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TIROSIN CHINASI CITOPLASMATICHE

TIROSIN CHINASI CITOPLASMATICHE. TIROSIN CHINASI CITOPLASMATICHE. Tec (Btk/Itk/Bmx). PH. SH3. KINASE DOMAIN. SH2. FAK/Pyk2. KINASE DOMAIN. SH2. KINASE DOMAIN. KINASE DOMAIN. Jak. SH3. SH2. KINASE DOMAIN. SH2. SH2. KINASE DOMAIN. ZAP70/Syk. SH3. SH2. KINASE DOMAIN.

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TIROSIN CHINASI CITOPLASMATICHE

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  1. TIROSIN CHINASI CITOPLASMATICHE

  2. TIROSIN CHINASI CITOPLASMATICHE Tec (Btk/Itk/Bmx) PH SH3 KINASE DOMAIN SH2 FAK/Pyk2 KINASE DOMAIN SH2 KINASE DOMAIN KINASE DOMAIN Jak SH3 SH2 KINASE DOMAIN SH2 SH2 KINASE DOMAIN ZAP70/Syk SH3 SH2 KINASE DOMAIN Focal adhesion targeting (paxillin binding) (FERM) Src (Blk, Lck, Lyn, Fyn, Fgr, Hck, Yes) Ac. miristico/palmitico Abl/Arg Ac. miristico

  3. G. Steven Martin : THE HUNTING OF THE SRC, Nature Reviews Molecular Cell Biology2, 467-475 (2001)  

  4. Integrine Src

  5. FAK e membri della famiglia del Src costituiscono un’unità di trasduzione del segnale da parte di molecole adesive definite INTEGRINE SH3 SH3 SH2 YP397 Src FAK Src SH2 SH1 SH1 FAK

  6. Outside-in signaling

  7. Autofosforilazione Interazioni con altre proteine (Src, PI3 chinasi, PLCg) Sito di interazione con Grb2 SH2 SH2 YP YP N dominiocatalitico dominio FAT C dominio FERM Poli-P SH3 Interazione con catena b delle integrine e fattori di crescita Interazione con fosfatidil inositolo (4,5) bisfosfato (PIP2) Interazione con proteine favorenti la polimerizzazione dell’actina (complesso Arp 2/3) Interazione con paxillina Interazione con Cas FAK F1 F2 F3

  8. Anche FAK come Src viene attivata in seguito alla rottura di interazioni intermolecolari che la mantengono in uno stato inibito.

  9. Il distacco da proteine della matrice extracellulare induce una forma di apoptosi (chiamata “anoikis”) che determina il fenomeno della cosiddetta “crescita dipendente dall’ancoraggio” SH3 SH2 YP397 Src FAK AKT PI3K PY PROLIFERAZIONE E SOPRAVVIVENZA

  10. Poli-P Poli-P Fosfatidilinositolo 4,5 fosfato Fosfatidilinositolo 3,4,5 fosfato P P P P SH3 P P P P P P PH SH2 PI3-chinasi YP397 Src IP3 PIP5-chinasi g FAK PLCg GEFs SH1 PY Cas paxillina PY PY YP925 Rac, Cdc42 Crk Dock180 Grb2 Sos Rac Interazione con altre Proteine cito- scheletriche e segna- lanti Ras PAK Polimerizzazione dell ’actina e protrusione del lamellopodio JNK Raf MAPK MAPK (ERK1/2) Movimento cellulare Proliferazione e sopravvivenza cellulare TRASCRIZIONE GENICA

  11. FAK, Src e altre chinasi vengono inibite da tirosin fosfatasi che sono sensibili all’azione inibitoria di ROIs FAK, Src, GF-Rs, MAP chinasi (JNK, ERKs) attive FAK, Src, GF-Rs, MAP chinasi (JNK, ERKs) inattive Protein fosfatasi inattiva Protein fosfatasi attiva

  12. La transizione epitelio-mesenchimale rappresenta una modificazione tipica dei tumori epiteliali metastatici An epithelial-mesenchymal transition (EMT) is a biologic process that allows a polarized epithelial cell, which normally interacts with basement membrane via its basal surface, to undergo multiple biochemical changes that enable it to assume a mesenchymal cell phenotype, which includes enhanced migratory capacity, invasiveness, elevated resistance to apoptosis, and greatly increased production of ECM components . The completion of an EMT is signaled by the degradation of underlying basement membrane and the formation of a mesenchymal cell that can migrate away from the epithelial layer in which it originated. (Kalluri and Weinberg, J. Clin. Invest. 119:1420, 2009)

  13. EPITHELIAL-MESENCHIMAL TRANSITION MESENCHIMAL-EPITHELIAL TRANSITION

  14. b cat. b cat. a cat. vinculina giunzioni serrate (tight junction) F-actina microtubuli p120 cat. Previene endocitosi e de- gradazione E- caderina giunzioni aderenti (adherens junctions) E-caderina p120 cat. E-caderina Rac F-actina Rho Movimento Migrazione nel nucleo e trascrizione geni implicati nella proliferazione cellulare

  15. b cat. a cat. vinculina L’aumentata stimolazione con fattori di crescita altera funzioni di E-caderine In diversi modi Recettori per fattori di crescita: TGFbRI e II, IGF-1R, EGFR, c-Met giunzioni serrate (tight junction) F-actina giunzioni aderenti (adherens junctions) p120 cat. E-caderina F-actina

  16. L’aumentata stimolazione con fattori di crescita altera funzioni di E-caderine In diversi modi • Il promotore del gene per le E-caderine viene represso da repressori trascrizionali • (Snail, Twist, Slug e altri) indotti da Recettori per GF e altre vie attivate nel corso di “progressione” • tumorale (compreso Nf-kB). 2. Tirosin chinasi recettoriali (EGFR, IGF-1R, c-Met, FGF) e non recettoriali (c-Src) inducono fosforilazione e successiva ubiquitinilazione di E-caderine e catenina con conseguente degradazione nel proteosoma. La fosforilazione di E-caderina comporta anche il distacco di a e b catenine. 3. Proteasi (metallo-proteasi – MMP) proteolizzano E-caderine alterando contatti cellula-cellula. 4. Gli stessi segnali che “down-regolano” espressione di E-caderine aumentano espressione di N-caderine (il cosiddetto “caderin-switch”), che sono espresse da cellule mesenchimali e regolano positivamente il movimento cellulare.

  17. b cat. a cat. vinculina movimento Sress fibers, focal adhesion Cdc42 Rac Rho p120 cat. E-caderina F-actina

  18. P P P b cat. b cat. Inibizione di GSK da parte di GF-Rs o altri stimoli favorisce la progressione neoplastica P Snail P GSK-3 E-caderina Repressione sintesi E-caderine Trascrizione geni implicati in proliferazione e progressione DEGRADAZIONE NUCLEO

  19. Un meccanismo aggiuntivo di regolazione della funzione di bcatenica è rappresentato da un suo aumento di espressione in cellule stimolate con proteine wnt (ampia famiglia di fattori di crescita poco caratterizzati) Gene onco-soppressore, la cui mutazione in cellule della linea germinale determina la Poliposi Adenomatosa Famigliare del Colon

  20. a | In the absence of WNT signalling, -catenin levels in the cytoplasm and nucleus are low as a result of continuous phosphorylation by the serine/threonine kinases CK1 (casein kinase 1) and GSK3 (glycogen synthase kinase 3), leading to binding of -transducin-repeat-containing protein (TRCP) and to ubiquitylation and degradation by the proteasome. The destruction complex is composed of CK1 and GSK3, as well as the anchor proteins AXIN1 (axis inhibition protein 1 ) and APC (adenomatous polyposis coli). In the nucleus, TCF (T-cell factor) molecules are bound by co-repressors such as GRG/TLE (Groucho/transducin-like enhancer) proteins that shut off expression of WNT target genes. Other components of the repressor complex include CTbP (C-terminal binding protein) and HDACs (histone deacetylases). -catenin in the nucleus is inhibited from binding TCF by ICAT (cell autonomous inhibitor of -catenin and TCF) . The Frizzled receptor complex — composed of Frizzled and LRP5 (LDL-receptor-related protein 5) or LRP6 — can also be actively inhibited by receptor-bound soluble inhibitors such as DKK1 (Dickkopf homologue 1). b | Upon binding of a lipid-modified WNT protein to the receptor complex, a signalling cascade is initiated. LRP is phosphorylated by CK1 and GSK3, and AXIN1 is recruited to the plasma membrane. The kinases in the -catenin destruction complex are inactivated and -catenin translocates to the nucleus to form an active transcription factor complex with TCF, leading to transcription of a large set of target genes. In the nucleus, -catenin binds to TCF and LEF factors and recruits co-factors such as legless (LGS; also known as BCL9) and Pygopus (PYGO), CBP/p300, Brahma and MED12 to initiate transcription99, 100, 101. DVL, mammalian homologue of Drosophila Dishevelled; PP2A, protein phosphatase 2A.

  21. Dipartimento di Patologia Sezione di Patologia Generale Università di Verona

  22. Granulocyte/macrophage progenitors Megakaryocyte/erythrocyte progenitors Common myeloid progenitors Haematopoietic stem cell Common lymphoid progenitors Chronic myeloid leukemia-blast phase (70%) Chronic myeloid leukemia-blast phase (30%) Chronic myeloid leukemia-chronic phase R. Ren, 5:172-183

  23. Weisberg et al. 7:345, 2007 With the aid of several mediator proteins, BCR-ABL associates with Ras and stimulates its activation. The adaptor protein, growth factor receptor-bound protein 2 (GRB2), interacts with BCR-ABL through the proximal SRC homology 2 (SH2)-binding site that develops when the tyrosine 177 (Y177) residue of BCR-ABL is autophosphorylated. GRB2, when bound to BCR-ABL, interacts with the son of sevenless (SOS) protein. The resulting BCR–ABL–GRB2–SOS protein complex activates Ras. The adaptor proteins CRKL (CRK-like) and SHC (SH2-containing protein) can also mediate the BCR-ABL activation of Ras. Ras and the mitogen activated protein kinase (MAPK) pathway are coupled by Raf (a serine/threonine kinase). Raf catalyses the phosphorylation of the mitogen-activated and extracellular-signal regulated kinase kinases 1 and 2 (MEK1 and MEK2); this results in their activation. Through the stimulation of the Ras–Raf pathway, BCR-ABL increases growth factor-independent cell growth. BCR-ABL also associates with and activates the phosphatidylinositol-3 kinase (PI3K) pathway, suppressing programmed cell death and increasing cell survival. BCR-ABL is associated with components of the focal adhesion(that is, actin, paxillin and focal adhesion kinase, or FAK); the activation of CRKL–FAK–PYK2 leads to a decrease in cell adhesion. BCR-ABL also associates with the Janus kinase and signal transducer and activator of transcription (JAK–STAT) pathway. Finally, BCR-ABL activates pathways that lead to atypical responses to chemotactic factors, which leads to an increase in cell migration. BCR-ABL also associates with survival proteins that interact with the mitochondrial-based BCL2 family. CAS, p130 CRK-associated substrate; GAB2, GRB2-associated binding protein 2; SHIP, SH2-containing inositol-5-phosphatase

  24. (Imatinib mesilato)

  25. TRASDUZIONE DEL SEGNALE DA PARTE DI RECETTORI IMMUNI ITAM (Immunoreceptor tyrosine-based inhibitory motif) : DExxYxxL/I(x)6-7YxxL/I

  26. FcgRIIA FcgRIIB ITIM: I/VxxYxxL/V ITAM Immunoreceptor tyrosine-based inhibitory motif

  27. TRASDUZIONE DEL SEGNALE DA PARTE DI RECETTORI IMMUNI

  28. attivazione

  29. C-type lectins

  30. Lyn/Hck/Fgr Syk Syk Lyn Syk Lyn Fyn/ Lck Syk ZAP70

  31. Abl

  32. Lipide fosfatasi

  33. Protein fosfatasi

  34. YP YP ANCHE ALCUNE FAMIGLIE DI INTEGRINE (b2 E b3) TRASDUCONO IL SEGNALE MEDIANTE UN MODULO BASATO SU ADATTATORI CON SEQUENZA ITAM E SYK ? SYK SYK ITAM SFK

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