Molecular mechanisms of

IR/IGFR Responses, 13
pS
pS
= activating
= inhibitory
How do we determine the
most important targets, the
timing, the cross regulation,
and the drugability of the
various pathways of a
signaling cascade?
Phosphoproteomics approaches
Dynamic Adipocyte
Phosphoproteome Reveals that Akt
Directly Regulates mTORC2
Sean J. Humphrey, Guang Yang, Pengyi Yang,
Daniel J. Fazakerley, Jacqueline Stockli, Jean Y.
Yang, and David E. James
Cell Metabolism 17, 10091020, June 4, 2013
Beavo Take Home Question 2013

Please read the posted review by Kahn and the research paper by James.
1) For the James paper briefly outline your opinion about at least two
strengths of the approach that they are using to help further refine the well-
studied insulin signaling pathway.
2) Identify what you feel are at least two limitations of the approach that they
use and briefly explain your reasoning for saying so. Your answer therefore
should have two parts and each part will be graded with approximately
equal weight. (One page total please).
SUMMARY
A major challenge of the post-genomics era is to define the
connectivity of protein phosphorylation networks. Here, we
quantitatively delineate the insulin signaling network in adipocytes by
high-resolution mass spectrometry-based proteomics. These data
reveal the complexity of intracellular protein phosphorylation. We
identified 37,248 phosphorylation sites on 5,705 proteins in this
single-cell type, with approximately 15% responding to insulin. We
integrated these large-scale phosphoproteomics data using a machine
learning approach to predict physiological substrates of several
diverse insulin-regulated kinases. This led to the identification of an
Akt substrate, SIN1, a core component of the mTORC2 complex. The
phosphorylation of SIN1 by Akt was found to regulate mTORC2
activity in response to growth factors, revealing topological insights
into the Akt/mTOR signaling network. The dynamic
phosphoproteome described here contains numerous phosphorylation
sites on proteins involved in diverse molecular functions and should
serve as a useful functional resource for cell biologists.
Phospho-peptide Enrichment Procedure
Protein extract is reduced, alkylated and digested with LysC/Trypsin. Peptides are desalted and
separated by SCX (strong cation exchange) chromatography. Each fraction is enriched by IMAC
(immobilized metal affinity chromatography) and analyzed by LC-MS/MS.
Fig 1. Quantification of the Insulin-Regulated Phospho-proteome
using SILAC Labeling & Tandem Mass Spectrometry
(A) Experimental design
of inhibitor screens.
PI3K screen
(B) Experimental design of temporal
phosphoproteome screen.
Insulin time course

8
(C) Workflow for
the proteome and
phosphoproteome
analysis
9
Dynamic Quantitative Analysis of
Akt/mTOR Networks
Fig 4. (A) Immunoblot analysis of adipocytes following different insulin-stimulated time points for proteins
known to belong to the Akt (blue) and mTOR (pink) pathways.
(B and C) Temporal profiles generated from SILAC-MS data for known direct Akt (B) and mTOR (C) substrates.
(D) Network model depicting the activation of Akt, mTORC1, and mTORC2 by growth factors.
10
Figure 5.
Temporal
Phosphorylation
in Response to
Insulin Reveals
Signaling
Network
Topology
Data from the literature
were used to construct a
cell signaling network.
Proteins identified in this
study were annotated with
their respective insulin-
dependent phosphorylation
sites color coded according
to the temporal patterns
derived from unsupervised
clustering (fuzzy c-means),
shown at the right.
Complete clusters (AR)
are shown in Fig S3 and
listed in Table S2. See also
Figs S4 and S5. Cell
Metabolism 17, 1009
1020, June 4, 2013
(B) Endogenous SIN1 is rapidly phosphorylated in response to insulin and blocked by the Akt allosteric inhibitor MK2206. 3T3-L1
adipocytes were treated with MK2206, and stimulated with insulin, and assessed by immunoblotting. (C) Insulin-stimulated
phosphorylation of endogenous SIN1 T86 is blocked by MK2206 and GDC-0068 (Akt competitive inhibitor), but not by rapamycin
(R). HEK293 cells were serum starved, treated with MK, GDC8, or rapamycin (50 nM) followed by insulin, and samples were
analyzed by immunoblotting. (D) Akt in vitro kinase assay using recombinant GST-Akt results in phosphorylation of SIN1 at T86 and
is blocked by GDC. (E) Expression of SIN1, but not SIN1 T86A mutant, in SIN1/ MEFs rescues mTORC2-dependent signaling. SIN1
WT or phosphomutants (T86A, T86E) were expressed in SIN1/ MEFs, selected by FACS, stimulated with insulin, and analyzed by
immunoblotting. (F) In vitro kinase activity of endogenous mTORC2 isolated from cells is enhanced by insulin stimulation and
blocked by pretreatment with MK2206, but not rapamycin. LY294002 was added directly to the in vitro kinase assay.
Fig 6. Akt is the
physiological kinase
for SIN1 T86, and its
phosphorylation
directly regulates
mTORC2 activity

(A) SIN1 domain structure
homology of the region
surrounding T86. TORC,
putative mTORC-binding
domain; CC, coiled-coil
domain; CRIM, conserved
region in the middle domain ;
RBD, Raf-like Ras-binding
domain; PH, pleckstrin
homology domain. Enlarged
is the region containing the
insulin-responsive
phosphorylation site, T86.
Residues surrounding
several other known Akt
substrates (AS160 T642,
FOXO1A S256, TSC2 S939
and BAD S99) are shown.
12
(G) mTORC2 isolated from SIN1/ MEFs reconstituted with SIN1 WT or phosphomutants (T86A, T86E)
displays differential growth factor-stimulated kinase activity in in vitro kinase assay, with enhanced mTORC2
activity isolated from T86E hosphomimetic mutants.
(H) Model depicting growth factor-dependent activation of mTORC2 mediated by Akt phosphorylation of SIN1.
See also Figure S6.
Fig 6 (cont). Akt Is the Physiological Kinase for SIN1 T86, and its
Phosphorylation Directly Regulates mTORC2 Activity
Fig S6. Identification of SIN1 as a Direct Akt
Substrate
(A) Time-course of SIN1 Thr86 phosphorylation in insulin-stimulated HEK-293T cells. Cells transiently expressing
Flag-SIN1 or empty vector were serum starved for 2 h followed by stimulation with insulin (100 nM) for the
indicated durations. Flag-SIN1 was immunoprecipitated from cell lysates using the Flag antibody.
Immunoprecipitated proteins and total cell lysates were analyzed by immunoblotting using the indicated
antibodies. (B) mTORC2 complex formation is not affected by SIN1 Thr86. mTORC2 was immunoprecipitated
from SIN1 -/- MEF cells stably expressing SIN1wild type (WT) or SIN1 phospho-mutant (T86A) and samples
were analysed by immunoblotting for components of mTORC2 complex. (C) Signalling in SIN1 -/- MEF cells
rescued with SIN1 phospho-mutants. Expression of SIN1but not SIN1 T86A mutant in SIN1 null MEFs rescues
mTORC2-dependent signalling. SIN1 wild type (WT) or phospho-mutants (T86A, T86E) were expressed in SIN1
-/- MEFs, and cells selected by FACS as described in Materials and Methods. Cell lines were serum starved for 2
h, stimulated with insulin (100 nM, 10 min) and samples analysed by immunoblotting.
14
How do other pathways talk to IRS-1? Functional
Interactions that can modify IRS-1 by phosphorylation
M. White Can J P Jul 06
Interrogating cAMP-dependent kinase signaling
in Jurkat T-Cells by a protein kinase A targeted
immune-precipitation phosphoproteomics
approach
P Giansanti, M P Stokes, J C Silva, A Scholten and A J R Heck
Molec & Cell Proteomics, Papers in Press. Published on
July 23, 2013
Workflow for the targeted analysis of PKA
substrates in Jurkat cells
Figure 1. A, Western blots of Jurkat clone E6.1 cells stimulated with PGE2 (10 M) over a 60 min time course using the phospho-
PKA substrate antibody (top) and a tubulin antibody (bottom) as control, supplemented by a densitometric analysis of the Western
blots. B, Quantitative Proteomics. Jurkat cells were either left unstimulated (Control) or activated with PGE2 (10 M) over two
different stimulation times (1 min and 60 min). After lysis and enzymatic digestion with Lys-C, peptides were differentially labeled with
three stable isotope dimethyl-labels and subsequently combined. Next, an immunoprecipitation with the immobilized phospho- PKA
substrate specific antibody was performed. After stringent washing, the eluate was analyzed by nanoLC-MS. Peptides and proteins
were identified by database search, and a functional analysis was performed using motif search algorithms as well as Ingenuity and
String
Molec & Cell Proteomics, July 23, 2013
Interaction map for PGE2 downstream substrates
Fig 6. Proteinprotein interactions were derived from both the STRING database using experimental evidence and
high confidence interactions (score > 0.700) while the substrate lines derived from the search function in the
PhosphoSitePlus database. The interaction map was generated using Cytoscape. Protein class information is
from PhosphoSitePlus. Color coding indicates sensitivity to PGE2 stimulation.
Molec & Cell Proteomics, July 23, 2013
Proteins not yet implicated in PKA
signaling
Molec & Cell Proteomics, Papers in Press. Published on July 23, 2013
Q: What PDEs regulate
which processes in this
steroid producing cell
type??
Currently
Recognized
Mechanism(s)
for regulation of
steroid
synthesis
HSL/CEH
AC
G
i
G
s

PKA
cAMP
P
P
P
P
P
P
PDEs ?
Cholesterol
Storage Droplet
PAT
PAT
P
AMP
HSL/CEH
P
Chol Storage
Droplet
P
Hormone (LH, ACTH etc.)
PKA
Pregnenolone
Progesterone
Androstenedione
TESTOSTERONE
3bHSD
P450c17
17bHSD
Corticosterone
PDE Superfamily
11 families, 21 genes, 100+ variants/isoforms
Viagra
Ca
2+
/CaM
cGMP hydrolysing
cAMP hydrolysing
cGMP and cAMP-
hydrolyzing
PDE11
PDE10
PDE9
PDE5
PDE6s
PDE1s
PDE4s
cGS PDE2
PDE8s
PDE7s
cGI PDE3s
PDE mRNA profiles in
enriched Leydig cell
preparation and MA10 cells.
In order to determine which PDEs are
expressed in Leydig cells, mRNA levels are
analyzed by real-time PCR reactions in Leydig
cell preparation obtained from WT testis and
MA10 cells. Total RNA from the cells was
isolated using a NucleoSpin RNA II kit
according to the manufactures protocol
(Macherey Nagel Inc., Bethlehem, PA). Then
cDNA samples were generated with
SuperScript III reverse transcriptase
(Invitrogen) using 1 g of total RNA for each
reaction. Relative gene expression was
determined by performing real- time PCR on a
MX3000P QPCR system (Stratagene/Agilent
Technologies, Santa Clara, CA) and analyzed
with Mx-Pro software. The sequences of
primers for PDEs are shown below and some
have been previously reported and verified
(Patrucco et al., 2010). RT-PCR reactions were
run with iTaq SYBR supermix (Biorad) with the
following thermal profile: denaturing at 95C
for 15 sec, annealing at 55C for 1 min,
extension at 72C for 1 min, for 40 cycles. The
levels of PDE mRNAs are shown as a relative
amount to -actin.

Shimizu-Albergine et al, Mol Pharmacol
81:556566, 2012

-Galactosidase expression for PDE8A and PDE8B
KOs in the interstitial area of the testes
PDE8A KO PDE8B KO WT
12
13
14
1 2
3
6 7 8 9 11
15
16
21 22 4
5
20
19
18 17 10
LAC-Z NEO
0.01 0.1 1 10 100
0
200
400
600
800
1000
none
PF-04957325
IBMX 30 M
PF + IBMX

Effect of PDE8 inhibitor on forskolin dose
response curve
NOTE; IBMX most effective in presence of PDE8 inhibitor or high forskolin
Forskolin (M)
PF-4957325 + IBMX
IBMX 30 uM
- PF-4957325 100 nM
vehicle

















150
100
30
20
10
50
0
P
r
o
g
e
s
t
e
r
o
n
e

(
n
g
/
m
g
)

Control
+Rol
+PF-04957325
40
+Rol +PF(200 nM)
Co-inhibition of PDE8 and PDE4 increases
progesterone in MA10 cells
0 10 100 1000
0
50
100
150
None
IBMX (50 uM)
Rolipram (10 uM)
REQUIRED FOR
PF 04957325 (nM)
Are there other, unknown, synergistic
mechanisms/pathways?
Unbiased approach: Phosphoproteomics
Martin Golkowski Shao-En Ong
SILAC Procedure
(Stable Isotope
Labeling with Amino
acids in Cell culture)

1. Cells are stimulated with factors of
interest (eg. PDE inhibitors) for
various times

2. Cells are lysed and enzymatically
digested.

3. Peptides are separated using ion
exchange chromatography.

4. Phosphopeptides are enriched
using phospho-specific antibodies,
or immobilized metal affinity
chromatography.

5. Phosphopeptides are analyzed
using mass spectrometry.
Identify and quantify by nanoLC-MS
Light Medium Heavy
m/z
I
n
t
e
n
s
i
t
y
PDE4
inhibitor
PDE4 + PDE8
inhibitors
Control
Proteolytic digestion
SCX-IMAC
enrichment of
phosphopeptides
Protein
abundance
profiling
95% 5%
PDE-regulated
changes in
phosphorylation
m/z
I
n
t
e
n
s
i
t
y
PDE-regulated
changes in
protein expression
MA10 cells labeled with Heavy, Medium, or Light
Isotopes are treated 1 hour with PDE inhibitors
(control, PDE4i, PDE8i, or both)
~ 8000 phospho-peptides identified

~ 320 increased >2 fold by combination of PDE8 and PDE4 inhibitors

Very few increased by individual PDE inhibitors alone

Over 30 with good consensus PKA sites ( >3x )

Several suggest new points for cAMP/PDE regulation of steroidogenic
function

Several suggest other previously unappreciated cAMP regulated
functions
Results
Protocol
PO
4

ratio
8i+4i/co
n
PO
4

ratio
8i+4i/co
n
PO
4

ratio
8i+4i/co
n
Protein/Gene
Name
PO
4
amino
acid
sequence
Major Functions

20.0 3.0 2.0 Linker Protein/
Clip1/2
RYARKISGT Binds & ends tubulin
15.0 6.0 nd Nesprin2/Syne2 WRKRRESE
E
Nuclear envelope/ actin
interaction
11.0 2.0 2.0 GPR107/GPR107 KKVKKVSNG Unknnown function
11.0 5.0 1.0 SH3&PX Protein
2A/Tks5
EGSRRGSAD Necessary for podosome
formation
8.0 nd 1.0 AKAP1/AKAP1 IKDRRLSEE Anchors signaling proteins
8.0 3.0 1.0 Chrebp/Mixpl GSERRLSGD Carbohydrate response
element binding protein
7.0 3.0 1.0 Oxysterol
BP/Osbp1
ISQRRPSQN Binds cholesterol metabolites
6.0 3.0 2.0 Salt Inducible
Kinase/Sik3
SPVRRFSDG cAMP Inhibits in Fat
7.0 2.0 1.0 RNAase/Dicer1 KMPKKASLG Formation of micro RNA
5.0 nd 0.9 Raf Kinase/Raf1 GYQRRASD
D
Upstream regulator of MEK
4.0 nd 0.3 HSL/CEH/Lipe FHPRRSSQG Major regulator of lipolysis
4.0 nd 0.8 SREBP cleavage
Act Protein/Scap
PGPRRDSCG Major regulator of cholesterol
synthesis
4.0 7.0 nd Perilipin/Plin1 PVVRRLSTQ Major regulator of lipolysis
PKA consensus site phosphorylation: +/- PDE4/8 inhibitors
Wnk1 (6x), Nav1 (6x), Tbc1d25 (5x), Ethe1 (5x), Sqstm1 (5x), Arhgef2 (4x), Arfgap1 (4x), Cgnl1 (3x), Ralgapa1 (3x), Nckap5
(3x), Ripk2 (3x), Slc24A3 (3x), Casp8 (3x), PDE8A&B (2.5x)
Increased phosphorylation of Raf-1 and HSL by
combined PDE4/8 inhibition in MA10 cells
p-Raf-1 (Ser43)
Raf-1
p-HSL (Ser660)
HSL
GAPDH
MA10 cells were treated with the inhibitors or 8Br-cAMP for 60 min
tmAC
Pregnenolone
Lipid
Droplet


cAMP/PKA regulates Leydig cell steroidogenesis
LHR
Nur77
EGF-R
cAMP
CREB
ERK
MEK
Ras
Raf
PDE4
PKA
StAR
StAR
p450
p450
Chol
AMPK
PDE8A
PDE8B
Perilipin-1
Progesterone
HSL
P
P
P
P
P
P
P
P
P
P
P
P
33
nuff for now