Intro

Molecular mechanisms of IR/IGFR Responses, 13pSpS= activating= inhibitoryFigure 2. Insulin and insulin-like signaling cascade. Two main branches propagate signals generated via the IRS-proteins: PI3K!PDK1!AKT and GRB2/SOS!RAS kinase cascades. Activation of the receptors for insulin and IGF-1 results in tyrosine phosphorylation of the IRS-proteins, which bind PI3K and GRB2/SOS. The GRB2/SOS complex promotes GDP/GTP exchange on p21ras, which activates the RAS!RAF!MEK!ERK1/2 cascade. Activated ERK stimulates transcriptional activity by direct phosphorylation of elk1 and by indirect phosphorylation of fos through p90rsk. The activation of PI3K by IRS-protein recruitment produces PI3,4P2 and PI3,4,5P3 (antagonized by the action of PTEN or SHIP2), which recruit PDK1 and AKT to the plasma membrane. AKT is activated via phosphorylation at T308 by PDK1 and at S473 by mTOR in complex with rictor. The mTOR kinase is activated by RhebGTP, which accumulates upon inhibition of the GAP activity of the TSC1TSC2 complex following PKB-mediated phosphorylation of TSC2. mTOR is also activated by AKT-mediated PRAS40 phosphorylation. The S6K is primed through mTOR-mediated phosphorylation for activation by PDK1. AKT phosphorylates many cellular proteins, and this inactivates PGC1a, p21kip, GSK3b, BAD and AS160, and activates PDE3b and eNOS. mTOR also promote cleavage and activation of SREBP1c (Clvd SREBP1C), which stimulates the expression of genes needed for lipid synthesis. AKT-mediated phosphorylation of forkhead proteins, including FOXO1, results in their sequestration in the cytoplasm, which inhibits their influence upon transcriptional activity. Insulin stimulates protein synthesis by altering the intrinsic activity or binding properties of key translation initiation and elongation factors (eIFs and eEFs, respectively) as well as crucial ribosomal proteins. Components of the translation machinery that are targets of insulin regulation include eIF2B, eIF4E, eEF1, eEF2 and the S6 ribosomal protein [117]. TNFa activates JNK which can phosphorylate IRS1, inhibiting its interaction with the insulin receptor and its subsequent tyrosine phosphorylation. IRS2 expression is promoted by nuclear FOXO, which increases IRS2 concentration in the fasted liver.

Red pS inhibition; Green pS activation I guess.

Split into multiple things that Erk can do ----- Feedback via PTEN on PI3K systemLarge increase in complexity for roles in mTOR pathway ---- Many more substrates/pathways for AKT/PKBFOXO1 roles

Split into multiple things that Erk can do ----- Feedback via PTEN on PI3K systemLarge increase in complexity for roles in mTOR pathway ---- Many more substrates/pathways for AKT/PKBFOXO1 roles1How do we determine the most important targets, the timing, the cross regulation, and the drugability of the various pathways of a signaling cascade?Phosphoproteomics approachesWhat pathways should we target in a given cell type to have the desired response.2Dynamic Adipocyte Phosphoproteome Reveals that Akt Directly Regulates mTORC2Sean J. Humphrey, Guang Yang, Pengyi Yang, Daniel J. Fazakerley, Jacqueline Stockli, Jean Y. Yang, and David E. JamesCell Metabolism 17, 10091020, June 4, 2013Beavo 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).SUMMARYA 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 ProcedureProtein 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

Figure 1. Quantification of the Insulin-Regulated Phosphoproteome using Tandem Mass Spectrometry(A) Experimental design of inhibitor screens.(B) Experimental design of temporal phosphoproteome screen.LY294002 is a morpholine derivative of quercetin ] It is a potent inhibitor of phosphoinositide 3-kinases (PI3Ks). Two of these are the proto-oncogene serine/threonine-protein kinase (PIM1) and the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma isoform] With an IC50 of 1.4M it is somewhat less potent than wortmannin, another well-known PI3 kinase inhibitor However, LY294002 is a reversible inhibitor of PI3K whereas wortmannin acts irreversibly].Akt inhibitor MK2206 An orally bioavailable allosteric inhibitor of the serine/threonine protein kinase Akt (protein kinase B) with potential antineoplastic activity. Akt inhibitor MK2206 binds to and inhibits the activity of Akt in a non-ATP competitive manner, which may result in the inhibition of the PI3K/Akt signaling pathway and tumor cell proliferation and the induction of tumor cell apoptosis. Activation of the PI3K/Akt signaling pathway is frequently associated with tumorigenesis and dysregulated PI3K/Akt signaling may contribute to tumor resistance to a variety of antineoplastic agents.78(C) Workflow for the proteome and phosphoproteome analysis

(C) Workflow for the proteome and phosphoproteome analysis.

89Dynamic 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.

Figure 4. Dynamic Quantitative Analysis of Akt/mTOR Networks (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.910Figure 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, 10091020, June 4, 2013

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 Figure S3 and listed in Table S2. See also Figures S4 and S5. Cell Metabolism 17, 10091020, June 4, 201310

(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. (A) SIN1 domain structure and sequence 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.(B) Endogenous SIN1 is rapidly phosphorylated in response to insulin and blocked by pretreatment with the allosteric Akt inhibitor MK2206. 3T3-L1 adipocytes were serum starved, treated with MK2206 (MK; 10 mM, 30 min), and stimulated with insulin (100 nM) for the indicated times, and samples were assessed by immunoblotting.(C) Insulin-stimulated phosphorylation of endogenous SIN1 T86 is blocked by MK2206 and GDC-0068 (competitive Akt inhibitor), but not by rapamycin. HEK293 cells were serum starved overnight, treated with MK2206 (MK; 10 mM and 1 mM), GDC-0068 (GDC;10, or rapamycin (50 nM) followed by insulin (200 nM, 10 min), and samples were analyzed by immunoblotting.(D) Akt in vitro kinase assay performed using recombinant GST-Akt results in specific phosphorylation of SIN1 at T86 and is blocked by GDC-0068 (GDC) added to the in vitro kinase reaction.(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, and cells were selected by FACS. Cell lines were serum starved and stimulated with insulin, and samples were analyzed by immunoblotting.(F) In vitro kinase activity of endogenous mTORC2 isolated from cells is enhanced by insulin stimulation (200 nM, 10 min) and blocked by pretreatment of cells with MK2206 (MK; 10 mM), but not rapamycin (R; 50 nM). LY294002 (LY*; 15 mM) was added directly to the in vitro kinase assay.

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(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 ActivityFigure 6. Akt Is the Physiological Kinase for SIN1 T86, and its Phosphorylation Directly Regulates mTORC2 Activity(A) SIN1 domain structure and sequence 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 insulinresponsive phosphorylation site, T86. Residues surrounding several other known Akt substrates (AS160 T642, FOXO1A S256, TSC2 S939 and BAD S99) are shown.(B) Endogenous SIN1 is rapidly phosphorylated in response to insulin and blocked by pretreatment with the Akt inhibitor MK2206. 3T3-L1 adipocytes were serum starved, treated with MK2206 (MK; 10 mM, 30 min), and stimulated with insulin (100 nM) for the indicated times, and samples were assessed by immunoblotting.(C) Insulin-stimulated phosphorylation of endogenous SIN1 T86 is blocked by MK2206 and GDC-0068, but not by rapamycin. HEK293 cells were serum starved overnight, treated with MK2206 (MK; 10 mM and 1 mM), GDC-0068 (GDC;10 mM), or rapamycin (50 nM) followed by insulin (200 nM, 10 min), and samples were analyzed by immunoblotting.(D) Akt in vitro kinase assay performed using recombinant GST-Akt results in specific phosphorylation of SIN1 at T86 and is blocked by GDC-0068 (GDC*; 10 mM) added to the in vitro kinase reaction.(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, and cells were selected by FACS. Cell lines were serum starved and stimulated with insulin, and samples were analyzed by immunoblotting.(F) In vitro kinase activity of endogenous mTORC2 isolated from cells is enhanced by insulin stimulation (200 nM, 10 min) and blocked by pretreatment of cells with MK2206 (MK; 10 mM), but not rapamycin (R; 50 nM). LY294002 (LY*; 15 mM) was added directly to the in vitro kinase assay.(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 S6.12Fig 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.Figure S6. Identification of SIN1 as a Direct Akt Substrate, Related to Figure 6(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.1314

How do other pathways talk to IRS-1? Functional Interactions that can modify IRS-1 by phosphorylationM. White Can J P Jul 061415Interrogating cAMP-dependent kinase signaling in Jurkat T-Cells by a protein kinase A targeted immune-precipitation phosphoproteomicsapproachP Giansanti, M P Stokes, J C Silva, A Scholten and A J R HeckMolec & Cell Proteomics, Papers in Press. Published on July 23, 2013Molec & Cell Proteomics, Papers in Press. Published on July 23, 2013

16Workflow 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 StringMolec & Cell Proteomics, July 23, 2013Figure 1. Experimental workflow used for the targeted and quantitative analysis of PKA substrates in Jurkat cells. 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, Papers in Press. Published on July 23, 2013

17

Interaction map for PGE2 downstream substratesFig 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, 2013Molec & Cell Proteomics, Papers in Press. Published on July 23, 2013

18Proteins not yet implicated in PKA signaling

Molec & Cell Proteomics, Papers in Press. Published on July 23, 2013What is major caveat here? Are these proteins really direct targets of PKA? Does it matter if they are downstream of PGE2?

Figure 6. Interaction map for regulated PGE2 downstream substrates. Proteinprotein interactions were derived from both the STRING database using experimental and database lines of 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, Papers in Press. Published on July 23, 2013

1920Q: What PDEs regulate which processes in this steroid producing cell type??Currently Recognized Mechanism(s) for regulation of steroidsynthesisHSL/CEHACGiGsPKAcAMPPPPPPPPDEs ?Cholesterol Storage Droplet PATPATPAMPHSL/CEHPChol StorageDropletPHormone (LH, ACTH etc.)PKAPregnenoloneProgesteroneAndrostenedioneTESTOSTERONE3bHSDP450c1717bHSDCorticosterone21ATGL = adipose triglyceride lipasePDE Superfamily11 families, 21 genes, 100+ variants/isoforms ViagraCa2+/CaM cGMP hydrolysing cAMP hydrolysing cGMP and cAMP- hydrolyzingPDE11PDE10PDE9PDE5PDE6sPDE1sPDE4s cGSPDE2PDE8sPDE7scGIPDE3s22

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

Q: Why does PDE profile in cell line differ a bit from primary cells?23

-Galactosidase expression for PDE8A and PDE8B KOs in the interstitial area of the testesPDE8A KOPDE8B KOWT12131412367891115162122452019181710LAC-Z NEO24LacZ gene containing a nuclear localization signal in the knockout cassette allows easy identification of the cells in a mixed cellular tissue that expresses the mRNA for the gene in question, in this case PDE8A.Testis cryo-sections were fixed with 4% PFA and incubated with rabbit anti-b-galactosidase antibody. The sections were then incubated with antirabbit Alexa546 secondary antibody and finally incubated with TOPRO3 for nuclear counterstaining.

Effect of PDE8 inhibitor on forskolin dose response curveNOTE; IBMX most effective in presence of PDE8 inhibitor or high forskolin Forskolin (M)Progesterone (ng / mg prot) PF-4957325 + IBMX IBMX 30 uM- PF-4957325 100 nM vehicle150100302010500Progesterone (ng/mg)Control+Rol+PF-0495732540 +Rol +PF(200 nM)Co-inhibition of PDE8 and PDE4 increases progesterone in MA10 cellsProgesterone (ng/mg)0101001000050100150NoneIBMX (50 uM)Rolipram (10 uM)REQUIRED FORPF 04957325 (nM)26Figure 5A. Co-inhibition of PDE8 and PDE4 increases progesterone production in MA10 Leydig cells.

Are there other, unknown, synergistic mechanisms/pathways?Unbiased approach: Phosphoproteomics

Martin GolkowskiShao-En Ong

SILAC Procedure (Stable Isotope Labeling with Amino acids in Cell culture)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.

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 functionsResultsProtocolPO4 ratio 8i+4i/conPO4 ratio 8i+4i/conPO4 ratio 8i+4i/conProtein/Gene NamePO4 amino acid sequence Major Functions20.03.02.0Linker Protein/ Clip1/2RYARKISGTBinds & ends tubulin15.06.0ndNesprin2/Syne2WRKRRESEENuclear envelope/ actin interaction11.02.02.0GPR107/GPR107KKVKKVSNGUnknnown function11.05.01.0SH3&PX Protein 2A/Tks5EGSRRGSADNecessary for podosome formation8.0nd1.0AKAP1/AKAP1IKDRRLSEEAnchors signaling proteins8.03.01.0Chrebp/MixplGSERRLSGDCarbohydrate response element binding protein7.03.01.0Oxysterol BP/Osbp1ISQRRPSQNBinds cholesterol metabolites6.03.02.0Salt Inducible Kinase/Sik3SPVRRFSDGcAMP Inhibits in Fat7.02.01.0RNAase/Dicer1KMPKKASLGFormation of micro RNA5.0nd0.9Raf Kinase/Raf1GYQRRASDDUpstream regulator of MEK4.0 nd0.3HSL/CEH/LipeFHPRRSSQGMajor regulator of lipolysis4.0nd0.8SREBP cleavage Act Protein/ScapPGPRRDSCGMajor regulator of cholesterol synthesis4.07.0ndPerilipin/Plin1PVVRRLSTQMajor regulator of lipolysisPKA consensus site phosphorylation: +/- PDE4/8 inhibitorsWnk1 (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)The 1st column shows the ratio (drug to control) of phosphorylation of PKA consensus sites increased by treatment of MA10 cells with a combination of PF 04957325 (200 nM) or Rolipram (10 uM). The 2nd column is PF alone and the 3rd column is rolipram alone. Column 5 shows the amino acid sequence context around the major phosphorylation site increased by the PDE inhibitor treatment. Values are rounded to the nearest whole number unless less than 1. 30Increased phosphorylation of Raf-1 and HSL by combined PDE4/8 inhibition in MA10 cells

p-Raf-1 (Ser43)Raf-1 p-HSL (Ser660)HSLGAPDHPF 200nM

Rol 10M

PF+Rol8Br-cAMP300MControlMA10 cells were treated with the inhibitors or 8Br-cAMP for 60 mintmACPregnenoloneLipidDropletcAMP/PKA regulates Leydig cell steroidogenesis LHRNur77EGF-RcAMPCREBERKMEKRasRafPDE4PKAStARStARp450p450CholAMPKPDE8A PDE8BPerilipin-1ProgesteroneHSLPPPPPPPPPPPP33nuff for now