Biomedical Engineering Reference
In-Depth Information
There are a large number of these, and the full range will not be
discussed here. It has been shown that there is considerable con-
servation of Sec genes from yeast to mammalian cells, and the
mammalian gene products have also been identifi ed [
6
].
The COPII coat is formed on the surface of the ER where
the small GTPase Sar1 and two complexes known as Sec23-Sec24
and Sec13-Sec31 assemble. Sar1 activation takes place via a GDP/
GTP exchange which is catalyzed by Sec12, a transmembrane gua-
nidine nucleotide exchange factor (GEF) [
7
]. Once activated, Sar1
recruits the Sec23/Sec24 heterodimer to the membrane, and
together these form the Sec23/Sec24/Sar1 complex, an approxi-
mately 15 nm long structure in the shape of a bow tie [
8
]. The side
that faces the membrane is concave and positively charged. This is
where cargoes bind to Sec24, and are included in the COPII vesi-
cle. The recruitment of Sec23/Sec24 in turn recruits the Sec13/
Sec31 heterotetramer, allowing for the export of proteins from the
ER in COPII-coated vesicles [
9
] (Fig.
1
). Sec23 acts as a GTPase-
activating protein (GAP), hydrolyzing the GTP from Sar1 to
release the vesicle from the membrane [
10
]. Sec24 is the selector
of the cargo proteins [
11
], and Sec13 and Sec31 are responsible
for forming the fl exible cage around the vesicle in a cuboctahedron
shape [
12
]. It has been suggested that to accommodate larger car-
goes, an icosidodecahedron shape may also be possible [
13
].
We have found that Sec24 is phosphorylated by Akt [
14
],
a kinase involved in cell survival and proliferation, and here we
describe the novel method we developed to investigate this. We used
an immunoprecipitation of overexpressed, epitope-tagged Sec24,
which is then incubated with commercially available Akt in a kinase
assay, followed by Western blotting with phosphorylated Akt sub-
strate antibody. To the best of our knowledge, this type of approach
is typically used only to assay for activity of immunoprecipitated
kinases on an added peptide substrate, rather than vice versa.
We also describe methods for identifying potential phosphory-
lation sites and kinases using a database and prediction programs.
We have recently used these to explore the role signaling processes
play in the activity of DHCR24, the fi nal enzyme in cholesterol
synthesis [
15
].
2
Materials
1. Protein G-Sepharose beads.
2. Dulbecco's Phosphate-buffered saline (PBS): 137 mM NaCl,
2.7 mM KCl, 8.1 mM disodium hydrogen phosphate,
1.47 mM potassium dihydrogen phosphate.
3. RIPA buffer: 20 mM Tris-HCl (pH 7.4), 0.1 % (w/v) sodium
dodecyl sulfate (SDS), 1 % (v/v) NP-40, 0.5 % (w/v)
Na-deoxycholate, 150 mM NaCl, 5 mM ethylenediamine-
tetraacetic acid (EDTA), 1 mM Na
3
VO
4
.
2.1 Immunopreci-
pitation (IP) of Protein
