Trasduzione del segnale da chinasi e lipidi

Dipartimento di Biologia e Biotecnologie, Università degli Studi di Pavia

Mauro Torti

V Corso Nazionale di aggiornamento PIASTRINE 2015

Milano, 11-12 Novembre 2015

ovvero:

Come può il Ca2+ intracellulare regolare le tirosin chinasi?

substrate

Y

protein kinase

ATP

ADP

Pi

protein phosphatase

PROTEIN TYROSINE KINASES

(and protein tyrosine phosphatases)

substrate

YP

substrate

Y

protein kinase

ATP

ADP

PROTEIN TYROSINE KINASES IN PLATELETS

EphR

Fyn

Tec

Yes

Csk

Jak2

Btk

Syk FAK

Fgr

Pyk2

Lck

Lyn

Src

substrate

YP

Axl Mer PDGFR

EGF-1R Sky

Src Family Kinases (SFKs)

Src  Family  Kinases  (SFKs)  

ü  Non  receptor  tyrosine  kinases      ü  MW  :  50-­‐60  kDa  

ü  Inac?ve  when  phosphorylated  on  Tyr  527  

ü  Ac?ve  when  phosphorylated  on  Tyr  416  

Okada,  Int  J  Biol  Sci,  2012  

GPVI/ FcR γ-chain

Collagen

ITA

M

Y Y

ITA

M

Y Y

P LAT Y

Y

Y

Y

P P P

Syk

SH2 SH2

Y P

Syk

SH2 SH2

Y P

P

P

P

P

Btk SH2

Y

PLCγ2 SH2

PLCγ2 Y P

PKC Ca2+ PKC

secretion, integrin αIIbβ3 activation platelet aggregation

Ca2+

Fyn

Lyn

Integrin αIIbβ3

fibrinogen

Src

PLCγ2 Y P

PKC Ca2+ PKC

adhesion, spreading

Ca2+

GPVI and integrin-mediated activation of tyrosine kinases

FNG

thrombin

Gαq β γ

GDP

Fyn

Lyn Src

Protein kinases in GPCRs-mediated platelet activation

thrombin  cause  the  ?me-­‐dependent  tyrosine  phosphoryla?on  of  several  proteins    

No  ADP  (+  apyrase  )  NO  TxA2  (+  aspirin)    NO  integrin  αIIbβ3  acAvaAon  (no  sArred)    

Src  family  kinases  

FNG

thrombin

Gαq β γ

GDP

secretion αIIbβ3 activation aggregation

Fyn

Lyn Src

Protein kinases in GPCRs-mediated platelet activation

Dorsam  et  al  ,  Blood  2002  Cho  et  al  ,  Blood  2005    Li  et  al,  J  Biol  Chem  2010  Xiang  et  al,  J  Biol  Chem  2012    

???

epinephrine

Gαi β γ

α2 adr

GDP

Src

Protein kinases in GPCRs-mediated platelet activation

Gαi GTP

TorA  et  al,  BBRC  1992   Ma  et  al,  Cell  2000  

FNG

thrombin

Gαq β γ

GDP

PLCβ

PKC

Ca2+ secretion αIIbβ3 activation aggregation

Fyn

Lyn Src ???

Protein kinases in GPCRs-mediated platelet activation

Dorsam  et  al  ,  Blood  2002  Cho  et  al  ,  Blood  2005    Li  et  al,  J  Biol  Chem  2010  Xiang  et  al,  J  Biol  Chem  2012     ???

The  focal  adhesion  kinase:  Pyk2    

Thrombin,  ADP,  TxA2  

Ca2+    AGGREGATION  

Integrin    α2β1  signalling  

Collagen  

PLCγ2  

Ca2+    

Akt  SFKs  

SPREADING  

Pyk2  

P

Fibrinogen  

PI3Kβ  

PLCβ

GPCRs  signalling  

Integrin    αIIbβ3  signalling  

Impaired  ac?va?on  in  thrombin-­‐s?mulated    Pyk2KO    murine  platelets    

Pyk2  and  SFKs  operate  on  the  same  pathway  for  thrombin-­‐induced  aggrega?on    

Focal  adhesion  kinase:  Pyk2  

FERM KINASE DOMAIN FAT

Y402   Y579   Y580   Y881  

PRR1 PRR2 PRR3

Ca2+ promoted phosphorylation

autophosphorylation site

SFKs SH2 domain binding site

phosphorylated by Src family

kinases

increased kinase activity

GRB2 SH2 domain binding site and

N-­‐   -­‐C  

ü   Pyk2  acAvaAon  is  mediated  both  by  increase  of  intracellular  Ca2+    and  by  Src  kinases  acAvaAon  

Ca2+  

Ca2+  

SFKs  

SFKs  

Thrombin-­‐induced  Pyk2    ac?va?on  and  autophosphoryla?on  on  Tyr402    is  Ca2+-­‐dependent    

thrombin  

GA23187  

Pyk2  Ca2+  

thrombin  

G

Ca2+   SFKs  

PROTEIN  T  Y  R  O  S  I  N  E  PHOSPHORYLATION  

SFKs  

Ca2+  

?

Ca2+  

SFKs  

SFKs  ac?vity  is  not  required  for  Ca2+  release…  

FURA  2-­‐AM  loaded  platelet  Preincubated  with  SFKs  inhibitors  PP2  

…but  Ca2+  contributes  to  SFKs  ac?vity  

P-­‐Src  (Y416)  :    recognises  the      phosphorylated  Src  (acAvatory  Tyr)  and  cross  reacts  with  all  SFKs  

thrombin  

G  

SFKs  

Ca2+  

thrombin  

Pyk2  

???  

Pyk2  regulates  thrombin-­‐induced  Src  kinases  phosphoryla?on     Pyk2KO  

thrombin  

G

A23187  

Pyk2   SFKs  Ca2+   PROTEIN  T  Y  R  O  S  I  N  E  PHOSPHORYLATION  

Pyk2  regulates  Ca2+  ionophore-­‐  and  thrombin-­‐induced  tyrosine  phosphoryla?on  

Pyk2  KO  mice  

thrombin  

G

A23187  

Pyk2   SFKs  Ca2+   PROTEIN  T  Y  R  O  S  I  N  E  PHOSPHORYLATION  

thrombin  

G

Pyk2  Ca2+  

Src  

Which  member  of  the  SFKs  is  ac?vated  by  Pyk2?  

Fyn  

Lyn  

Fyn  

Lyn  

Focal  adhesion  kinase:  Pyk2  

FERM KINASE DOMAIN FAT

Y402   Y579   Y580   Y881  

PRR1 PRR2 PRR3

Ca2+ promoted phosphorylation

autophosphorylation site

Src SH2 domain binding site

phosphorylated by Src kinase

increased kinase activity

GRB2 SH2 domain binding site and

N-­‐   -­‐C  

ü   Pyk2  acAvaAon  is  mediated  both  by  increase  of  intracellular  Ca2+    and  by  Src  kinases  acAvaAon  

Ca2+  

Ca2+  

SFKs  

SFKs  

thrombin  

G

Pyk2  

Thrombin-­‐induced  Pyk2  ac?va?on  is  mediated  by  Ca2+  ,  but  also  by  Src  kinases    

SFKs  Ca2+  

Fyn  KO

 Fyn  regulates    Pyk2  ac?va?on    

Lyn  KO

 

thrombin  

G

Pyk2  Ca2+  Fyn  

Lyn  

Pyk2

Thrombin

PROTEIN-TYROSINE PHOSPHORYLATION

Ca2+ ionophore

Ca2+

Fyn

Lyn

secre?on  

TxA2  

ADP  

αIIbβ3 ac?va?on    

The  focal  adhesion  kinase:  Pyk2    

Thrombin,  ADP,  TxA2  

Ca2+    AGGREGATION  

Integrin    α2β1  signalling  

Collagen  

PLCγ2  

Ca2+    

Akt  SFKs  

SPREADING  

Pyk2  

P

Fibrinogen  

PI3Kβ  

PLCβ

GPCRs  signalling  

Integrin    αIIbβ3  signalling  

thanks  to  :    Ilaria  Canobbio  Gianni  Guidef  Lina  Cipolla  

Daria  Manganaro  Caterina  Visconte  

Alessandra  Consonni