Download (SREBP 1c) is strongly expressed in MIN6 beta cells

Biochemical Society Transactions (200 I ) Volume 29, Part 3
23
Regulation of Protein Kinase B activity by p Adrenergic
Agonists in Rat Epididymal Fat Cells.
S.K.Moule
University of Bristol, Department of Biochemistry, School of
Medical Sciences, University Walk, Bristol, BS8 1T D
w,
Protein Kinase B (PKB, also known as Akt) is an important
signalling molecule which has been shown to become activated in
response to many stimuli, including insulin, growth factors and a
variety of survival promoting agents. The signalling pathway by
which insulin activates PKB has been well characterised. Insulin
receptor ligation results in Phosphatidylinositol3' kinase
activation, which leads to phosphorylation of PKB on its
activatory sites (Thr 308 and Ser 473) following its translocation
to the membrane. PKB is also activated by p adrenergic agonists
(such as isoproterenol) in fat cells by an as yet unidentified
mechanism. This is particularly interesting since these agonists
usually produce opposing physiological effects to insulin. In
order to elucidate the mechanism by which p adrenergic agonists
activate PKB, we stimulated primary rat adipocytes with isoproterenol and other agents such as CPT-CAMPand forskolin. We
then measured the activity of PKB and one of its downstream
effectors, Glycogen Synthase Kinase 3 (GSK-3), in the presence
and absence of inhibitors such as wortmannin and the PKA
inhibitor H-89. We present evidence for the involvement of
several different pathways in the regulation of PKB and GSK-3
by isoproterenol.
A7 I
25 Sterol Regulatory Element Binding Protein lc (SREBP lc)
is strongly ex ressed in MIN6 p cells.
C.Andreolas[C.Zhao$,G.da Silva
Xavier$,A.Varadi$,F.Diraison$,F.Foufelle$$,P.Ferre$$,G.A.Ru
tter
$.
$University of Bristo1,School of Medical
Sciences,C&on, Bristol,BS8 1 TD; "INSER M Unite-342,Centre
Biomedical des Cordeliers lI,rue de I'ecole de Medecine,71270
Paris,France.
SREBP Ic plays an important role in the regulation by glucose
and insulin of lipogenic gene transcription in liver and adipose
tissue.Glucose also enhances the expression of similar genes in p
cells, but the role of SREBP lc in these cells is unknownwe show
that SREBP I c j n unprocessed and mature forms,is present in the
p cell line MIN6,and its expression increased by elevated glucose
concentrations (3 versus 30 mM, 24h).Exogenously added insulin
or blockage of insulin secretion did not affect these
changes.Microinjection of a dominant negative form of SREBP l c
impaired transcription from the L-pyruvate kinase,acetyl CoA
carboxylase,fatty acid synthase and preproinsulin promoters at
elevated glucose.Injection of a constitutively active form of
SREBP l c increased transcription from the same promoters at
elevated but not low glucose concentrations.These data suggest a
critical role of SREBP l c in glucose regulated gene expression in
p cells.However, overexpression of SREBP lc may cause lipid
accumulation in p cells and defective insulin secretion in some
forms of diabetes mellitus.
24 Characterisation of a PTEN homologue from Drosophila
W c C o n n a c h i e , I.Pass, N.Leslie, C.P.Downes
Division of Cell Signalling, MSI/WTB Complex, University
of Dundee, DDI IEH
Mutations in PTEN are common in gliomas, breast and prostate
cancers; as well as three autosomal disorders (Parsons et al, 1997).
PTEN encodes a phosphatase that resembles mixed function
protein phosphatases and selectively hydrolyses the 3-phosphate
in inositol phospholipid second messengers (Dixon et al, 1998). A
mutant form of PTEN -G129E- found in Cowden's sufferers has
a significantly reduced activity towards PtdIns(3,4,5)P3, whereas
its activity towards polyGluTyrP is not adversely affected (Tonks
et al, 1998). This indicates that the lipid phosphatase activity of
PTEN is required for its tumour suppressor function. A
homologue of PTEN in Drosophila (dPTEN) has a role in controlling cell number and size, as well as regulating the subcellular
organisation of the actin cytoskeleton in flies, but has not been
analysed biochemically (Wilson et a1,1999). Here we demonstrate
that dPTEN is an inositol phospholipid phosphatase that specifically dephosphorylates the D3 position of the inositol ring, and
has a similar effect as mammalian PTEN on protein kinase B
activity in mammalian cells in vivo. In contrast, we have found
that dPTEN does not display protein tyrosine phosphatase
activity against a standard acidic phosphopeptide substrate. This
demonstrates further the importance of the lipid phosphatase
activity for the function of PTEN.
26 Nanoscale Behavior of Ions in Narrow Channels
W.L. Duax, B.M. Burkhart and V. Pletnev
Hauptman- Woodward Inst., Inc., 73 High St., Buffalo, N Y
14203 USA and Shemyakin Inst., Moscow, RUSSIA
The crystal structures of ion complexes of gramicidin (CsCI,
TINO,, KCI, KI, KSCN, and RbCI) provide information
concerning the distribution of ions and water in a single file
channel. The crystallographically observed structures agree with
those based on NMR measurements in organic solvents and lipid
bilayers. Analysis reveals 7~ coordination of the ions with the
peptide bonds, asymmetry in the position of water molecules on
either side of the ions, and ion specific distribution within the
channel. When there is a water molecule on either side of an ion,
one is approximately 2.4A and the other 3.2A from the ion. Each
ion makes three or more contacts to the 7~ clouds of the carbonyl
oxygens and nitrogens of backbone peptide bonds. The ion coordination distances range from 2.6 to 3.9A. The torsion angles
defining the relationship of the ion and the plane of the peptide
bonds range from 54" to 104". The peptide angles of residues
involved in ion coordination exhibit significant non-planarity (as
much as 18"). ')C NMR chemical shift measurements attributed
to Na+ ion interactions with leucine residues in oriented
gramicidin channels in dimyristoyl phosphatidylcholine bilayers
correlate with these observations.
0 2001 Biochemical Society