Have We Overlooked the Importance

vinvay Kumar Parameswara, Aditi Jagdish Sule, Victoria Esser
Department of Internal Medicine, University of Texas Southwestern Medical Center.
Dallas, TX, USA
Summary
Genetic predisposition and environmental
influences insidiously converge to cause
glucose intolerance and hyperglycemia. Beta-
cell compensates by secreting more insulin
and when it fails, overt diabetes mellitus
ensues. The need to understand the
mechanisms involved in insulin secretion
cannot be stressed enough. Phosphorylation
of proteins plays an important role in
regulating insulin secretion. In order to
understand how a particular cellular process is
regulated by protein phosphorylation the
nature of the protein kinases and protein
phosphatases involved and the mechanisms
that determine when and where these
enzymes are active should be investigated.
While the actions of protein kinases have
been intensely studied within the beta-cell,
less emphasis has been placed on protein
phosphatases even though they play an
important regulatory role. This review focuses
on the importance of protein phosphatase 2A
in insulin secretion. Most of the present
knowledge on protein phosphatase 2A
originates from protein phosphatase inhibitor
studies on islets and beta-cell lines. The
ability of protein phosphatase 2A to change
its activity in the presence of glucose and
inhibitors provides clues to its role in
regulating insulin secretion. An aggressive
approach to elucidate the substrates and
mechanisms of action of protein phosphatases
is crucial to the understanding of
phosphorylation events within the beta-cell.
Characterizing protein phosphatase 2A within
the beta-cell will certainly help us in
understanding the mechanisms involved in
insulin secretion and provide valuable
information for drug development.
INTRODUCTION
Diabetes mellitus is a multifactorial disease
characterized primarily by absolute or relative
deficiency of insulin that leads to
hyperglycemia. There are two main forms of
diabetes mellitus. In type 1 diabetes mellitus,
there is an absolute insulin insufficiency
caused by the immunological destruction of
pancreatic beta-cells that produce and secrete
insulin, and it accounts for approximately
10% of all cases of diabetes mellitus in the
United States. However, type 2 diabetes
mellitus, which constitutes approximately
90% of the cases, is characterized by insulin
resistance in peripheral tissues and/or relative
deficiency of insulin due to the failure of
pancreatic beta-cells to secrete insulin [1].
Insulin resistance, frequently associated [1, 2]
with but not exclusive to obesity [3],
generates excessive stress on the beta-cells to
hypersecrete insulin to compensate.
Eventually when beta-cells are unable to cope

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up with the sustained need for a state of
hyperinsulinemia, overt diabetes mellitus
ensues [4]. Although insulin resistance is
critical in the development of type 2 diabetes
mellitus, it is not always the first step in the
cascade of events that leads to disease. Recent
studies have shown that primary defect in
insulin secretion can instigate hyperglycemia
and ultimately lead to type 2 diabetes mellitus
[5, 6, 7].
Protein Phosphorylation and Insulin
Secretion
Irrespective of the cause of hyperglycemia,
insulin secretion is without a doubt, a key
cellular event that needs to be characterized in
order to treat diabetes mellitus effectively. For
example, the classic group of drugs,
sulfonylureas, utilized routinely to treat
hyperglycemia in type 2 diabetes mellitus act
primarily by enhancing insulin secretion [8].
Therefore, understanding the complex
mechanisms involved in insulin secretion is
crucial for understanding this disease. Many
decades of extensive research from all over
the world have shown that there are multiple
pathways involved in regulating this complex
stimulus-secretion coupling in the beta-cells.
One such mechanism is via activation of
phospholipases and protein kinase C [9].
Hormones such as acetylcholine activate this
pathway via phosphoinositide, which causes
an increase in intracellular Ca2+ levels and
diacylglycerol, which then activates protein
kinase C that subsequently phosphorylates
various substrates. Another mechanism is by
stimulation of G protein coupled adenyl
cyclase activity and activation of protein
kinase A [10]. Hormones such as vasoactive
intestinal peptide and glucagon like peptide-1
activate these pathways. Mechanistically, the
most well studied pathway is the KATP
channel-dependent pathway, whereby,
increased concentrations of glucose and other
nutrients cause depolarization of the beta-cells
via closure of the KATP channel. Closure of
this channel increases Ca2+ entry and this rise
in intracellular calcium concentrations
stimulates insulin release [11, 12]. In the KATP
channel-independent pathway, a site distal to
the elevation of intracellular calcium levels is
involved. The exact mechanisms of action of
this pathway have not been clearly defined,
and several candidate mechanisms exist.
Calcium-dependent kinases have been
implicated to play a role in phosphorylation
and subsequent signal transduction [13, 14]. It
is apparent that multiple signaling pathways
are able to modulate insulin secretion, but the
molecular mechanisms may be differentially
regulated by protein phosphorylation. This
phosphorylation event involves several
different protein kinases such as calmodulin-
dependent protein kinase II (CaMKII), protein
kinase A (PKA), protein kinase C (PKC) and
mitogen-activated protein kinases (MAPK)
acting alone or in concert on different
substrates leading to an increase in insulin
release [15, 16, 17, 18]. The activation of
these kinases, in turn, will lead to
phosphorylation of various substrates that will
ultimately result in insulin exocytosis.
Although kinases act on different substrates,
phosphorylation seems to be an important
orchestrator of insulin release. Hitherto,
investigators have tried hard to resolve the
importance of one kinase over another and to
find out how kinases play specific roles in
regulating this phosphorylation event. Beta-
cell phosphatases, on the other hand, have not
been extensively researched. Phosphorylation
events can be regulated by two sets of
opposing enzymes-kinases and phosphatases.
Protein Phosphatases
Reversible phosphorylation regulates almost
all aspects of cell life, from metabolic
pathways to cell death [19]. At least one-third
of human proteins contain covalently bound
phosphate [20]. More than 98% of protein
phosphorylation occurs on serine and
threonine residues. The degree of
phosphorylation is modulated by changes in
the activities of protein kinases and protein
phosphatases [21]. Because a single
phosphatase catalytic moiety associates with
several different regulatory or targeting
subunits, the total number of functional

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phosphatase holoenzymes is expected to be
similar to the number of protein kinases [22].
Protein phosphatases are divided into three
families namely: phospho protein phosphatase
(PPP), protein phosphatase magnesium
dependent (PPM), and protein tyrosine
phosphatase (PTP) according to amino acid
sequence homology, protein structure, and
sensitivity to inhibitors [23]. PPP and PPM
comprise phosphoserine and phospho-
threonine specific enzymes, whereas, the PTP
includes phosphotyrosine specific and dual
specificity phosphatases that can de-
phosphorylate all three phosphoresidues [24].
The PPP class includes PP1, PP2A, PP2B,
PP4, PP5, PP6 and PP7 as well as their
various isoforms. The PPM includes PP2C
and related isoforms [25, 26]. Even within the
same PPP family significant structural
diversity is present. The catalytic domains of
these phosphatases have a high degree of
identity. However, their ability to form
heteromeric complexes with a variety of
regulatory subunits makes them unique.
These regulatory domains or subunits localize
the protein complexes to a specific subcellular
compartment, modulate the substrate
specificity, or alter catalytic activity [27, 28].
The recent heightened interest in
phosphatases has led to the discovery of a
large number of isoforms and targeting
subunits. Limited evidence exists as to where
the various subunits are expressed and what
substrates are involved in the beta-cells. There
is a pressing need for understanding the
mechanisms involved in the regulation of
protein phosphorylation in the beta-cells
through a phosphatase perspective. Insulin
secretagogues
that
increase
the
phosphorylation state within the beta-cell by
activating kinases have been shown to
suppress beta-cell phosphatase activities.
Certain secretagogues such as glucose that
increase L-glutamate concentrations within
the beta-cell and subsequent insulin secretion
[29], have been shown to act by regulating
protein phosphatase activity [30]. Inositol
hexakisphosphate, the dominant inositol
phosphate in beta-cells, has been shown to
inhibit serine threonine phosphatases in a
concentration dependent manner and increase
insulin secretion [31]. Another insulin
secretagogue, L-arginine, a metabolic
precursor to polyamines, has been shown to
cause a rapid and transient decrease in protein
phosphatase activity in beta-cells [32]. Also,
sulfonylureas, used routinely to reduce
hyperglycemia in type 2 diabetes mellitus
subjects, inhibits these phosphatases [33]
although some studies have argued that
sulfonylureas do not affect phosphatases [34].
The main function of the beta-cell is to
synthesize and secrete insulin. This paper
focuses on the role played by phosphatases,
particularly PP2A, in regulating the secretion
aspect of beta-cells (Figure 1).
Protein Phosphatase 2A (PP2A)
PP2A holoenzyme consists of a constant
dimeric core, i.e. the catalytic subunit
(PP2AC) and the A subunit (PP2AA),
associated with one of the family of the B
(PP2AB), subunit. PP2AC is the
enzymatically active component and it has
two isoforms: alpha and beta. The expression
of the PP2AC is tightly controlled resulting in
Figure 1. Model showing PP2A’s role in insulin
secretion from the beta-cells. Upon stimulation,
multiple pathways converge to increase insulin
secretion. One such pathway is via the kinases, which
are activated by insulin secretagogues and thus increase
the phosphorylation state within the beta-cells. A
complimentary regulatory pathway is via PP2A, which
dephosphorylates and maintains a dephosphorylated
state. Certain insulin secretagogues inactivate PP2A
thus inhibiting the dephosphorylation event. However,
the complex mechanisms involved in the regulation of
this inactivation of PP2A and the substrate(s) through
which PP2A increases insulin secretion within the beta-
cell is not clearly defined.

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a constant level of PP2A [35]. The PP2AA
appears to function primarily as a scaffolding
protein that serves to assemble the
holoenzyme complex. Two distinct PP2AA
isoforms are present which share 86%
homology [36]. The PP2AB is thought to act
as a targeting module that directs the enzyme
to various intracellular locations and also
provides distinct substrate specificity. All of
these subunits exist in various isoforms within
the body, so that the ABC holoenzyme is a
structurally diverse enzyme enabling a single
catalytic subunit to associate with a wide
array of regulatory subunits. The PP2AB is
made up of four unrelated families named B,
B', B'' and B''' with several different members,
all of which are able to bind to the PP2AA in
a mutually exclusive manner to form a
distinct ABC holoenzyme complex. While the
PP2AA and PP2AC are present in all cells,
some of the PP2AB are expressed in a tissue-
specific fashion and at distinct developmental
stages [37]. The B family of the regulatory
PP2AB consists of four members with
differential expression in the brain [38]. The
structural feature of this family is the presence
of WD-40 repeats (tryptophan-aspartate
(WD)), and they mediate various protein-
protein interactions [39]. The B' family
contains at least five members and each
member has more than one isoform with
different localizations within the cell. These
members are unique because of their ability to
be phosphorylated [37]. The B'' family has at
least four members and is present in many
organs within the body. The B''' family
consists of at least two members and they all
contain the WD-40 repeats. They are
predominantly nuclear in localization and
may play a role in Ca2+ dependent signaling
[40].
PP2A has been implicated in the regulation of
a multitude of cellular functions, such as
metabolism, transcription and translation,
RNA splicing and DNA replication,
development and morphogenesis, as well as
cell cycle progression and transformation
[41]. Within the beta-cell PP2A has been
suggested, via a circuitous route, to play a
role in protein synthesis [42], and perhaps it is
involved in a multitude of other cellular
functions. For purposes of simplicity, this
paper focuses on the secretion aspect in the
beta cell.
In the body, PP1, PP2A and PP2B constitute
the majority of the phosphatases [43]. Also, in
the beta-cell other serine threonine protein
phosphatases have not been identified. So,
most beta-cell studies performed on protein
phosphatases have grouped PP1 and PP2A
since they are inhibited by a group of
inhibitors [44, 45]. Understanding their
functional roles is predominantly based on
studies that have utilized a number of
naturally occurring inhibitors. However, due
to the differences in the effects of these
inhibitors in vitro and in vivo, concentrations
of inhibitors far in excess of the [IC]50 for
most enzymes must be used, and so
interpretation of results is difficult. In
addition, due to differences in subcellular
targeting the local concentrations of
individual phosphatases in distinct regions of
the beta cell would be quite different from
one another. Differential accessibility of the
inhibitors to different regions of the cell also
makes interpretation of in vivo effects
difficult.
The majority of these inhibitors have a degree
of specificity for different members of the
PPP family. Okadaic acid inhibits all
members of the PPP family with a degree of
selectivity for PP2A and the closely related
PP4. PPM phosphatases are unaffected by
okadaic acid or any other natural inhibitor.
Calyculin A is also a potent inhibitor of the
PPP family, but in contrast with okadaic acid,
shows little specificity for individual family
members. Tautomycin, on the other hand, has
a higher affinity for PP1 over other family
members and may therefore be useful in
conjunction with other inhibitors to identify
roles for PP1 inside cells [46]. Fostriecin is
unique among inhibitors in that it has strong
selectivity for PP2A and PP4 over other PPP
family members and is therefore a particularly
useful tool in delineating functional roles for
these phosphatases [47, 48]. To differentiate
the action of PP1 and PP2A, other PP2A
inhibitors such as endothall can be used [49].

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However, tissue or species variations of
inhibition constants can profoundly hamper
interpretation of data. The development of
compounds that alter the activity of specific
phosphatases is rapidly emerging as an
important area in drug discovery.
Beta-cell studies using phosphatase inhibitors
have shown that inhibition of PP1 and PP2A
enhances insulin secretion from islets and
insulin secreting cell lines (INS-1, RIN-5,
MIN-6 and HIT-T15). Although using the
inhibitors alone may not be sufficient to elicit
insulin response; but, in the presence of
glucose the inhibitors enhance insulin
response. However, some studies have
demonstrated that inhibiting protein
phosphatases alone is sufficient for insulin
secretion [50]. Nevertheless, there is a general
consensus that upon glucose stimulation or
Ca2+
influx [51], inhibition of protein
phosphatases enhances insulin secretion.
Some studies have shown that inhibition of
phosphatases reduce insulin secretion [52,
53]. This inconsistency in the studies could be
because long-term exposure to these
inhibitors or using high concentration of
inhibitors reduces insulin secretion by being
toxic to beta-cells. Surprisingly, PP2A
specific inhibitors have not been used within
the beta-cell to demonstrate its involvement in
insulin secretion. Okadaic acid and nodularin,
for example, respectively have over 100-fold
and 50-fold higher potency of inhibition
towards PP2A than towards PP1 [54]. Due to
lack of specificities, and in order to gain
insight as to which phosphatase is involved,
the subcellular co-localization of PP1 and
PP2A needs to be addressed.
Besides phosphatase inhibitors, stimulation of
beta-cells with the insulin secretagogues such
as L-arginine, L-glutamine, as well as KCl
and ATP decreases PP1 and PP2A activities.
Interestingly, ATP and ADP inhibit PP2A
more than PP1 suggesting that PP2A is
primarily involved in insulin secretion [55]. In
the beta-cell, ATP is known to increase upon
uptake and metabolism of stimulatory
concentrations of glucose as well as amino
acids. In fact, increased ATP is an indicator of
high-energy state which physiologically leads
to an increase in insulin secretion to signal
target tissues to store excess energy into fats.
However, addition of cAMP, cGMP, or
prostaglandins E2 and F1 alpha at widely
different concentrations failed to affect
protein phosphatase activities although insulin
secretion was enhanced [55]. This suggests
that protein dephosphorylation is not
necessarily the sole mechanism through
which insulin secretion occurs. This could
explain the discrepancies between data where
one group shows an increase in insulin
secretion upon inhibiting phosphatases and
another group shows the exact opposite.
Nevertheless, during an event where protein
phosphatases are involved in insulin
secretion, PP2A appears to be playing the
primary role. Perhaps, upon very strong
stimuli, other phosphatases may be involved
in regulating the phosphorylation state.
Studies have shown direct inhibition of PP2A
by glucose and its metabolites by membrane-
depolarizing concentrations of KCl in beta-
cells [56]. Fructose-2,6-bisphosphate and
glucose-1,6-bisphosphate, which are known
to allosterically activate phosphofructokinase,
one of the rate-limiting enzymes in the
glycolytic pathway, have been reported to
have inhibitory effects on porcine heart PP2A
[56, 57]. It has also been shown that 3-
phosphoglycerate and phosphoenolpyruvate
increase protein phosphorylation in
permeabilized beta-cells [58] to support this
model. Inositol hexakisphosphate, which
increases insulin secretion by inhibiting
protein phosphatases, has been shown to
preferentially inhibit PP2A at lower
concentrations [34], once again supporting the
model that PP2A primarily regulates the
phosphorylation state that regulates insulin
secretion. As shown in Figure 1, upon
stimulation, multiple pathways converge to
increase insulin secretion from the beta-cells.
One such pathway is via the kinases, which
are activated by certain insulin secretagogues
and thus increase the phosphorylation state
within the beta-cells. Certain stimuli that
increase insulin secretion lead to an
inactivation of PP2A probably in conjunction
with the activation of kinases and presumably

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regulate this phosphorylation state. The
complex mechanisms involved in the
regulation of this inactivation of PP2A and
the substrate(s) through which PP2A exerts its
role is not clearly defined.
One mechanism through which the activity of
PP2A is regulated is by covalent
modifications.
PP2AC
undergoes
modifications such as carboxymethylation at
its C-terminal leucine (Leu-309) [59], which
leads to an increase in insulin secretion.
Ebelactone, an inhibitor of PP2AC
demethylation, markedly reduced nutrient-
induced insulin secretion from normal rat
islets. Taken together, these data seem to
suggest a key modulatory role for PP2A in
insulin secretion [60]. Carboxymethylation
not only dictates its interaction with other
subunits of the PP2A but also its substrate
specificity, subunit assembly, its subcellular
localization and association with regulatory
proteins [61]. Consistent with the PP2A
regulating insulin secretion model is the
finding in beta-cells that the PP2AC
undergoes carboxymethylation, an effect
accompanied by increased PP2A activity and
suppressed insulin secretion [60, 62]. Some
studies have shown that carboxymethylation
has either no effect [63] or decreased effect
on the activity of PP2A [64] suggesting that
modifications of PP2AC may affect other
characteristics of PP2A. However, these
effects were observed in non-insulin secreting
cells. The presence of numerous PP2AB
subunits and the lack of studies on these
within the beta-cell illustrates the importance
of characterizing the role played by PP2A in
the beta-cell.
Furthermore, heat stable inhibitors [65],
several other proteins [66], as well as certain
lipid second messengers such as ceramide
[67], have been implicated in the regulation of
PP2A function. Ceramides are formed from
the hydrolysis of sphingomyelin by
membrane-bound
sphingomyelinase.
Localization of such an enzyme activity was
reported in isolated rat islets, mouse islets and
clonal beta-cells [68]. Ceramides either
delivered exogenously or generated
endogenously, inhibit insulin secretion by
activating PP2A. This is not mediated via
activation of the carboxyl methylation of the
catalytic subunit of PP2A, suggesting yet
another PP2A regulatory locus in the beta-
cells [69]. This inhibition of insulin secretion
was rescued by the addition of okadaic acid in
the beta-cell. Saturated fats like palmitate and
stearate if given in excess lead to the
accumulation of ceramides [70]. Also, long-
term exposure of isolated beta-cells to
ceramides significantly reduced glucose and
carbachol induced insulin secretion from
these cells [71]. Some studies have
demonstrated beta-cell necrosis in the
presence of ceramides [72]. This could be the
reason why obesity and lipotoxicity leads to
decrease in insulin secretion although fatty
acids have been shown to increase insulin
secretion [73]. Another method of regulating
PP2A is by phosphorylation of its tyrosine
and serine threonine residues in the PP2AC
[74, 75]. Kinases could be regulating this
pathway (Figure 1). Alternatively, the action
of kinases can be regulated by PP2A and thus
regulate the overall phosphorylation state
within the beta-cells. However, this has not
been shown to be the case in the beta-cells.
Clearly, multiple signaling pathways can
modulate the phosphorylation aspect of
insulin secretion, but little is known, however,
about how these various regulatory forces are
coordinated and integrated to direct the
function of PP2A towards its multifaceted
tasks. The different isoforms of the A, B or C
subunit of PP2A have not been identified
within the beta-cell. The subcellular
localization is another important piece of
information that is missing. This information
will provide valuable insight on its function
and resolve discrepancies.
Protein Phosphatase 1 (PP1)
PP1 consists of a constant catalytic subunit
and one or two variable regulatory subunits
that target the phosphatase to a particular
cellular compartment and/or act as substrate
specifiers [76, 77]. Dozens of different
regulatory subunits of PP1 have been
described and for some of them a function ha

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