The nerve growth factor NGF has been shown to cause cell fate decisions toward either differentiation or proliferation depending on the relative activity of downstream pERK, pAKT, or pJNK signaling. However, how these protein signals are translated into and fed back from transcriptional activity to complete cellular differentiation over a time span of hours to days is still an open question. Comparing the time-resolved transcriptome response of NGF- or EGF-stimulated PC12 cells over 24 h in combination with protein and phenotype data we inferred a dynamic Boolean model capturing the temporal sequence of protein signaling, transcriptional response and subsequent autocrine feedback. Network topology was optimized by fitting the model to time-resolved transcriptome data under MEK, PI3K, or JNK inhibition. The integrated model confirmed the parallel use of MAPK/ERK, PI3K/AKT, and JNK/JUN for PC12 cell differentiation. Redundancy of cell signaling is demonstrated from the inhibition of the different MAPK pathways. As suggested in silico and confirmed in vitro, differentiation was substantially suppressed under JNK inhibition, yet delayed only under MEK/ERK inhibition. Most importantly, we found that positive transcriptional feedback induces bistability in the cell fate switch. De novo gene expression was necessary to activate autocrine feedback that caused Urokinase-Type Plasminogen Activator (uPA) Receptor signaling to perpetuate the MAPK activity, finally resulting in the expression of late, differentiation related genes. Thus, the cellular decision toward differentiation depends on the establishment of a transcriptome-induced positive feedback between protein signaling and gene expression thereby constituting a robust control between proliferation and differentiation. Crowther Audrey audrey.crowther@huskers.unl.edu University of Nebraska-Lincoln 2016-08-17T11:57:51Z 2017-03-03T13:57:42Z

nerve growth factor

 2016-08-21T08:34:21Z

protein kinase B

 2016-08-19T13:35:02Z

Cyclic AMP-responsive element-binding protein

Gene: CREB1

UniProt ID: P16220

Gene ID: 1385

 2016-08-18T12:46:18Z

SHC-transforming protein

 2016-08-17T16:14:33Z

Early growth response protein 1

Gene: EGR1

UniProt ID: P18146

Gene ID: 1958

 2016-08-28T16:37:59Z

Dual specificity protein phosphatase 6

Gene: DUSP6

UniProt ID: Q16828

Gene ID: 1848

 2016-08-18T13:21:55Z

Activity-regulated cytoskeleton-associated protein

Gene: ARC

UniProt ID: Q7LC44

Gene ID: 23237

 2016-08-28T16:37:59Z

Growth factor receptor-bound protein 2

Gene: GRB2

UniProt ID: P62993

Gene ID: 2885

 2016-08-17T16:14:58Z

Transcription factor jun-D

Gene: JUND

UniProt ID: P17535

Gene ID: 3727

 2016-08-17T20:28:19Z

Proto-oncogene c-Fos

Gene: FOS

UniProt OD: P01100

Gene ID: 2353

 2016-08-19T13:23:47Z

Serum response factor

Gene: SRF

UniProt ID: P11831

Gene ID: 6722

 2016-08-17T19:56:34Z

Mitogen-activated protein kinase kinase kinase 3

Gene: MAP3K3

UniProt D: Q99759

Gene ID: 4215

 2016-08-17T19:40:12Z

Urokinase plasminogen activator/Tissue-type plasminogen activator

Gene: PLAU/PLAT

UniProt ID: P00749/P00750

Gene ID: 5328/5327

 2016-08-17T18:49:52Z

Activator Protein-1

AP1 is a general term for a dimeric transcription factor composed of Jun, Fos, or ATF.

 2016-08-28T16:45:12Z

Dual specificity mitogen-activated protein kinase kinase 7

Gene: MAP2K7

UniProt ID: O14733

Gene ID: 6885

 2016-12-15T15:11:43Z

Telomeric repeat-binding factor 2-interacting protein 1

Gene: TERF2IP

UniProt ID: Q9NYB0

Gene ID: 54386

 2016-09-15T14:00:55Z

Urokinase plasminogen activator receptor.

 2016-08-17T18:49:52Z

phosphoinositide 3-kinase

 2016-08-17T16:38:38Z

extracellular signal-regulated kinase

 2016-08-19T13:35:02Z

Integrin, Alpha 1

Gene: ITGA1

UniProt ID: P56199

Gene ID: 3672

 2016-08-17T18:54:44Z

Fos-related antigen 1

Gene: FOSL1

UniProt ID: P15407

Gene ID: 8061

 2016-08-28T16:45:12Z

protein kinase C

 2016-08-17T18:40:35Z

Cyclic AMP-dependent transcription factor ATF-2

Gene: ATF2

UniProt ID: P15336

Gene ID: 1386

 2016-08-19T13:27:44Z

Calcium ion

 2016-08-17T18:40:35Z

matrix metallopeptidase 3/matrix metallopeptidase 10 (stromelysin 2)

Stromelysin-1/Stromelysin-2

Gene: MMP3/MMP10

UniProt ID: P08254/P09238

Gene ID: 4314/4319

 2016-08-17T18:49:52Z

Krueppel-like factor 10

Gene: KLF10

UniProt ID: Q13118

Gene ID: 7071

 2016-08-18T12:07:38Z

Rap guanine nucleotide exchange factor 1

Gene: RAPGEF1

UniProt ID: Q13905

Gene ID: 2889

 2016-08-17T16:24:57Z

diacylglycerol

 2016-08-17T18:40:35Z

Krueppel-like factor 4

Gene: KLF4

UniProt ID: O43474

Gene ID: 9314

 2016-08-17T20:33:51Z

Ribosomal protein S6 kinase alpha-5/Ribosomal protein S6 kinase alpha-4

Gene: RPS6KA5/RPS6KA4

UniProt ID: O75582/O75676

Gene ID: 9252/8986

 2016-08-17T20:20:56Z

Ras-related C3 botulinum toxin substrate 1

Gene: RAC1

UniProt ID: P63000

Gene ID: 5879

 2016-08-17T19:20:16Z

Krueppel-like factor 5

Gene: KLF5

UniProt ID: Q13887

Gene ID: 688

 2016-08-17T20:39:03Z

tyrosine kinase receptor A

Gene: NTRK1

UniProt ID: P04629

Gene ID: 4914

 2016-08-21T08:34:21Z

Dual specificity mitogen-activated protein kinase kinase 6

Gene: MAP2K6

UniProt ID: P52564

Gene ID: 9064

 2016-12-15T15:10:31Z

mitogen-activated protein kinase kinase

Gene: MAP2K1

UniProt ID: Q02750

Gene ID: 5604

 2016-08-17T16:29:32Z

Son of sevenless

Gene: SOS1

UniProt ID: Q07889

Gene ID: 6654

 2016-08-17T16:15:35Z

Plasminogen

Gene: PLG

UniProt ID: P00747

Gene ID: 5340

 2016-08-17T18:49:52Z

Tristetraprolin

Gene: ZFP36

UniProt ID: P26651

Gene ID: 7538

 2016-08-18T13:06:02Z

c-Jun N-terminal kinase

 2016-08-19T13:35:02Z

Transcription factor jun-B

Gene: JUNB

UniProt ID: P17275

Gene ID: 3726

 2016-08-17T20:28:19Z

Focal adhesion kinase 1

Gene: PTK2

UniProt ID: Q05397

Gene ID: 5747

 2016-09-15T14:00:55Z

Transcription factor MafF

Gene: MAFF

UniProt ID: Q9ULX9

Gene ID: 23764

 2016-08-19T13:27:44Z

Krueppel-like factor 6

Gene: KLF6

UniProt ID: Q99612

Gene ID: 1316

 2016-08-18T11:57:16Z

Family of heterotrimeric G-proteins

 2016-08-17T19:14:58Z

ribosomal s6 kinase

 2016-08-17T20:20:56Z

Cbp/p300-interacting transactivator 2

Gene: CITED2

UniProt ID: Q99967

Gene ID: 10370

 2016-08-18T12:51:42Z

Mitogen-activated protein kinase kinase 1

Gene: MAP3K1

UniProt ID: Q13233

Gene ID: 4214

 2016-08-17T19:34:55Z

Signal transducer and activator of transcription 3

Gene: STAT3

UniProt ID: P40763

Gene ID: 6774

 2016-08-19T13:24:51Z

Protein C-ets-1

Gene: ETS1

UniProt ID: P14921

Gene ID: 2113

 2016-08-18T13:21:55Z

extracellular matrix

 2016-08-17T18:52:55Z

Pro-neuropeptide Y

Gene: NPY

UniProt ID: P01303

Gene ID: 4852

 2016-08-17T19:14:58Z

Neuropeptide Y receptor type 1

Gene: NPY1R

UniProt ID: P25929

Gene ID: 4886

 2016-08-17T19:14:58Z

GTPase Ras

 2016-08-17T19:20:16Z

Mitogen-activated protein kinase kinase 4

Gene: MAP3K4

UniProt ID: Q9Y6R4

Gene ID: 4216

 2016-08-17T19:40:12Z

Myc proto-oncogene protein

Gene: MYC

UniProt ID: P01106

Gene ID: 4609

 2016-08-18T13:10:15Z

Krueppel-like factor 2

Gene: KLF2

UniProt ID: Q9Y5W3

Gene ID: 10365

 2016-08-17T20:36:41Z

phospholipase C

 2016-08-17T18:38:11Z

BTG Family Member 2

Gene: BTG2

UniProt ID: P78543

Gene ID: 7832

 2016-08-18T12:54:26Z

P38 mitogen-activated protein kinases

 2016-08-17T19:53:57Z

Fibroblast growth factor receptor substrate 2

Gene: FRS2

UniProt ID: Q8WU20

Gene ID: 10818

Gene ID: 10818

 2016-08-17T16:24:57Z

RAF proto-oncogene serine/threonine-protein kinase

Gene: RAF1

UniProt ID: P04049

Gene ID: 5894

 2016-08-17T16:27:39Z

Cellular tumor antigen p53

Gene: TP53

UniProt ID: P04637

Gene ID: 7157

 2016-08-18T12:51:42Z

PI3K activates AKT.

PI3K activates AKT by catalyzing the production of PtdIns-3,4-P2 and PtdIns-3,4,5-P3, which help recruit AKT to the membrane.

 S_17 1

RSK activates CREB.

MSK1/2 activates CREB.

AKT activates CREB.

RSK activates CREB through phosphorylation.

AKT induces CREB phosphorylation via SKF38393.

MSK1/2 is required for the stress-induced phosphorylation of CREB.

 S_29 1 S_1 1 S_45 1

FAK activates SHC.

TrkA activates SHC.

TrkA activates SHC through phosphorylation.

The SHC PI domain binds to FAK.

 S_32 1 S_40 1

JNK activates Egr1.

AKT activates Egr1.

ERK activates Egr1.

H2O2-induced upregulation of Egr1 mRNA and protein was inhibited in the presence of agents inhibiting the JNK pathway

AKT phosphorylates the Egr1 inhibitor, FoxO1.

H2O2-induced upregulation of Egr-1 mRNA and protein was inhibited in the presence of agents inhibiting the ERK pathway.

 S_38 1 S_18 1 S_1 1

ETS1 activates Dusp6.

ETS1 activates Dusp6 through binding to the Dusp6/MKP-3 promoter.

 S_49 1

Egr1 activates ARC.

CREB activates ARC.

CREB binds to the critical synaptic activity-responsive elements (SARE) of the ARC gene.

Egr1 activates ARC by binding to a ERE sequence in the ARC promoter.

 S_2 1 S_4 1

SHC activates GRB2.

Phosphorylated SHC binds to GRB2.

 S_3 1

ERK activates JUND.

JNK activates JUND.

JNK activates JUND through phosphorylation.

ERK activates JUND through phosphorylation.

 S_38 1 S_18 1

AKT activates FOS.

ERK activates FOS.

JNK activates FOS.

Akt activated the SRE region of the FOS promoter through the TCF site.

JNK activates FOS through phosphorylation.

ERK activates FOS through phosphorylation.

 S_1 1 S_38 1 S_18 1

RSK activates SRF.

RSK activates SRF through phosphorylation at Ser-103.

 S_45 1

MEKK4 activates Mapk3k.

MEKK4 indirectly activates Mapk3k.

 S_54 1

uPAR activates uPA/PLAT.

uPAR binds to uPA/PLAT.

 S_16 1

FOS activates AP1.

Fosl1 activates AP1.

JUNB activates AP1.

JUND activates AP1.

FOS binds to AP1

JUND binds to AP1.

JUNB binds to AP1.

Fosl1 binds to AP1.

 S_39 1 S_8 1 S_20 1 S_9 1

MEKK1 activates MKK7.

MEKK1 activates MKK7 through phosphorylation.

 S_47 1

C3G activates RAP1.

C3G stimulates the nucleotide exchange of RAP1.

 S_26 1

AP1 activates uPAR.

AP1 binds to uPAR.

 S_13 1

TrkA activates PI3K.

PI3K binds to the Tyr751 residue of TrkA.

 S_32 1

MEK activates ERK.

MEK activates ERK through phosphorylation.

 S_34 1

ECM activates Itga1.

ECM components bind to Itga1.

 S_50 1

JNK activates Fosl1.

ERK activates Fosl1.

AKT activates Fosl1.

AKT activates Fosl1 through phosphorylation.

JNK activates Fosl1 through phosphorylation.

ERK activates Fosl1 through phosphorylation.

 S_1 1 S_18 1 S_38 1

Ca2+ activates PKC.

DAG activates PKC.

DAG binds to the C1 domain of PKC.

Ca2+ indirectly activates PKC.

 S_27 1 S_23 1

JNK activates ATF2.

ERK activates ATF2.

P38 activates ATF2.

P38 activates ATF2 through phosphorylation.

JNK activates ATF2 through phosphorylation.

ERK activates ATF2 through phosphorylation.

 S_59 1 S_18 1 S_38 1

PLC activates Ca2+.

PLC activates Ca2+ by hydrolyzing phosphatidylinositol 4,5-bisphosphate to generate IP3 and DAG. IP3 then induces an increase in intracellular Ca2+.

 S_57 1

Plasmin activates Mmp3/10.

Plasmin-dependent branching was prevented by both plasmin and MMP inhibitors, suggesting that plasmin activates MMPs.

 S_36 1

AKT activates KLF10.

ERK activates KLF10.

JNK activates KLF10.

AKT activates KLF10 through phosphorylation.

JNK indirectly activates KLF10.

ERK indirectly activates KLF10.

 S_1 1 S_18 1 S_38 1

FRS2 activates C3G.

Phosporylated FRS2 scaffolds the assembly of a stable complex involving C3G, Crk, Rap1, and B-Raf.s..

 S_60 1

PLC activates DAG.

PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate to produce IP3 and DAG.

 S_57 1

JNK activates KLF4.

AKT activates KLF4.

ERK activates KLF4.

JNK indirectly activates KLF4.

AKT activates KLF4 through phosphorylation.

ERK activates KLF4 through phosphorylation.

 S_38 1 S_18 1 S_1 1

ERK activates MSK1/2.

P38 activates MSK1/2.

P38 activates MSK1/2 through phosphorylation.

ERK activates MSK1/2 through phosphorylation.

 S_18 1 S_59 1

RAS activates RAC1.

RAS stimulate the activity of the RAC1-specific GEF, Tiam1.

 S_53 1

AKT activates KLF5.

ERK activates KLF5.

P53 activates KKLF5.

The causal relationship between uPAR signaling and late gene expression (such as KLF5) remains unclear.

P53 binds to KKLF5.

ERK activates KLF5 through phosphorylation.

 S_62 1 S_1 1 S_18 1

NGF binds to TrkA.

NGF binds to TrkA through phosphorylation.

 S_43 1

MEKK4 activates MEKK6.

MEKK4 activates MEKK6's downstream target, p38. MEKK4 and MEKK6 are also share an activating regulatory protein, MTK1.

 S_54 1

RAF activates MEK.

MEKK1 activates MEK.

RAF binds to MEK.

MEKK1 activates MEK through phosphorylation.

 S_47 1 S_61 1

GRB2 activates SOS.

GRB2 directly binds to SOS.

 S_7 1

uPA/PLAT activates Plasmin.

uPA/PLAT catalyzes conversion of plasminogen to Plasmin.

 S_12 1

ERK activates ZFP36.

JNK activates ZFP36.

JNK activates ZFP36 through phosphorylation.

ERK indirectly activates ZFP36.

 S_38 1 S_18 1

MEKK4 activates JNK.

MEKK7 activates JNK.

MEKK4 indirectly activates JNK due to environmental stress.

MEKK7 activates JNK through phosphorylation.

 S_54 1 S_14 1

AKT activates JUNB.

ERK activates JUNB.

JNK activates JUNB.

AKT activates JUNB via AIP4/Itch ubiquitination of JUNB.

JNK activates JUNB through phosphorylation.

ERK activates JUNB through phosphorylation.

 S_18 1 S_1 1 S_38 1

Itga1 activates FAK.

RAP1 activates FAK.

Itga1 activates FAK by promoting tyrosine phosphorylation.

RAP1 activity blocked the effect of Csk, a negative regulator of the Src family.

 S_19 1 S_15 1

ATF2 actvivates Maff.

ERK activates Maff.

JNK activates Maff.

ATF2 forms a binding complex with Maff.

JNK may need to be active for NGF to stimulate Maff.

Maff is a target of ERK signaling.

 S_18 1 S_38 1 S_22 1

P53 activates KLF6.

JNK activates KLF6.

Inhibition of JNK activation decreased PMA-induced KLF6 induction.

P53 is suggested to enhances KLF6 stability.

 S_62 1 S_38 1

NPYY1 activates G(i/o).

NPYY1 is a receptor for G-proteins and binds to G(i/o).

 S_52 1

ERK activates RSK.

ERK activates RSK through phosphorylation.

 S_18 1

ERK activates CITED2.

CREB activates CITED2.

P53 activates CITED2.

CREB binds to CITED2.

P53 is generally indepentent of CITED2 regulation, but can be detected during CITED2 regulation.

ERK activates CITED2 through phosphorylation.

 S_2 1 S_18 1 S_62 1

RAC1 activates MEKK1.

RAC1 indirectly activates MEKK1 through interaction with PAK.

 S_30 1

ERK activates Stat3.

JNK activates Stat3.

JNK activates Stat3 through phsophorylation.

ERK activates Stat3 through phosphorylation.

 S_18 1 S_38 1

JNK activates ETS1.

ERK activates ETS1.

JNK activates ETS1 through phosphorylation.

ERK activates ETS1 through phosphorylation.

 S_38 1 S_18 1

Mmp3/10 activates ECM.

Mmp3/10 play a role in ECM degradation. Loss of Mmp3 blocks side branching

 S_24 1

AP1 activates Npy.

AP1 binds to the Npy promoter.

 S_13 1

Npy activates NPYY1.

Npy binds to NPYY1.

 S_51 1

SOS activates RAS.

SOS binds to RAS.

 S_35 1

RAC1 activates MEKK4.

RAC1 binds to MEKK4.

 S_30 1

AKT activates MYC.

ERK activates MYC.

JNK activates MYC.

AKT activates MYC through phosphorylation.

JNK activates MYC throguh phosphorylation.

ERK activates MYC through phosphorylation.

 S_38 1 S_1 1 S_18 1

ERK activates KLF2.

AKT activates KLF2.

JNK activates KLF2.

JNK indirectly activates KLF2.

AKT activates KLF2 through phosphorylation.

ERK activates KLF2 through phosphorylation.

 S_18 1 S_38 1 S_1 1

TrkA activates PLC.

G(i/o) activates PLC.

TrkA activates PLC through phosphorylation.

Experimental data showed that PLC-β1 and PLC-β2 were stimulated by free G protein βγ subunits.

 S_44 1 S_32 1

AKT activates BTG2.

JNK activates BTG2.

AKT indirectly activates BTG2.

JNK indirectly activates BTG2.

 S_1 1 S_38 1

MKK6 activates P38.

Mapk3k activates P38.

MKK6 activates P38 through phosphorylation.

Mapk3k activates P38 through phosphorylation.

 S_33 1 S_11 1

TrkA activates FRS2.

TrkA binds to FRS2.

 S_32 1

RAS activates RAF.

PKC activates RAF.

RAS binds to RAF.

PKC activates RAF through phosphorylation.

 S_53 1 S_21 1

ERK activates P53.

AKT activates P53.

JNK activates P53.

JNK activates P53 through phosphorylation.

AKT activates P53 through phosphorylation.

ERK activates P53 through phosphorylation.

 S_1 1 S_18 1 S_38 1