Download The mitogen-activated protein kinase cascade in rat islets of

BiochemicalSociety Transactions (1995) 23 221S
The mitogen-activatedprotein kmase cascade in rat islets of
Langerhans.
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eMAPK-P
MAPK-deP
S.J. PERSAUD', C.P.D. WHEELER-JONES' AND P.M. JONESt
'The Randall Institute, *Vascular Biology Research Centre, and
+Physiology Group, Biomedical Sciences Division, King's
College London, UK.
Mitogen-activated protein kinases (MAPKs), a family of
proteins ranging in molecular weight from 41-44kDa, are
stimulated in response to a variety of extracellular growth factors
and one major role of MAPKs is the regulation of cell
proliferation [reviewed in I]. However, it is becoming apparent
that MAPKs also function to transduce non-mitogenic signals,
and they have been implicated in stimulus-secretion coupling in
several cell types 12-41.
The roles played by the major serinehhreonine protein
kinases (CAMK, PKC, PKA) in signal transduction processes in
pancreatic p cells have been examined in detail in recent years
[reviewed in 51, but there is little information on the expression,
regulation or function of MAPKs in islets of Langerhans. Like
CAMK, PKC and PKA, MAPKs phosphorylate substrate
proteins on serine and/or threonine residues. However, rather
than being activated directly by second messengers, MAPK
activities are regulated by another family of kinases (MEKs; 4446kDa) which phosphorylate MAPKs on tyrosine and threonine
residues. Further upstream, the phosphorylation state of MEKs
is regulated by MEK kinases which include ruf--1,coupled to
receptor tyrosine kinases, and MEKK, thought to be coupled to
G protein-linked receptors [6].
In the present studies we have examined whether MEKs
and MAPKs are present in rat islets and whether the
phosphorylation state and activity of MAPKs can be correlated
with insulin secretory responses. Rat islets of Langerhans,
isolated from the pancreas by collagenase digestion, were used
for all of the following studies. We investigated the expression
of the families of MEKs and MAPKs by immunoblotting of
fractionated islet proteins with appropriate antisera. The
phosphorylation state of MAPK(s) was assessed using both an
anti-phosphotyrosine antibody and gel shift assays. MAPK
activity was determined by phosphorylation of myelin basic
protein (MBP), after immunoprecipitationof MAPKs from islet
extracts. Insulin secretion from islets was measured by
radioimmunoassay.
Three isoforms of both MEK (44, 45 and 46kDa) and
MAPK (42, 43, 44kDa) were detected in rat islets. In
unstimulated islets (2mM glucose), MAPK was largely (> 95 %)
in a dephosphorylated state. The tyrosine phosphatase inhibitor
sodium pervanadate (IOOpM) stimulated an increase in MAPK
phosphorylation, as assessed both by a shift in its electrophoretic
mobility (Figure 1) and by increased phosphotyrosine
immunoreactivity. The 42kDa isoform of MAPK (ERK2) showed
the most pmounced increase in tyrosine phosphorylation in
response to sodium pervanadate. The serinelthreonine
phosphatase inhibitor okadaic acid (10pM) caused a small
increase in the tyrosine phosphorylation state of ERK2. Insulin
secretion was significantly stimulated by exposure of islets to
20mM glucose (percentage increase: 5 min, 143*24%; 15 min,
622+114%, n=4, P<0.05) or to 500nM 48PMA (5 min,
54+6%; 15 min, 497+73%, n=4, P<0.05), but neitheragonist
caused a significant increase in phosphorylation of ERK2 at
either 5 (Figure 1) or 15 minutes, as assessed by mobility shift
assays using a monoclonal anti-ERK2 antibody. The lack of
effect of 4PPMA was somewhat surprising since it has been
reported to stimulate MAPK phosphorylation in several cell types
[e.g. 7.81, but even prolonged (30 min) exposure of islets to
1
2
3
4
Figure 1. MAPK phosphorylation by sodium pervanadate.
In unstimulated islets (2mM glucose, lane 1) the 42kDa isoform
of MAPK (ERK2) was not phosphorylated (MAPK-deP), as
determined by a mobility shift assay. Incubation of islets for 5
minutes in the presence of 20mM glucose (lane 2) or 500nM 40
PMA (lane 3) did not affect the phosphorylation state of ERK2.
However, stimulation of islets with 100pM sodium pervanadate
for 5 minutes (lane 4) caused a marked increase in ERK2
phosphorylation (MAPK-P).
4PPMA caused no change in MAPK phosphorylation. In parallel
experiments, using human umbilical vein endothelial cells,
4PPMA (100nM) stimulated a dramatic (-5-fold) increase in
MAPK phosphorylation within 10 minutes.
The increase in MAPK phosphorylation stimulated by
sodium pervanadate was not coupled to an increase in MAPK
activity. However, okadaic acid (IOpM), either alone or in the
presence of sodium pervanadate (100pM). caused an increase in
MAPK activity, as assessed by phosphorylation of MBP.
Exposure of islets for 15 minutes to glucose (20mM) or to
4PPMA (500nM) did not result in stimulation of MAPK activity.
Okadaic acid (IOpM) had no effect on unstimulated insulin
release at 2mM glucose (127*20% basal, P>0.2). Similarly,
sodium pervanadate (100pM) did not significantly affect basal
insulin secretion, either alone (l07+15% basal, P>0.2), or in
combination with 10pM okadaicacid (133*15% basal, P>O.l).
The results from our studies indicate that both MEK and
MAPK families are present in islets, but that the insulin
secretagogues glucose and 4PPMA do not stimulate MAPK
activation. Phosphoprotein phosphatases may affect the
phosphorylation state/activity of MAPK(s), but have no effect on
the secretory capacity of /3 cells. The role(s) of the MAPK
family in islets remains to be determined.
I , Marshall, C. J. (1994) Current Opinion in Genetics & Development
4, 82-89
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Sturgill, T.W. and Parsons, S.J. (1990) J. Cell Biol. 110, 731-742
3. Santini, F. and Beaven, M.A. (1993) J. Biol. Chem. 268, 2271622722
4. Mitchell, R., Sim, P.J., Johnson, M.S. and Thomson, F.J. (1994)
J. Endocrinol. 140, R15-Rl8
5 . Persaud, S.J., Jones, P.M. and Howell, S.L. (1994) In: Frontiers
of insulin secretion and pancreatic Bcell research (eds P.R. Flatt and
S. Lenzen), chapter 34
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and Johnson, G.L. (1993) Science 260, 315-319
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SIP is a Wellcome Trust Research Fellow (grant number 039057).