Ras family G proteins

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Belén Ponce Martin
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Ras family G proteins RAS RHO Rap2A Rap2B Rap1A Rap2B TC 21 R-Ras N-Ras K-Ras RhoA H-Ras RhoC RalA RalB Ras-Apl DRas3 DRas2 DRas1 RhoB Rho-Ap Rac3 Rsr1 Ras2 Ras1 Rac2 Rnd2 C dc42 Rac1 Rnd1 RhoG TC 10

1 Ras family G proteins RAS RHO Rap2A Rap2B Rap1A Rap2B TC 21 R-Ras N-Ras K-Ras RhoA H-Ras RhoC RalA RalB Ras-Apl DRas3 DRas2 DRas1 RhoB Rho-Ap Rac3 Rsr1 Ras2 Ras1 Rac2 Rnd2 C dc42 Rac1 Rnd1 RhoG TC 10 RHO1 DdRas Ras-S p M-Ras Rit Rin RheB RHO2 Gem Arf Spi1 TTF RhoD Ran Rnd3 RhoE RHY YPT3 Sas YPT1 Rab9 Rab7 Rab6 YPT2 YPMT Ara Rab2 Rab5 Rab4A Rab4B Rab11 Rab10 Rab8 Sec4 Rab1A Rab1B Rab3A Rab3B Rab3C RAB INDEPENDENCIA PÉRDIDA DE DE FACTORES INHIBICIÓN DE CRECIMIENTO POR CONTACTO AUMENTO EN AUMENTO EN ENDOCITOSIS LA MOTILIDAD CAMBIOS EN AUMENTO EN CITOESQUELETO METABOLISMO SÍNTESIS ENTRADA EN DE DNA CICLO CELULAR - PROLIFERACIÓN - INHIBICIÓN APOPTOSIS* - INHIBICIÓN DIFERENCIACIÓN* - INVASIÓN / METÁSTASIS 1

2 Cytological Effects H-Ras K-Ras A,B N-Ras - Cell cycle progression - DNA synthesis - Energy metabolism - Endocytosis - Autocrine regulation - Actin cytoskeleton - Morphology - Anchorage - Cell-to-cell contact - Cell-specific functions Biological Effects - Proliferation - Differentiation - Apoptosis - Motility - Cell-specific roles - Transformation - Metastasis ONCOGEN ORIGEN GEN CROMOSOMA (h) H- ras K- ras N- ras Retrovirus de sarcoma de rata (Harvey) Retrovirus de sarcoma de rata (Kirstein) Neuroblastoma humano (no retroviral) 3 kb 35 kb 7 kb H- Ras N- Ras K- Ras4A K- Ras4B / % 90% 10-15% HYPERVARIABLE REGION H K L R K L N P P D E S G P G C M S C K C V L S Farnesyl, palmitate Y R M K K L N S S D D G TQ G C M G L P C V V M Farnesyl, palmitate Y R L K K I S K E E K T P G C V K I K K C I I M Farnesyl, palmitate H K E K MS K D G K K K K K K S K T K C V I M Farnesyl LINKER DOMAIN MEMBRANE-TARGETING MOTIF Main differences among H-, K-, and N-Ras - Distinct patterns of expression in tissues and during development. - Distinct involvement in human carcinogenesis. - Distinct transforming potential in different cell types. - Differences in their anti-apoptotic apoptotic potential. - Differences in their sensibility towards NF1- GAP. - Differentially activated by some exchange factors. - Quantitative differences in their ability to activate Raf and PI-3K. - Distinct phenotypes of ras knock-out mice. 2

3 Processing and trafficking of Ras proteins Endoplásmic reticulum CAVEOLAE LIPID RAFTS DISORDERED MEMBRANE + CaaX C-COO - C-COO-CH 3 C C-COO-CH 3 H-Ras N-Ras H-Ras K-Ras4B Proteolysis Methylation Palmitoylation (H-Ras, N-Ras) H-Ras Farnesylation CaaX K-Ras4B Golgi apparatus H-Ras K-Ras4A?? Ras Cytoplasm CH 3 -OOC-C KKKK CH 3 -OOC-C C H-Ras Plasma membrane GOLGI APP. ENDOPLASMIC RETICULUM RAS CYCLE E F F E C T O R S Ras family GEFs (2001) PDZ-GEF PDZ-GEF2 LinkII MR-GEF Epac2 Epac1 LTE1 BAB31280 BAB31297 PLCε Grasp-1 BUD5 NSP1 BCAR3 SHEP1 Sos1 Sos2 C3G GRF1 GRF2 Rgr Rlf CDC25 SCD25 RalGDS Rgl Rgl3 STE1 GRP1 GRP3 GRP2 GRP4 RalGPS1 RalGPS2 3

Ras Mammalian Exchange Factors AAs EGFR 1244 1336 795 1489 P SMG 21 GDS 558

Ras family GEFs *=splice variants catalytic domain REM scr1 scr2 scr3 Sos Dbl

loop (10-17) Residuos 60-74 Unión del GEF: - Desestabiliza la unión a Mg 2+ -

4 Ras Mammalian Exchange Factors AAs EGFR P SMG 21 GDS 558 Ras-GDP Ras-GDP-GEF Ras-GEF Ras-GTP-GEF Ras-GTP GTP Residuos GEF Ras Ras family GEFs *=splice variants catalytic domain REM scr1 scr2 scr3 Sos Dbl PH PxxP 2 GRF PH C C IQ Dbl PH 2* GRASP-1 RA PDZ 1 GRP/CalDAG-GEF EF - C 1 4 P- loop (10-17) Residuos Unión del GEF: - Desestabiliza la unión a Mg 2+ - Desestabiliza el P- loop Rap Ral PLase Cε X Y C 2 RA RA 1* C3G PxxP 1* Epac DEP camp-binding RA 2 PDZ-GEF camp-binding? PDZ RA PPXY SAV 2* MR-GEF/GFR RA 1 LINK-II RA 1 BAB-GEFs 2 BCAR3/NSP1-3 SH2 PxxP 3 RalGDS RA 5 RalGPS PxxP PH 2* SmgGDS

5 Ras-GDP Ras-GTP Gln 61 N N C C Switch I (32-40) Switch II (60-76) TIQ LIQNHFVDEYDPTIEDSYRKQVV specificity C 2A C 2B general recognition specificity P H BTK C AP RI RAS AL GAP 1 IP4BP Actividad GTPasa intrínseca de Ras: 0.03 mmol min-1 mol-1 (25 min / 37º) 5

2 µ M Rab5 Mst1 Rac Rho Cdc42 AF-6 Rin NORE Rac Rho Cdc42 Fosfolípido GRUP O P OLAR

6 Arg789 Incremento de 105 veces Actividad GTPasa Ras-GAP: 19 mmol s -1 mol-1, Kd Kd:: 9.7 µ M Actividad GTPasa Ras-NF 1: 10 mmol s -1 mol-1, Kd Kd:: 0.2 µ M Rab5 Mst1 Rac Rho Cdc42 AF-6 Rin NORE Rac Rho Cdc42 Fosfolípido GRUP O P OLAR Ral-BP TIAM-1 PLC ε PLD P Ral-BP PLC c Filamin ε MEK - Transformación células neurales c PLC PLA1? 6

7 Akt - v-akt, presente en virus transformantes - Sobreexpresada o activada en distintos tipos de tumores (mama, t. cerebrales) Raf Bad Iκ-K forkhead G3K p70s6k MEK Translocación a mitocondrias Inhibición Bcl2 / Bcl Xl Activación Transcripción Nf-kB genes pro-apotóticos? Transcripción genes anti-apotóticos apotóticos POTENTE SEÑAL ANTIAPOPTOTICA Ras Sur-8 CNK Hsp 90 BRaf Ksr p14 MEK RKIP Mp-1 Mxi2/p38 Morg Proliferación Diferenciación Senescencia Apoptósis 7

p68 Raf p115 Ral-GDS p Raf PI3K Ral-GDS p21 Ras p110 PI3K p120 GAP Ras - Effectors Interactions Ras - Raf Kd = 50 nm Ras - PI3K Kd = 150 nm Ras - Ral-GDS Kd = 1 µm Ras - p120 GAP Kd = 2 µm Ras-GAP

Stomach - <50% <5% Pancreas - 90% - Cholangio & gall bladder - 90% - C olorectal - 40% 10% Urogenital Kidney <10% - - Urothelial <10% <10% - Prostate <5% - - Ovarian - rare - Endometrial - 40% -

8 p68 Raf p115 Ral-GDS p Raf PI3K Ral-GDS p21 Ras p110 PI3K p120 GAP Ras - Effectors Interactions Ras - Raf Kd = 50 nm Ras - PI3K Kd = 150 nm Ras - Ral-GDS Kd = 1 µm Ras - p120 GAP Kd = 2 µm Ras-GAP complex Ras-Raf complex Breast - <5% - Lung Small cell Non-small - 30%(adeno) <5% Head & kneck Squamous 35% - - Skin Melanoma - - <20% Non-Melanoma 30-50% <5% - Digestive track Esophagus Stomach - <50% <5% Pancreas - 90% - Cholangio & gall bladder - 90% - C olorectal - 40% 10% Urogenital Kidney <10% - - Urothelial <10% <10% - Prostate <5% - - Ovarian - rare - Endometrial - 40% - cervical 20-30% - - Estimated Ras-GTP half life in vivo: 1 s. Ras-Raf complex dissociation rate constant: 10 s-1 Ras-Raf complex half life in vivo: ms. Endocrine Thyroid 90% 90% 90% Pheocromo Medullary Lymphomas Hodgkins Non-Hodgkins Myeloma % Leukemias Acute myeloid % Acute lymphoid % Chronic myeloid - - rare Chronic lymphoid % C MML - 65% MDS % C hildhood T. Neuroblastoma

9 Ras-specific GAPs Mutations in codons 12, 13 or 61 E F F E C T O R S Overlay of G α -GDP-AlF 4 - with the Ras-GDP-AlF 3 -Gap complex Cis-Arginine Trans-Arginine 9

10 Rac Rho Cdc42 Rho CYCLE GDIs E F F E C T O R S Rho GTPase (active) Rho GEF Rho-GDI Rho-GDI Rho GTPase (inactive) 10

11 Ras GTP 11

12 Ral-BP MEK Regulación / atenuación de un potente activador? La ciencia es aceptable siempre que no contradiga la fe. Todas las ciencias deben tender a la búsqueda de la verdad en su orden y su lícita autonomía metodológica, si bien sus conclusiones no pueden contradecir la fe, ya que tanto el mundo como el saber teológico tienen su origen en Dios. Pío XII; encíclica Humani Generi,, 1950 Eeeeeeleeeeeeeee. El Cordobés, Las Ventas

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