Signal Transduction II.ppt

1、,Signal Transduction II,BICH202,Signal transduction through the MAPK pathways Protein-protein interactions Protein kinase cascades,.,Overview,The MAPK signalling pathways, Cells in an organism receive a variety of extracellular stimuli for cell proliferation. The best characterized mitogenic pathway

2、 is the mitogen-activated protein kinase (MAPK) cascades. Many growth stimulation converges on the kinase cascade that activates the MAPK (also called ERK1 and ERK2 = extracellular signal-regulated kinase 1 and 2).,The MAPK signalling pathways, The RAS-activated MAPK pathway: RASRAFMEKMAPK represent

3、s the first example where all the steps in a complete signalling cascade from the cell surface receptor PTK, to the nuclear transcription is known., Upon activation, MAPK then translocate into the nucleus and induce the transcription of the immediate early genes (e.g. MYC)., Immediate early genes: g

4、enes that are expressed early during stimulation (in contrast to late response genes). They are usually transcription factors,The MAPK signalling pathways, It should be noted that the RAS-RAF-MEK-MAPK pathway is only one example of so called “MAPK pathways”. Two other mammalian MAPK pathways involvi

5、ng JNK1 and p38, are involved in stress responses (they are also “MAPK pathways).,The MAPK signalling pathways,RAS-RAF-MEK-MAPK, Ligand binds receptor PTK, Ligand binds receptor PTK Autophosphorylation on tyrosine,RAS-RAF-MEK-MAPK, Ligand binds receptor PTK Autophosphorylation on tyrosine GRB2 (a SH

6、2- and SH3-containing protein) binds to the receptor phosphotyrosine motif Y-V/L-N-X via its SH2 domain,RAS-RAF-MEK-MAPK, Ligand binds receptor PTK Autophosphorylation on tyrosine GRB2 (a SH2- and SH3-containing protein) binds to the receptor phosphotyrosine motif Y-V/L-N-X via its SH2 domain The SH

7、3 of GRB2 binds constitutively to the proline-rich sequence in the C-terminus of SOS (a guanine nucleotide exchange factor for RAS).,SOS,RAS-RAF-MEK-MAPK, Recruitment of SOS to the close proximity of RAS in the membrane,SOS,RAS-RAF-MEK-MAPK, RAS becomes activated by exchanging GDP for GTP,SOS,GDP,GT

8、P,RAS,RAS-RAF-MEK-MAPK,GTP, The RAS-GTP interacts with the N-terminal regulatory region of the RAF (serine/threonine protein kinase), hence recruiting RAF to the membrane and changing its conformation,SOS,RAS,RAF,RAS-RAF-MEK-MAPK,GTP,14-3-3, Activation of RAF (localization to membrane and conformati

9、on changes induced by RAS-GTP: cause phosphorylation of RAF and binding to the scaffold protein 14-3-3),SOS,RAS,RAF,RAS-RAF-MEK-MAPK,GTP,14-3-3, Activation of RAF (localization to membrane and conformation changes induced by RAS-GTP: cause phosphorylation of RAF and binding to the scaffold protein 1

10、4-3-3),SOS,RAS,RAF,RAS-RAF-MEK-MAPK,GTP,14-3-3, Activated RAF in turn activates MEK (also called MAPK kinase; a dual specificity kinase) by phosphorylation on two conserved serine residues in MEK.,SOS,RAS,RAF,MEK,RAS-RAF-MEK-MAPK,GTP,14-3-3, Activated RAF in turn activates MEK (also called MAPK kina

11、se; a dual specificity kinase) by phosphorylation on two conserved serine residues in MEK.,SOS,RAS,RAF,MEK,RAS-RAF-MEK-MAPK,GTP,14-3-3, Activated MEK activates MAPK (a serine/threonine protein kinase) by phosphorylation of conserved threonine and tyrosine residues.,SOS,RAS,RAF,MEK,MAPK,RAS-RAF-MEK-M

12、APK,GTP,14-3-3, Activated MEK activates MAPK (a serine/threonine protein kinase) by phosphorylation of conserved threonine and tyrosine residues.,SOS,RAS,RAF,MEK,MAPK,RAS-RAF-MEK-MAPK,GTP,14-3-3, Activated MAPK phosphorylates a number of substrates in the plasma membrane and the cytoplasm;,SOS,RAS,R

13、AF,MEK,MAPK,Substrates,Substrates,RAS-RAF-MEK-MAPK,GTP,14-3-3, Activated MAPK phosphorylates a number of substrates in the plasma membrane and the cytoplasm; it also translocated into the nucleus (within minutes) where it phosphorylates nuclear transcription factors.,SOS,RAS,RAF,MEK,Substrates,RAS-R

14、AF-MEK-MAPK,MAPK,GTP,14-3-3, Activated MAPK phosphorylates a number of substrates in the plasma membrane and the cytoplasm; it also translocated into the nucleus (within minutes) where it phosphorylates nuclear transcription factors.,SOS,RAS,RAF,MEK,Substrates,RAS-RAF-MEK-MAPK,GTP,14-3-3, Activated

15、MAPK phosphorylates a number of substrates in the plasma membrane and the cytoplasm; it also translocated into the nucleus (within minutes) where it phosphorylates nuclear transcription factors. Transcription of genes important for cell proliferation.,SOS,RAS,RAF,MEK,RAS-RAF-MEK-MAPK,Substrates,14-3

16、-3, More detail for each components,SOS,RAS,GTP,RAF,MEK,Substrates,RAS-RAF-MEK-MAPK,14-3-3,SOS,RAS,GTP,RAF,MEK,MAPK,GRB2 24 kDa adaptor molecule. Only contains an SH2 domain between two SH3 domains (only one is shown in the diagram).,RAS-RAF-MEK-MAPK,GRB2 The SH2 of GRB2 binds to phosphotyrosine in

17、a number of receptor PTK: for EGF, PDGF, colony-stimulating factor-1 (CSF-1), stem cell factor (SCF); all lead to the activation of RAS pathway. i.e. GRB2 functions as a point of convergence for Ras-activated signaling pathways.,RAS-RAF-MEK-MAPK,14-3-3,SOS,RAS,GTP,RAF,MEK,MAPK,14-3-3,SOS,RAS,GTP,RAF

18、,MEK,MAPK,SOS 180 kDa guanine nucleotide exchange factor for RAS; stimulate the formation of active GTP-bound RAS.,RAS-RAF-MEK-MAPK,14-3-3,SOS,RAS,GTP,RAF,MEK,MAPK,RAS-RAF-MEK-MAPK,SOS SOS belongs to the DBL family of oncogenes (14 members). Most of these activate the RHO family of GTP-binding prote

19、ins involved in cytoskeletal reorganization. SOS is one of the few known member of the family for RAS.,14-3-3,SOS,RAS,GTP,RAF,MEK,MAPK,RAS 21 kDa GTPase protein. A special case of G protein monomer GTP-bound form: active GDP-bound form: inactive. originally identified as oncogenes in several retrovi

20、ruses (rat sacroma), and are activated in a variety of human tumors. Amino acid substitution in one of two positions impairs its GTPase actvity and the activity cannot be shut off.,RAS-RAF-MEK-MAPK,14-3-3,SOS,RAS,GTP,RAF,MEK,MAPK,RAS Oncogenic forms of RAS often have point mutations that lock RAS in

21、 the active GTP-bound form. Evidence: 3T3 cells were induced to proliferate by + PDGF and EGF. Microinjection of anti-RAS antibodies into the cells blocked the cell proliferation. Microinjection of a constitutively active mutant of RAS caused cells to proliferate in the absence of PDGF and EGF.,RAS-

22、RAF-MEK-MAPK,14-3-3,RAS,GTP,RAF,MEK,MAPK,RAF RAF is a serine/ threonine protein kinase RAF is a MAPK kinase kinase (MAPKKK). Can transform cells when constitutively active or when overexpressed.,SOS,RAS-RAF-MEK-MAPK,14-3-3,RAS,GTP,RAF,MEK,MAPK,SOS,RAF The 14-3-3 family of scaffold proteins interacts

23、 constitutively with RAF via the phosphorylated Ser621 in RAF - both 14-3-3 and RAS are required for activation of RAF.,RAS-RAF-MEK-MAPK,14-3-3,RAS,GTP,RAF,MEK,MAPK,SOS,RAF RAS recruits RAF to the membrane. Membrane targeting of RAF is necessary to fully activate RAF. (Expression of mutant RAF that

24、cannot bind RAS no stimulation of cell proliferation by a constitutively active RAS).,RAS-RAF-MEK-MAPK, Interaction between RAS and RAF (or between any two proteins in general) can be demonstrated by:,(1) Co-immunoprecipitation,Methods, Interaction between RAS and RAF (or between any two proteins in

25、 general) can be demonstrated by:,(1) Co-immunoprecipitation,Methods,Antibodies against B on a solid matrix (e.g. beads coated with Staphylococcal Protein A - which binds IgG), Interaction between RAS and RAF (or between any two proteins in general) can be demonstrated by:,(1) Co-immunoprecipitation

26、,A,B,C,Methods,Wash out unbound proteins,The presence of protein A (or absence of C) in the immunoprecipitates can then be detected by immunoblotting.,Immunoblotting (Western blot),SDS-PAGE,-,+,View from the side,Methods,Immunoblotting (Western blot),SDS-PAGE,-,+,View from side,Methods,Immunoblottin

27、g (Western blot),SDS-PAGE,-,+,View from side,Methods,Immunoblotting (Western blot),-,View from side,+,Transfer to membrane,Methods,Immunoblotting (Western blot),-,View from side,+,Transfer to membrane,Methods,Immunoblotting (Western blot),Methods,Immunoblotting (Western blot),Probe with primary anti

28、bodies,Methods,Immunoblotting (Western blot),Wash away unbound antibodies,Methods,Immunoblotting (Western blot),Probe with secondary antibodies (HRP-conjugated),Methods,Immunoblotting (Western blot),Wash away unbound secondary antibodies,Methods,Immunoblotting (Western blot),Add substrate for HRP, e

29、xpose to film,Methods,Immunoblotting (Western blot),Add substrate for HRP, expose to film,Methods,Immunoblotting (Western blot),Develop film,Methods,-,+,?,A,B,(immunoprecipitation),(Immunoblotting),What is this?,(2) yeast two-hybrid system,Transcription factor,DNA-Binding Domain,Activating domain,Re

30、porter gene,Methods,B,Activating domain,DNA-Binding Domain,A,Methods,(2) yeast two-hybrid system,Transcription factor,DNA-Binding Domain,Activating domain,Reporter gene,DNA-Binding Domain,A,C,Activating domain,No interaction,Interaction,Methods,(2) yeast two-hybrid system,Reporter gene,B,Activating

31、domain,DNA-Binding Domain,A,Reporter gene,?, What controls are needed for designing immunoprecipitation experiments? What are the disadvantages of yeast two-hybrid?,14-3-3,RAS,GTP,RAF,MEK,MEK Also called MAPK kinase (MAPKK). Phosphorylated on Ser218 and Ser222 by activated RAF. Mutation of MEK that

32、leads to constitutive activity (by replacing the two Ser with glutamic acids or aspartic acids - by mimic phosphorylation) MAPK activation, mitogenicity, and cellular transformation.,SOS,MAPK,RAS-RAF-MEK-MAPK,14-3-3,RAS,GTP,RAF,MEK,SOS,MAPK,MEK The first true dual-specificity kinase to be discovered

33、.,RAS-RAF-MEK-MAPK,14-3-3,RAS,GTP,RAF,MEK,MAPK Five isoforms of ERK have been identified, but ERK1 and ERK2 have been most studied. Activated by phosphorylation on the 183T-E-Y185 - located similarly to the conserved Ser in MEK.,SOS,MAPK,RAS-RAF-MEK-MAPK,14-3-3,RAS,GTP,RAF,MEK,SOS,MAPK,MAPK MAPK act

34、ivation is biphasic: a transient peak within 5-10 min, and a sustained peak lasting several hours. The different activation kinetics different cellular response (e.g. in PC12 cells, EGF induces only the transient response and stimulate cell growth; but NGF (nerve growth factor) induces the sustained

35、 response and stimulate differentiation).,RAS-RAF-MEK-MAPK,14-3-3,RAS,GTP,RAF,MEK,SOS,MAPK,MAPK Inactivation of MAPK is achieved by several phosphatases: the serine/threonine phosphatase PP2A (may be for short term inactivation of MAPK); the dual-specificity phosphatase MKP1 (and the related PAC) an

36、d PTP1 (a tyrosine phosphatase, may be for long term negative feedback control of MAPK).,RAS-RAF-MEK-MAPK,MKP1: MAPK phosphatase 1,MAPK phosphorylates the consensus motif: P-X-S/T-P. In the cytoplasm, MAPK phosphorylates its upstream components in a negative feedback loop - MAPK phosphorylates SOS,

37、RAF, MEK inhibition of MAP kinase pathway.,Substrates of MAPK:, In the nucleus, MAPK phosphorylates a number of transcription factors (e.g. Elk1) increase transcription (e.g. of c-Fos mRNA). Many other substrates of MAPK probably unknown - identification is difficult as in the case of CDKs - see Cel

38、l cycle lectures).,Substrates of MAPK:,JNK pathway,Generic pathway,MAPKKK,MAPKK,MAPK,ERK/MAP kinase pathway,RAF1,MEK1,2,ERK1,2,RAS,GRB2/SOS,Receptor PTK,JNK/SAPK pathway,RAC/CDC42,MEKK1-3,MEK4,JNK/SAPK,TAK,MEK3,6,p38,p38 pathway,PAK,Stress responses,Proliferation/differentiation,JNK pathway, In resp

39、onse to extracellular stresses like cycloheximide treatment, UV irradiation, heat shock, or TNF-a treatment, a family of MAPK relatives known as JNKs (also called stress-activated protein kinase (SAPKs) become activated.,JNK pathway, RAC1 and CDC42 are two members of the RHO family of GTP-binding pr

40、oteins., RAC1 can also be activated by RAS, explaining why receptor PTK can sometimes contribute to JNK activation. However, this is a minor pathway. RAC1 and CDC42 are mainly activated by stress response independent of RAS.,JNK pathway, GTP-bound form of RAC1 and CDC42 bind and activate the serine/

41、threonine protein kinase PAK, PKN, and PtdIns kinases.,PAK,PAK: p21-activated protein kinase PKN: protein kinase N PtdIns kinase: phosphatidylinositol kinase,JNK pathway, GTP-bound form of RAC1 and CDC42 bind and activate the serine/threonine protein kinase PAK, PKN, and PtdIns kinases.,PAK,JNK path

42、way, these kinases phosphorylate and activate MEKK1-3,PAK,MEKK1-3,MEKK1-3,JNK pathway,PAK,MEKK1-3,MEK4, MEKK1-3 phosphorylate and activate MEK4 (also called JNKK) ( MEK in MAPK pathway; 45% identical in sequence with MEK),MEK4,JNK pathway, MEK4 phosphorylates JNK at two similar sites as in ERK (but

43、T-P-Y in JNK instead of T-E-Y in MAPK) i.e. conservation between the ERK and JNK pathways at the level of proteins and mode of regulation!,PAK,MEKK1-3,MEK4,JNK,JNK,JNK pathway, JNK translocation into the nucleus phosphorylation of the transcription factor c-JUN at the N-terminal residues (Ser63 and

44、Ser73) activation of transcription by c-JUN,PAK,MEKK1-3,MEK4,JNK,c-JUN,c-JUN,PAK,14-3-3,SOS,RAS,GTP,RAF,Specificity of MAP kinase pathways, When cells are treated with mitogenic agents (e.g. growth factors), ERK(MAPK) become strongly activated but JNK is poorly activated. Conversely, when cells are

45、challenged with stress, JNK is activated but ERK(MAPK) is only weakly activated. i.e. it seems JNK and ERK pathways are biologically distinct. However, they are both protein kinases with similar substrate specificity - most in vitro substrates are the same for both. Yet these pathways must result in

46、 unique transcriptional activity - because stress and mitogen must elicit different responses., Similarly, there are at least five parallel MAP kinase pathways in mammalian cells. How is specificity achieved? One way is by scaffold proteins.,Specificity of MAP kinase pathways,MAPKKK,MAPKK,MAPK,Scaff

47、old protein A,MAPKKK,MAPK,MAPKK,Scaffold protein B,Specificity of MAP kinase pathways,MAP kinase pathways in yeast, Most, perhaps all, eukaryotic cells contain kinase cascades that activate MAP kinase. In multicellular organisms, MAP kinase pathway is activated by ligand binding to receptor PTK. But there is no receptor PTK in yeast. How is MAP kinase pathways activated in yeast?, At least five independent MAPK-related cascades exist in yeast. Various functions: Mating (related to p