MAP (mitogen-activated protein) kinase (also called
Erk 1/2) plays a crucial role in cell proliferation and
differentiation. Its impact on secretory events is less
well established. The interplay of protein kinase C
(PKC), PI3-kinase nd cellular tyrosine kinase with
MAP kinase activity using inhibitors and compounds
such as glucose, phorbol 12-myristate 13-acetate
(PMA) and agonists of G-protein coupled receptors
like gastrin releasing peptide (GRP), oxytocin (OT)
and glucose-dependent insulinotropic peptide (GIP)
was investigated in INS-1 cells, an insulin secreting
cell line. MAP kinase activity was determined by
using a peptide derived from the EGF receptor as a
MAP kinase substrate and [P32]ATP. Glucose as well
as GRP, OT and GIP exhibited a time-dependent
increase in MAP kinase activity with a maximum at
time point 2.5 min. All further experiments were
performed using 2.5 min incubations. The flavone PD
098059 is known to bind to the inactive forms
of MEK1 (MAPK/ERK-Kinase) thus preventing activation
by upstream activators. 20 μM PD 098059
(IC50=51 μM) inhibited MAP kinase stimulated by
either glucose, GRP, OT, GIP or PMA. Inhibiton
(“downregulation”) of PKC by a long term (22h) pretreatment
with 1 μM PMA did not influence MAP
kinase activity when augmented by either of the
above mentioned compound. To investigate whether
PI3-kinase and cellular tyrosine kinase are involved
in G-protein mediated effects on MAP kinase, inhibitors
were used: 100 nM wortmannin (PI3-kinase
inhibitor) reduced the effects of GRP, OT and GIP
but not that of PMA; 100 μM genistein (tyrosine
kinase inhibitor) inhibited the stimulatory effect of
either above mentioned compound on MAP kinase
activation. Inhibition of MAP kinase by 20 μM PD
098059 did not influence insulin secretion modulated
by either compound (glucose, GRP, OT or GIP).
[H3]Thymidine incorporation, however, was severely
inhibited by PD 098059. Thus MAP kinase is important
for INS-1 cell proliferation but not for its insulin
secretory response with respect to major initiators
and modulators of insulin release. The data indicate
that MAP kinase is active and under the control
of MAP kinase. PKC is upstream of a genisteinsensitive
tyrosine kinase and probably downstream
of a PI3-kinase in INS-1 cells.
