*** *** rafrules 5-20-03 Maude alpha80 *** ******************************************************************* mod RAFMOD is inc PROTEINOPS . *** This is the high detail version of rules 876, 830, and 280 *** It is based on a hypothesis by Dhillon and Kolch *** 22122191(R) vars cyto nuc cm nm lyso endo ccp out proteo : Soup . var ?Pak : Pak . var ?Ras : Ras . var ?Slk : Slk . ***** Proteins op 14-3-3a : -> Protein . *** These are not isoforms of 14-3-3 op 14-3-3b : -> Protein . *** The alphabetical stuff is to distinquish op 14-3-3c : -> Protein . *** one copy from another until the actual op 14-3-3d : -> Protein . *** isoforms used are determined **** Atts used for Raf1 *** S43, S259, S338, Y341, T491, S494, S621 *** RBD - Ras Binding Domain *** RBD IPR003116 *** C1 - Cysteine Rich Domain *** DAG_PE-bind IPR002219 *** PABM - Phosphatidic Acid Binding Motif *** Prot_kinase IPR000719 *** Ser_thr_pkinase IPR002290 *** Tyr_pkinase IPR001245 ***** The kickoff 14-3-3a 14-3-3b *** rl[Raf1#0.init]: op Raf1.domains : -> Soup . eq Raf1.domains = [Raf1 | (S 43), (S 259 - phos - bound), (S 338), (Y 341), (S 621 - phos - bound), RBD, C1, PABM] [14-3-3a | (SBD - bound), (DMD - bound)] [14-3-3b | (SBD - bound), (DMD - bound), (T 141)] e((Raf1,(S 621)), (14-3-3a,SBD)) e((Raf1,(S 259)), (14-3-3b,SBD)) e((14-3-3a,DMD), (14-3-3b,DMD)) . ***** Rules rl[Raf1#1.PKCz]: {CM | cm [PKCz - act] {cyto [Raf1 | (S 43), (S 259 - phos - bound), (S 338), (Y 341), (S 621 - phos - bound), raf1:Atts] [14-3-3a | (SBD - bound), (DMD - bound), 1a:Atts] [14-3-3b | (SBD - bound), (DMD - bound), (T 141)] e((Raf1,(S 621)), (14-3-3a,SBD)) e((Raf1,(S 259)), (14-3-3b,SBD)) e((14-3-3a,DMD), (14-3-3b,DMD)) }} => {CM | cm [PKCz - act] {cyto [Raf1 | (S 43), (S 259 - phos), (S 338), (Y 341), (S 621 - phos - bound), raf1:Atts] [14-3-3a | (SBD - bound), (DMD - bound), 1a:Atts ] [14-3-3b | SBD , (DMD - bound), (T 141 - phos)] e((Raf1,(S 621)), (14-3-3a,SBD)) e((14-3-3a,DMD), (14-3-3b,DMD)) }} . *** 22122191(R) says that *** Activated Ras recruites the RAf1 complex to the CM and breaks *** the bond between phos S259 and 14-3-3 *** 22072572(D) 21642148(D) 21864215(D) *** revised hypothesis based on 20088663(D) *** PKCz phoses 14-3-3beta (in vitro) on S130 and T141 which decreases *** binding of PKCz, Raf1, and 14-3-3 complex *** PKCz also phoses 14-3-3theta (in vitro) which only has T141 *** PKCz does not phos or bind to 14-3-3zeta (in vitro) *** 21061396(D) Raf1 reqs S621 for activation by PMA or v-Src *** also evidence that 14-3-3 stays attached to Raf1 ****** rl[Raf1#2.PP2A]: {CM | cm {cyto PP2A [Raf1 | (S 43), (S 259 - phos), (S 338), (Y 341), (S 621 - phos - bound), raf1:Atts] [14-3-3a | (SBD - bound), (DMD - bound), 1a:Atts ] [14-3-3b | SBD, (DMD - bound), (T 141 - phos)] e((Raf1,(S 621)), (14-3-3a,SBD)) e((14-3-3a, DMD), (14-3-3b,DMD)) }} => {CM | cm {cyto PP2A [Raf1 | (S 43), (S 259), (S 338), (Y 341), (S 621 - phos - bound), raf1:Atts] [14-3-3a | (SBD - bound), (DMD - bound), 1a:Atts ] [14-3-3b | SBD, (DMD - bound), (T 141 - phos)] e((Raf1,(S 621)), (14-3-3a,SBD)) e((14-3-3a,DMD), (14-3-3b,DMD)) }} . *** 22122191(R) 21864215(D) *** S259 is no longer bound to 14-3-3 and can be dephos by PP2A *** PP2A is assumed to be cytoplasmic (Swiss Protein) but no *** real data found - so it is more probable that Raf#2 happens *** before Raf#3 *** 22499937(D) Mutation S259A/D constitutively activates Mek, Erk *** but is no tumorigenic like v-Raf ***** rl[Raf1#3.PS.PA]: {CM | cm PS PA {cyto [Raf1 | (S 43), (S 259), (S 338), (Y 341), (S 621 - phos - bound), C1, PABM, raf1:Atts] [14-3-3a | (SBD - bound), (DMD - bound), 1a:Atts] [14-3-3b | SBD, (DMD - bound), (T 141 - phos)] e((Raf1, (S 621)), (14-3-3a,SBD)) e((14-3-3a, DMD ), (14-3-3b,DMD)) }} => {CM | cm PS PA [Raf1 | (S 43), (S 259), (S 338), (Y 341), (S 621 - phos - bound), (C1 - bound), (PABM - bound), raf1:Atts] [14-3-3a | (SBD - bound), (DMD - bound), 1a:Atts] [14-3-3b | SBD , (DMD - bound), (T 141 - phos)] e((Raf1, (S 621)), (14-3-3a,SBD)) e(b(PS),(Raf1, C1 )) e(b(PA),(Raf1, PABM )) e((14-3-3a,DMD ), (14-3-3b,DMD)) {cyto }} . *** 21278045(R-20) *** 20379031(D) for PA *** 99426181(D) for PS *** 21278045(R) 99426181(D) Raf1 is no longer held in the cyto by 14-3-3 *** and is free to migrate to CM where the C1 domain attaches to *** DAG and/or phosphatidylserine (requires raf R143,K144,K148 in the *** C1 domain). *** 21278045(R) 20379031(D) Raf1 can also be recruited to the CM in the *** absence of a RBD by Phosphatidic Acid via the PABM aa 389-413 in *** the kinase domain *** 22122191(R) Activated Ras also recruites the RAf1 complex to the CM *** 22072572(D) 21642148(D) 21864215(D) *** 21916673(A) 94193580(A) PS binds to Raf-C1 better than DAG *** 99373021(A) after membrane binding 14-3-3 is totally displaced (?) ***** rl[Raf1#4.Ras]: {CM | cm PS PA [?Ras - GTP] [Raf1 | (S 43), (S 259), (S 338), (Y 341), (S 621 - phos - bound), (C1 - bound), (PABM - bound), RBD, raf1:Atts] [14-3-3a | (SBD - bound), (DMD - bound), 1a:Atts] [14-3-3b | SBD , (DMD - bound), (T 141 - phos)] e((Raf1, (S 621)), (14-3-3a,SBD)) e(b(PS),(Raf1, C1 )) e(b(PA),(Raf1, PABM )) e((14-3-3a,DMD ), (14-3-3b,DMD)) {cyto }} => {CM | cm PS PA [?Ras | GTPbound, (RafBD - bound)] [Raf1 | (S 43), (S 259), (S 338), (Y 341), (S 621 - phos - bound), (C1 - bound), (PABM - bound), (RBD - bound), raf1:Atts] [14-3-3a | (SBD - bound), (DMD - bound), 1a:Atts] [14-3-3b | SBD , (DMD - bound), (T 141 - phos)] e((Raf1, (S 621)), (14-3-3a,SBD)) e(b(PS),(Raf1, C1 )) e(b(PA),(Raf1, PABM )) e( (?Ras, RafBD ), (Raf1, RBD )) e((14-3-3a, DMD ), (14-3-3b,DMD)) {cyto }} . *** There is a great debate here: *** Is Ras required only to recruit Raf to the membrane? *** Refs 94255771(A) *** Or does it also do something else? *** Refs 97256741(D) 97390455(D) Raf Zinc Finger (C1) required for act *** 21369894(D) N-ras, Raf1, and PKCe are found in a complex in the CM *** of quiescent C3H10T1/2 when raf1 is not active by kinase of Mek ****** rl[Raf1#5.S338phos]: {CM | cm PS PA [?Pak - act] [?Ras | GTPbound, (RafBD - bound)] [Raf1 | (S 43), (S 259), (S 338), (S 621 - phos - bound), (C1 - bound), (PABM - bound), (RBD - bound), raf1:Atts] [14-3-3a | (SBD - bound), (DMD - bound), 1a:Atts] [14-3-3b | SBD , (DMD - bound), (T 141 - phos)] e((Raf1, (S 621)), (14-3-3a,SBD)) e(b(PS),(Raf1, C1 )) e(b(PA),(Raf1, PABM )) e( (?Ras, RafBD ), (Raf1, RBD )) e((14-3-3a, DMD ), (14-3-3b,DMD)) {cyto }} => {CM | cm PS PA [?Pak - act] [?Ras | GTPbound, (RafBD - bound)] [Raf1 | (S 43), (S 259), (S 338 - phos), (S 621 - phos - bound), (C1 - bound), (PABM - bound), (RBD - bound), raf1:Atts] [14-3-3a | (SBD - bound), (DMD - bound), 1a:Atts] [14-3-3b | SBD , (DMD - bound), (T 141 - phos)] e((Raf1, (S 621)), (14-3-3a,SBD)) e(b(PS),(Raf1, C1 )) e(b(PA),(Raf1, PABM )) e( (?Ras, RafBD ), (Raf1, RBD )) e((14-3-3a, DMD ), (14-3-3b,DMD)) {cyto }} . *** 22122191(R) *** phos on Y341 is also required - it may be Src or a SLK but no one *** has proved it yet *** 21160274(D) shows that activated Ras + Src is sufficient to activate *** Raf1 and that Pak3 is able to phos Raf1 S338 but not to activate it *** by kinase assay or translocation to CM nor does it influence the *** activation by either Src or Ras *** unbinding of S259 from 14-3-3 opens up Raf1 so that S338 is now *** acessable to phos by Pak and Src. *** This rule could happen before or after Raf#6 so Y341 is an var:Att *** 21369894(D) PMA leads to phos of p338 in C3H10T1/2 (not necessarily *** direct) but shows a requirement somewhere for cPKC or nPKC. ****** rl[Raf1#6.Y341phos]: {CM | cm PS PA [?Slk - act] [?Ras | GTPbound, (RafBD - bound)] [Raf1 | (S 43), (S 259), (Y 341), (S 621 - phos - bound), (C1 - bound), (PABM - bound), (RBD - bound), raf1:Atts] [14-3-3a | (SBD - bound), (DMD - bound), 1a:Atts] [14-3-3b | SBD , (DMD - bound), (T 141 - phos)] e((Raf1, (S 621)), (14-3-3a,SBD)) e(b(PS),(Raf1, C1 )) e(b(PA),(Raf1, PABM )) e( (?Ras, RafBD ), (Raf1, RBD )) e((14-3-3a, DMD ), (14-3-3b,DMD)) {cyto }} => {CM | cm PS PA [?Slk - act] [?Ras | GTPbound, (RafBD - bound)] [Raf1 | (S 43), (S 259), (Y 341 - phos), (S 621 - phos - bound), (C1 - bound), (PABM - bound), (RBD - bound), raf1:Atts] [14-3-3a | (SBD - bound), (DMD - bound), 1a:Atts] [14-3-3b | SBD , (DMD - bound), (T 141 - phos)] e((Raf1, (S 621)), (14-3-3a,SBD)) e(b(PS),(Raf1, C1 )) e(b(PA),(Raf1, PABM )) e( (?Ras, RafBD ), (Raf1, RBD )) e((14-3-3a, DMD ), (14-3-3b,DMD)) {cyto }} . *** 22122191(R) *** phos on Y341 is also required - it may be Src or a SLK but no one *** has proved it yet *** 21160274(D) shows that activated Ras + Src is sufficient to activate *** Raf1 and that Pak3 is able to phos Raf1 S338 but not to activate it *** by kinase assay or translocation to CM nor does it influence the *** activation by either Src or Ras *** unbinding of S259 from 14-3-3 opens up Raf1 so that S338 is now *** acessable to phos by Pak and Src. *** This rule could happen before or after Raf#5 so S338 is an var:Att *** but 21061396(D) and Ref22 give evidence that S338 gets phosed first *** 21061396(D) more evidence that Src is needed for Raf1 act ************************************************************************* *** This is the end of the activation half of the raf mod. *** Need to figure out what to do with 14-3-3 - is it an integral part of *** Raf1 all the time? rl[Raf1#7.Raf1.is.act]: [?Ras | GTPbound, (RafBD - bound)] [Raf1 | (S 43), (S 259), (S 338 - phos), (Y 341 - phos), (S 621 - phos - bound), (C1 - bound), (PABM - bound), (RBD - bound)] [14-3-3a | (SBD - bound), (DMD - bound), 1a:Atts] [14-3-3b | SBD, (DMD - bound), (T 141 - phos)] e((Raf1, (S 621)), (14-3-3a, SBD )) e(b(PS),(Raf1, C1 )) e(b(PA),(Raf1, PABM )) e( (?Ras, RafBD ), (Raf1, RBD )) e((14-3-3a, DMD ), (14-3-3b, DMD )) => [Raf1 - act] [?Ras - GTP] . ************************************************************************** endm ****** mod ALLBP is inc RAFMOD . endm ****** ************************************************************************** ***( Things to think about: 1. Question: Why does PMA act Raf1 Mek kinase activity? see 21061396 for discussion - increase in Ras - GTP - reqs phos of S621 - increased act of Pak 2. Hypothesis: PKCz phoses and binds to 14-3-3 beta or theta which breaks up the complex between PKCz, Raf1, and ?14-3-3. PKCz does not bind to 14-3-3zeta. Could 14-3-3 be a dimer of beta or theta (which would bind to S259 and zeta which would bind to S621)? Then PKCz would release the S259 end but not S621. - argument for: 14-3-3z req intact S621 to bind to CR3 fragment of Raf1 - argument against: 14-3-3z binds lots more wtRaf1 than CR3 fragment 21061396(D) 3. Question: Could 14-3-3 S259 end bind to Ksr? Or could 14-3-3 dimer break up after release from S259 and 14-3-3-S621-Raf1 dimerize with 14-3-3-Swhichever-Ksr1? )