20, 35), we speculate that PKA may
affect PP-2B activity by changes in PP-2B targeting to AKAP79. AKAP79
inhibition of PP-2B would explain our finding that expression of AKAP79
results in lower PP-2B cellular activity. AKAP targeting of PKA and
PP-2B also occurs in native cells confirmed by our finding AKAP150,
the rat homologue of AKAP79, in pancreatic cells and showing
colocalization of a portion of PKA and PP-2B in these cells.
Furthermore, it is possible that PKA activation may alter the activity
or distribution of PP-2B. Previously, Dell’Aqua et al.
(35) found that PKA and PKC phosphorylate AKAP79 resulting
in an increase in AKAP79 in the cell soluble fraction. Our data
confirms this change in distribution of AKAP79 and 150. In addition, we
show that expression of AKAP79 diminished the translocation of PP-2B
following forskolin stimulation. At this time we do not know whether
the movement of PP-2B or AKAP150 alters reversible phosphorylation
events that affect insulin secretion. However, our data supports an
effect of PKA on PP-2B that is partially regulated via a common
targeting protein, AKAP 79/150.
Independent of the mechanism by which PKA affects PP-2B, our data
suggests that the two enzymes are involved in an intracellular feedback
loop whereby a kinase promotes the activation of a phosphatase
resulting in reversible phosphorylation of specific proteins. The net
effect of such a feedback loop would be a return to a basal state of
phosphorylation, resetting the cell for additional stimulations.
The reciprocal function of these enzymes will ultimately affect shared
substrates including proteins involved in endocytosis
(46), desensitization of channels (25, 47) or
other mechanisms involved in regulating insulin secretion (37, 48). Disrupting the equilibrium between the phosphorylation and
dephosphorylation of these substrates could result in abnormal insulin
secretion, which may explain why insulin secretion was lower in the
AKAP79 expressing cells. For example, development of Alzheimer’s
disease is associated with increased phosphorylation of Tau resulting
from altered targeting of PKA and PP-2B by AKAP79 (49).
AKAP targeting of PKA and PP-2B in cells represents a mechanism for
regulating insulin secretion through balanced reversible
phosphorylation of specific substrates.
In summary, we have found that insulin secretion can be regulated by
the reversible phosphorylation of cell proteins through the
targeted effects of PKA and PP-2B.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received May 4, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
cells. J Biol Chem 271:8075–8081 cells. Proc Natl Acad
Sci USA 91:4343–4347This article has been cited by other articles:
 |
|
 |
|
  O. M. Faruque, D. Le-Nguyen, A.-D. Lajoix, E. Vives, P. Petit, D. Bataille, and E.-H. Hani Cell-permeable peptide-based disruption of endogenous PKA-AKAP complexes: a tool for studying the molecular roles of AKAP-mediated PKA subcellular anchoring Am J Physiol Cell Physiol, February 1, 2009; 296(2): C306 - C316. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Kim and J. M. Egan The Role of Incretins in Glucose Homeostasis and Diabetes Treatment Pharmacol. Rev., December 1, 2008; 60(4): 470 - 512. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Edemir, S. Reuter, R. Borgulya, R. Schroter, U. Neugebauer, G. Gabriels, and E. Schlatter Acute Rejection Modulates Gene Expression in the Collecting Duct J. Am. Soc. Nephrol., March 1, 2008; 19(3): 538 - 546. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. J. Martin, H. Chen, X. Liao, H. Allayee, D. M. Shih, G. S. Lee, D. N. Hovland Jr, W. A. Robbins, K. Carnes, R. A. Hess, et al. FK506, a Calcineurin Inhibitor, Prevents Cadmium-Induced Testicular Toxicity in Mice Toxicol. Sci., December 1, 2007; 100(2): 474 - 485. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. S. Goehring, B. S. Pedroja, S. A. Hinke, L. K. Langeberg, and J. D. Scott MyRIP Anchors Protein Kinase A to the Exocyst Complex J. Biol. Chem., November 9, 2007; 282(45): 33155 - 33167. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Costes, C. Broca, G. Bertrand, A.-D. Lajoix, D. Bataille, J. Bockaert, and S. Dalle ERK1/2 Control Phosphorylation and Protein Level of cAMP-Responsive Element-Binding Protein: A Key Role in Glucose-Mediated Pancreatic {beta}-Cell Survival. Diabetes, August 1, 2006; 55(8): 2220 - 2230. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Oynebraten, N. Barois, K. Hagelsteen, F.-E. Johansen, O. Bakke, and G. Haraldsen Characterization of a Novel Chemokine-Containing Storage Granule in Endothelial Cells: Evidence for Preferential Exocytosis Mediated by Protein Kinase A and Diacylglycerol J. Immunol., October 15, 2005; 175(8): 5358 - 5369. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Seino and T. Shibasaki PKA-Dependent and PKA-Independent Pathways for cAMP-Regulated Exocytosis Physiol Rev, October 1, 2005; 85(4): 1303 - 1342. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-Y. Ku, R. A. Word, and B. M. Sanborn Differential Expression of Protein Kinase A, AKAP 79, and PP2B in Pregnant Human Myometrial Membranes Prior to and During Labor Reproductive Sciences, September 1, 2005; 12(6): 421 - 427. [Abstract] [PDF]
|
|
|
|
 |
|
 C. Longuet, C. Broca, S. Costes, E. H. Hani, D. Bataille, and S. Dalle Extracellularly Regulated Kinases 1/2 (p44/42 Mitogen-Activated Protein Kinases) Phosphorylate Synapsin I and Regulate Insulin Secretion in the MIN6 {beta}-Cell Line and Islets of Langerhans Endocrinology, February 1, 2005; 146(2): 643 - 654. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. TASKEN and E. M. AANDAHL Localized Effects of cAMP Mediated by Distinct Routes of Protein Kinase A Physiol Rev, January 1, 2004; 84(1): 137 - 167. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Arnette, T. B. Gibson, M. C. Lawrence, B. January, S. Khoo, K. McGlynn, C. A. Vanderbilt, and M. H. Cobb Regulation of ERK1 and ERK2 by Glucose and Peptide Hormones in Pancreatic {beta} Cells J. Biol. Chem., August 29, 2003; 278(35): 32517 - 32525. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. E. Doyle and J. M. Egan Pharmacological Agents That Directly Modulate Insulin Secretion Pharmacol. Rev., March 1, 2003; 55(1): 105 - 131. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Nakatomi and M. Yazawa Identification and Characterization of a Novel Calcineurin-Binding Protein in Scallop Testis J. Biochem., February 1, 2003; 133(2): 159 - 164. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Affaitati, L. Cardone, T. de Cristofaro, A. Carlucci, M. D. Ginsberg, S. Varrone, M. E. Gottesman, E. V. Avvedimento, and A. Feliciello Essential Role of A-kinase Anchor Protein 121 for cAMP Signaling to Mitochondria J. Biol. Chem., January 31, 2003; 278(6): 4286 - 4294. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Nakazaki, A. Crane, M. Hu, V. Seghers, S. Ullrich, L. Aguilar-Bryan, and J. Bryan cAMP-Activated Protein Kinase-Independent Potentiation of Insulin Secretion by cAMP Is Impaired in SUR1 Null Islets Diabetes, December 1, 2002; 51(12): 3440 - 3449. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Alto, J. J. Carlisle Michel, K. L. Dodge, L. K. Langeberg, and J. D. Scott Intracellular Targeting of Protein Kinases and Phosphatases Diabetes, December 1, 2002; 51(90003): S385 - 388. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-Y. Ku and B. M. Sanborn Progesterone Prevents the Pregnancy-Related Decline in Protein Kinase A Association with Rat Myometrial Plasma Membrane and A-Kinase Anchoring Protein Biol Reprod, August 1, 2002; 67(2): 605 - 609. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. G. Vila Petroff, J. M. Egan, X. Wang, and S. J. Sollott Glucagon-Like Peptide-1 Increases cAMP but Fails to Augment Contraction in Adult Rat Cardiac Myocytes Circ. Res., August 31, 2001; 89(5): 445 - 452. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
), forskolin only (
), Glucose + FK506 (
)
and Forskolin + FK506 (
) are shown. Statistical significance was
determined by t test using Prism, GraphPad Software, Inc. *, P < 0.01 for forskolin
and FK506 vs. forskolin alone.
PS) or antisynapsin 1 antibody in the
lower panel (
), RIN
transfected with pcDNA vector
(
) and RIN transfected
with AKAP79 (