Vias de sinalização na regulação da expressão génica

(1)

Peter Jordan,

(peter.jordan@insa.min-saude.pt)

Departamento de Genética Humana,

Instituto Nacional de Saúde Doutor Ricardo Jorge, Lisboa

Vias de sinalização

na regulação

da expressão génica

Mestrado Tecnologias Moleculares em Saúde, ESTeSL

(2)

Signal transduction Gene expression plasma-membrane nucleus Long term response

Extracellular signals and cellular response

Rapid response cytoplasm Signal Cell movement, Glucose uptake, Ion transport, … Proliferation, Differentiation, … Receptor

(3)

Levels of gene

expression

regulation

(4)

Alternative splicing ….

species nb genes % alt. splice

S. cerev. 6 000 0,002

C. elegans 19 000 15

D. melanog. 14 000 40

H. sapiens 25 000 >90

…can occur in most human protein coding genes,

…tissues (or tumours) express specific sets of variants, lessons learned from the study of RAC1b

(5)

Alternative splicing ….

~40%

~8%

~18%

~3%

~50%

(6)

RAS RAS nucleus TF MEK ERK

The MAP kinase pathway

Small GTPase “Molecular Switch” receptor membrane EGF EGF BRAF Tyrosine kinase

(7)

inactive active Signal propagation to effector proteins GEFs GAPs in vivo GDP GTP

The RAS superfamily

and their GTPase cycle

in vitro

Cellular response

(8)

GTPase mutations found in tumors

inactivo _{constitutively} active Signalling stimulation GAPs GDP _GTP GEFs

X

Single nucleotide point mutations: -GGT-GGC- = -G12-G13--GTT=G12V; -GAC=G13D

(9)

(10)

Signalling dynamics through Cross-talk – integrating complex signals

(11)

RAS RAS TF MEK ERK Receptor BRAF

Modulation of ERK activation through cross-talk

PAK Cell adhesion, Signal 2 S218 S222 S298 Growth factor, Signal 1 Rac1 GTP

(12)

ECM Plasmamembrane nucleus Focal adhesion actin lamellipodia actin G1 S

RAC1 signaling

-adhesion

-motility

-proliferation

-gene expression

PAK Adherens junction actin JNK

RAC1

GTP NF-kB STATs

_Oncogenic

RAC1 mutations

in tumours ??

(13)

RAC1 expression in colorectal cancer (CRC):

discovery of RAC1b

RAC1 (27 kb) utr 5’ 2 3 3’ utr 1 4 56 Meta A1 B2 C2 C2 Muc D Tumor stage RAC1 Jordan 1999, Oncogene 18:6835-39

RAC1b

3b

(57 bp) Matos 2000, BBRC 277:741-51

→ RAC1 alternative splicing

1 Switch I Switch II

RAC1b

protein 19 a.a. 211 75

(14)

RAC1b expression and genotype of CRC cell lines

Expression Genotype

Cell line RAC1 RAC1b BRAF KRAS

V9P + - wt wt SW48 + - wt wt LS174 + - wt G12D Lovo + - wt G13D TC71 + - wt G12D HCT116 + - wt G13D HCT15 + - wt G13D IS513 + - wt G12D IS2 + - wt G12D Colo320 + - wt G12D SW480 + - wt G12V CO115 + + V600E wt HT29 + + V600E wt RKO + + V600E wt Colo205 + + V600E wt Caco2 + + wt wt

BRAF

MEK ERK

KRAS

EGFR Muc

+

Matos 2008, Gastroenterology 135:899-906

(15)

0 50 100 KRAS high MSI-H MSS/MSI-L BRAF CIMP-H serrated adenomatous negative or CIMP-L

instable (CIN) stable

proximal distal

wt

percentage of all sporadic CRC cases low Localization in colon Polyp of origin Karyotype DNA sequence stability CpG island methylation RAS/RAF mutation RAC1b expression

Matos 2016, BBA-Rev Cancer 1866:51–63

% RAC1b overexpression characterizes a subtype CRC classification:

(16)

nucleus TF EGF B-Raf ERK nucleus TF Ras mutant nucleus TF Ras B-Raf mutant ~15% ~30%, distal colon ~10%, proximal colon B-Raf ERK ERK receptor membranae EGF Ras RAC1b Gastroenterology (2008), 135: 899-906 EGFR inhibitors Anti-EGFR membrane

(17)

RAC

GDP RAC GTP

Pi

Inactive

Active

Study RAC1b in mammalian cell lines

RT-PCR Total lysates (-RAC1) RAC1b RAC1 RAC1b RAC1

(18)

CRIB domain

(19)

RAC

GDP RAC GTP

Pi

Inactive

Active

PAK pull-down Active GTPase

RAC1b

RAC1

Inactive

GDP Rac Pi RAC GTP

RAC1b in mammalian cell lines

RT-PCR Total lysates (-RAC1) RAC1b RAC1 RAC1b RAC1

Matos 2003, J Biol Chem 278:50442–48.

(20)

co-IP

Plasma membrane

Regulation of RAC1 activation:

RAC1 GDP

Inactive

RAC1 GTP

Active

Pi

GEFs

Guanine-nucleotide Exchange Factors

GAPs GTPase Activating Proteins Rho-GDI RAC1 GDP Signal Receptor active RAC 0,0 0,05 0,125 0,25 HA-Rho GDI (g) RAC1b RAC1 HA-Rho-GDI: + - + --HA -Rac IP -Myc Total lysates RAC1b RAC1 Myc-RAC1b Myc-RAC1 HA-GDI Transfection of HT29 cells

(21)

(22)

Regulation of RAC1 activation:

RAC1 GDP

Inactive

RAC1 GTP

Active

Pi

GEFs

GAPs GTPase Activating Proteins Rho-GDI RAC1 GDP Signal Receptor

(23)

Regulation of RAC1 activation:

RAC1 GDP

Inactive

RAC1 GTP

Active

Pi

GEFs

GAPs GTPase Activating Proteins Rho-GDI RAC1 GDP Signal Receptor

(24)

GTP RAC1b PAK JNK c-Jun Gene transcription Multiple pathways lamellipodia Cytoplasmic Sequestration GDP RAC1

GDI

Summary of the unique properties of

alternative spliced RAC1b

Inactive

Active

GDP GAPs RAC1b p50 RelA p50 RelA ROS kB GEFs Plasma membrane

(25)

Imagem: http://www.nature.com/nrm/poster/signalling/green.html

important research tools:

1. anti-phospho-antibodies

Anti-pThr183-Glu-pTyr185 CTRL EGF P-ERK1/2 ERK1/2 P P Fos Fos Jun

Membrane recruitment

(26)

important research tools:

2. GTPase mutants

inactivo _{constitutively} active Signalling stimulation GAPs GDP _GTP Mutations G12V G13D Q61L GEFs

X

Single nucleotide point mutations: -GGT-GGC- = -G12-G13--GTT=G12V; -GAC=G13D Mutation

T17N

X

Dominant negative

(27)

Myc-Rac1b Myc-Rac1 -Myc -active PAK -Flag Flag-PAK    PAK JNK Rac1b L61 Actin Merge Merge Rac1 L61 Actin p50 RelA IkB p50 RelA IkB

X

(28)

-Myc -active JNK -Flag Myc-Rac1b Myc-Rac1 Flag-JNK    PAK JNK Rac1b L61 Actin Merge Merge Rac1 L61 Actin p50 RelA IkB p50 RelA IkB

X

RAC1 but not RAC1b

(29)

Activation of the transcription factor NF-kB

Gene expression • Cell cycle progression (e.g. Cyclin D1)

• Resistence to apoptosis (e.g. IAPs, BCL-XL)

Nucleus p50 RelA IkB Inactive NF-kB Phosphorylation p50 RelA active NF-kB Degradation P P p50 RelA IkB IkB IKK Kinases proteasome ubiquitylation RAC GTP RAC GDP

(30)

RAC1b-induced phosphorylation of IkB Myc-RAC1b Myc-RAC1   Myc-IkB p-IkB P P p50 RelA IkB Kinases RAC GTP

(31)

P P p50 RelA IkB Kinases RAC GTP 0,00 2,00 4,00 6,00 8,00 10,00 12,00

Vector Rac1-L61 Rac1b-L61 Control 10 uM DPI 25 uM DPI NF-kB reporter 0,00 2,00 4,00 6,00 8,00 10,00 12,00

Vector Rac1-L61 Rac1b-L61 Control 10 uM DPI 25 uM DPI NF-kB reporter RAC1b-induced activation of NF-kB transcriptional reporter gene activity GGGAATTCCC

(32)

GTP RAC1b PAK JNK c-Jun Gene transcription Multiple pathways lamellipodia Cytoplasmic Sequestration GDP RAC1

GDI

Summary of the unique properties of

alternative spliced RAC1b

Inactive

Active

GDP GAPs NF- kB-mediated cell survival and proliferation RAC1b p50 RelA p50 RelA ROS P P IkB GEFs Plasma membrane

(33)

0 20 40 60 80 100 120 0h 24h 48h % o f V ia b le c e ll s siCtrl siRac1b siBRafVE siBRafVE+siRac1b 0 20 40 60 80 100 120 0h 24h 48h

%

o

f

V

ia

b

le

c

e

ll

s

siCtrl siRac1b siBRafVE siBRafVE+siRac1b siCtrl siRac1b siBRafVE siBRafVE+siRac1b 0 20 40 60 80 100 120 0h 24h 48h % o f V ia b le c e ll s siCtrl siRac1b siBRafVE siBRafVE+siRac1b 0 20 40 60 80 100 120 0h 24h 48h

%

o

f

V

ia

b

le

c

e

ll

s

siCtrl siRac1b siBRafVE siBRafVE+siRac1b siCtrl siRac1b siBRafVE siBRafVE+siRac1b siCt rl siRac1 b siRafVE siRafVE + siRac1 b 48h

Effect of suppressing RAC1b and B-Raf on cell viability (HT29)

(34)

Summarizing so far…

● Overexpression of RAC1b ocurrs in a subtype of CRC

characterized by mutation in BRAF (10-15% of cases)

● Alternative splicing of RAC1 pre-mRNA generates

the highly activated variant RAC1b

● RAC1b protein differs in regulation and signaling

● Overexpression of RAC1b cooperates with mutant

(35)

RAC1 (27 kb) utr 5’ 2 3 3’ utr 1 4 56

How is

alternative splicing of RAC1b

regulated

in colorectal cells ?

3b

(36)

How can the expression of alternative splicing variants

become altered in cells?

GT AG 5’ splice site 3’ splice site YYYYYYYY Polypyrimidine tract A Branch point Intron Exon Exon Mutation ISS ISE GU A YYYYYYYY AG U1 snRNP hnRNP U2AF 65 ₃₅ SF1 Regulated by relative SF concentration ISS SR Regulated by post-translational SF modifications P Nucleus Cytoplasm ESE ESS ESE ESS

(37)

3 3b ₄ H A 4.3 kb 1.4 kb pcDNA3 3 3b ₄ H A 4.3 kb 1.4 kb 3 3b ₄ H A 4.3 kb 1.4 kb 3 3b ₄ H A 4.3 kb 1.4 kb pcDNA3 pcDNA3 pcDNA3-HA-RAC1 Minigene

Dissection of splicing regulation with a RAC1

minigene

RAC1(27 kb) utr 5’ 2 3 3’ utr 1 3b 4 56 (57 bp)

Gonçalves 2009, Hum Mol Genet 18:3696-3707

HT29 SW480 Endogenous expression Rac1b Rac1 Rac1b Rac1 HT29 SW480 Transfected RAC1 minigene

(38)

Two regulatory sequence elements

determine exon 3b inclusion or skipping

-1,0 -0,5 0,0 0,5 1,0 A lternat iv e v s. constituti v e tr an scr ipt (L og 10 ) E 5 .0 E 4 .0 E 3 .0 E 2 .C E 2 .1 E 2 .0 E 1 .C E 1 .2 E 1 .1 E 1 .0 C trl

conserved:GTTGGAGAAACGTACGGTAAGGATATAACCTCCCGGGGCAAAGACAAGCCGATTGCC

E1.0 - GTTGGACAAATGTACGGTAAGGATATAACCTCCCGGGGCAAAGACAAGCCGATTGCC

E1.1 – GTTGGACAAACGTACGGTAAGGATATAACCTCCCGGGGCAAAGACAAGCCGATTGCC

E1.2 - GTTGGAGAAATGTACGGTAAGGATATAACCTCCCGGGGCAAAGACAAGCCGATTGCC

E1.C - GTTGGAGACACGTACGGTAAGGATATAACCTCCCGGGGCAAAGACAAGCCGATTGCC

E2.0 - GTTGGAGAAACGTACGGCAAGGATATAACCTCCCGGGGCAAAGACAAGCCGATTGCC

E2.1 - GTTGGAGAAACGTACGGTCAGGATATAACCTCCCGGGGCAAAGACAAGCCGATTGCC

E2.C - GTTGGAGAAACGTACGGTAAAGATATAACCTCCCGGGGCAAAGACAAGCCGATTGCC

E3.0 - GTTGGAGAAACGTACGGTAAGGATATAACCACCCCGGGCAAAGACAAGCCGATTGCC

E4.0 - GTTGGAGAAACGTACGGTAAGGATATAACCTCCCGGTGCTAAGACAAGCCGATTGCC

E5.0 - GTTGGAGAAACGTACGGTAAGGATATAACCTCCCGGGGCAAAGACATGCCGATTGCC

EMSA

Gonçalves 2009, Hum Mol Genet 18:3696-3707

(39)

Model for regulation of RAC1 alternative

splicing in CRC

Exon 3b inclusion AG ESS SRSF1_ESE SRSF3 3b Exon 3b skipping AG SRSF1 ESE SRSF3ESS 3b RAC1(27 kb) utr 5’ 2 3 3’ utr 1 3b4 56 (57 bp)

(40)

Active Rac Blot: -Rac SW480 DLD-1 HT29 Endogenous expression Rac1b Rac1 RT-PCR gene RAC1 (27 kb) utr 5’ 2 3 3’ utr 1 3b 4 5 6 (57 bp)

Cell lines have identical genomic RAC1 sequence

Do genomic mutations cause increased exon 3b

inclusion?

(41)

Model for regulation of RAC1 alternative

splicing in CRC

Exon 3b inclusion AG ESS SRSF1_ESE SRSF3 3b Exon 3b skipping AG SRSF1 ESE SRSF3ESS 3b RAC1(27 kb) utr 5’ 2 3 3’ utr 1 3b4 56 (57 bp)

The changes in RAC1 alternative splicing in colorectal cells are not caused by gene mutations….

(42)

How can the expression of alternative splicing variants

become altered in cells?

GT AG 5’ splice site 3’ splice site YYYYYYYY Polypyrimidine tract A Branch point Intron Exon Exon Mutation ISS ISE GU A YYYYYYYY AG U1 snRNP hnRNP U2AF 65 ₃₅ SF1 Regulated by relative SF concentration ISS SR Regulated by post-translational SF modifications P Nucleus Cytoplasm ESE ESS ESE ESS

(43)

Protein kinases and phosphatases previously described to regulate alternative splicing events

CLK1 CLK2 CLK3 CLK4 DYRK1a DYRK1b DYRK2 DYRK3 DYRK4 CDK11 CDK12 CDK13 GSK3alpha GSK3beta SRPK1 SRPK2 MSSK1 PRPF4B PRP4 TOPOI AKT1 AKT2 AKT3 PP1alpha PP1beta PP1gamma Kinase Group CMGC Depletion by shRNAs Differences in RAC1b protein levels in CRC cell lines ?? Gonçalves 2014, RNA 20:474-82

(44)

ADP protein X phosphoprotein X ATP

inactive

active

Ser Thr Tyr Protein kinase PO₄

ө ө

‘Molecular switch’

- alters 3D conformation and activity

- creates or masks protein interaction motifs

-

EGF + .

pSer 89% 85%

pThr 10% 12%

(45)

Hanks SK (2003) Genome Biology 4 (5), Article 111

518

protein kinase genes

478 ePKs

conserved eucaryotic domain

40 aPKs

atypical catalytic domain

428 ePKs with known or likely kinase activty 8 subgroups:

TK- 84; CAMK- 66; AGC- 61; CMGC- 61; STE- 45; TKL- 37; CK1- 11; Other- 63

(46)

The human kinome tree:

clustering by sequence similarity in the kinase

domain led to identification of different subfamilies

(incl. 60 receptor kinases)

(47)

Protein kinases and phosphatases previously described to regulate alternative splicing events

CLK1 CLK2 CLK3 CLK4 DYRK1a DYRK1b DYRK2 DYRK3 DYRK4 CDK11 CDK12 CDK13 GSK3alpha GSK3beta SRPK1 SRPK2 MSSK1 PRPF4B PRP4 TOPOI AKT1 AKT2 AKT3 PP1alpha PP1beta PP1gamma Kinase Group CMGC Depletion by shRNAs Differences in RAC1b protein levels in CRC cell lines ?? Gonçalves 2014, RNA 20:474-82 5 protein kinases affected RAC1b

(48)

Validation of 5 protein kinases affecting RAC1b splicing

At RAC1b protein level: Secondary screen with

commercial siRNAs

clearly reproduced the effects of 4 out of 5 shRNAs, (with exception of siAKT1)

At RAC1b transcript level: only the depletion of

GSK3β and SRPK1 decreased

endogenous RAC1b expression

(49)

Candidate 1: role of protein kinase SRPK1 in RAC1b splicing Protein

Exon 3b

inclusion

AG ESS SRSF1_ESE SRSF3

3b

(50)

SRSF1 SRPK1

RAC1B p-SRSF1

Candidate 1: role of protein kinase SRPK1 in RAC1b splicing

Inhibition of SRPK1 decreases phosphorylation of SRSF1 (but not its transcription), leading to decreased

SRSF1-dependent alternative splicing of RAC1b

Kinase inhibitor

(gift from M Hagiwara)

(51)

siCtrl siSRPK1

Tag-SRSF1

Actin DAPI Role of protein kinase SRPK1 in RAC1b splicing

3 4 RAC1 pre-mRNA 3b ESS ESE pSRSF1 SRSF3 4 3 3b 3 4 inclusion skipping SRPK1 Gonçalves 2014, RNA 20:474-82 nuclear translocation

(52)

SRSF3 GSK3β αtub SRPK1 SRSF1 Rac1b RAC1B

Candidate 2: role of protein kinase GSK3β in RAC1b splicing

Inhibition of GSK3β decreases RAC1b alternative splicing through SRSF1 Gonçalves 2014, RNA 20:474-82 Exon 3b inclusion AG ESS SRSF1_ESE SRSF3 3b

(53)

Signaling pathways regulating RAC1b splicing 3 4 RAC1 pre-mRNA 3b ESS ESE pSRSF1 SRSF3 4 3 3b 3 4 inclusion skipping SRPK1 nuclear translocation GSK3β ???? Inflammatory Signaling ?? βcat Gonçalves 2008, RNA 14:2538–49

(54)

Summary-2

 SRSF1 is a key splicing factor required for inclusion of

alternative exon 3b to generate RAC1b

 Protein kinases GSK3β and SRPK1 are upstream regulators

of SRSF1-mediated RAC1b overexpression in CRC

 Small molecule protein kinase inhibitors can correct RAC1

(55)

Paulo Matos

Vânia Gonçalves

Joana Pereira

Andreia Henriques

Luís Moita – IMM, Lisbon

Raquel Seruca, Carla Oliveria – IPATIMUP, Porto Eric Chastre, Larissa Kotelevets – INSERM, Paris