Biom600 f lee 12 5-12
77
SERINE AND THREONINE PHOSPHORYLATION: PROTEIN KINASE C, MAP KINASES, AND NF-κB Frank S. Lee 605 Stellar Chance Labs 898-4701 [email protected]
Transcript of Biom600 f lee 12 5-12
SERINE AND THREONINE PHOSPHORYLATION:!PROTEIN KINASE C, MAP KINASES, AND NF-κB!
!!
Frank S. Lee!605 Stellar Chance Labs!
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PROTEIN! O -! PROTEIN! O! +!
ATP ADP
PROTEIN KINASES:!The Phosphotransfer Reaction!
N-terminal!lobe!
C-terminal!
lobe!
Active!site!
PROTEIN KINASE STRUCTURE (PKA)!
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2 lobes N= beta pleated sheets C=alpha helices cleft = active site
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PROTEIN KINASES:!Eleven Subdomains!
• Subdomain I: GXGXXG forms ATP binding pocket!
• Subdomain II: Lys interacts with α and β phosphates of ATP!
• Subdomain VI: Asp deprotonates hydroxyl of Ser or Thr of protein substrate!
• Subdomain VII: Asp chelates Mg++, which orients γ phosphate of ATP!
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promote nucleophilic attack on gamma phosphate - to inact catalytic act of kinase, highly conserved site for mutagenesis
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-why Mg typically includd in protein kinase rxns
Stimulus!
Ras!DAG! Ca++!
MAP KINASES!PKC!
PLC!
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PROTEIN KINASE C!
MEMBRANE!
C1! C2!
C3/4!AI!
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C3 = Nterminal lobe C4 = cterminal lob c1/2 = distinct
PROTEIN KINASE C:!CATALYTIC (C3/4) AND AUTOINHIBITORY DOMAINS!
CATALYTIC!AI!
Orr and Newton, 1994, JBC 269, 8383!
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AI = portion of PKC that blocks enz activity of PKC in resting state
PROTEIN KINASE C:!AUTOINHIBITORY DOMAIN!
! ! -3 -2 -1 +1 +2!
Substrate ...R K G S L R...!AI domain ...R K G A L R...!
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site of phosphorylation = designated PKC = pref for bulky residues at -3 prim structure of AI matches closely seq of substrate - exception is A instead of S - A cannot be phosphorylated - peptide motif can sit in PKC site and block enz activity
Stimulus!
Ras!DAG! Ca++!
MAP KINASES!PKC!
PLC!
PROTEIN KINASE C!
MEMBRANE!
C1! C2!
C3/4!AI!
PROTEIN KINASE C:!C2 DOMAIN!
Verdaguer et al., 1999, EMBO J 18, 6329!
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calcium sensor also binds to phosphotidylserine in addition to Ca
PROTEIN KINASE C:!Ca++ ACTIVATION!
MEMBRANE!
C1! C2!
C3/4!AI!
PS! Ca!
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how it activates PKC in response to these second messengers
PROTEIN KINASE C:!C1 DOMAIN!
Zhang et al., 1995, Cell 81, 917!
GREEN=HYDROPHOBIC!WHITE=HYDROPHILIC!BLUE=ACIDIC!RED=BASIC!
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globular structure hydrophobic = green white = hydrophilic large hydrophobic patch w/ hydrophilic surface DAG = promotor that's analog DAG would bind to same location (?) - create continuous hydrophobic surface and translocation of PKC to membrane
PROTEIN KINASE C:!AUGMENTATION OF PKC AFFINITY FOR PS BY DAG!
Newton & Keranen, 1994, Biochem 33, 6651!
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PS content is varied - composition varied - dose dependent binding of PKC to PM - done in absence of DG if you add DG, shifted to left - greater binding of PKC to PM
PROTEIN KINASE C:!DAG ACTIVATION!
MEMBRANE!
C1! C2!
C3/4!
AI!
PS! Ca!DAG!
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DAG binds to C1 domain C2 - promotoes translocation of PKC to PM, results in translocationof A1/movement = catalytic activity of enzyme
MARCKS !(myristoylated alanine-rich !protein kinase C substrate)!
S!K!K!
K! K! K!
S! S!
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middle = binds to actin - on 1 facce, + charged residues, adjacent phase = serine - site of phosphorylation of PKC - change overall charge, binding to actin if mutated
MARCKS REGULATION OFACTIN!
MARCKS !PEPTIDE!
+!ACTIN!
MARCKS !PHOSPHOPEPTIDE!
+!ACTIN!
Hartwig et al., 1992, Nature 356, 618!
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in presence of native peptide, promote actin polymerization
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if you take peptide, phosphorylate residues, this peptide is defective in promoting actin polymerization ??
PKC REGULATION OF MARCKS !
PKC!
MARCKS!
ACTIN!
MARCKS!
ACTIN!
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stimulation of PKC = MRCKS phosph - disrupts actin binding
PROTEIN KINASE C
• Autoinhibitory domains as pseudosubstrates!
• Integration of signals!• Subcellular localization
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translocation of PKC from cyt to PM is imp component of enzyme
Stimulus!
Ras!DAG! Ca++!
MAP KINASES!PKC!
PLC!
ERK (MAPK): An Insulin-Stimulated Kinase!Phosphorylated on Thr and Tyr!
Ray & Sturgill, 1988, PNAS 85, 3753!
ERK!
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cells stimulated w/ insulin in p32 then fractionate, run sds page, appearance of band at around 43 kD = ERK. take protein, study, has prot kinase acti - hydrolyze to AA - TLC run to identify sites of phosph (run standards that contain phosph version of AA - phoph occus on thr and tyr
Activation of the!Serum Response Element!
SRF! Elk-1!
S 383!
SRF! Elk-1!
ERK!
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Elk is component of ternanry complex DNA element - binds to SRF and binds to Elk. phosph of ELk (on ser/thr residue) results in potentiation of transcriptional activity of protein
Canagarajah et al., 1997, Cell 90, 859!
ACTIVATION LOOP!PHOSPHORYLATION IN ERK!
UNPHOSPHORYLATED! PHOSPHORYLATED!
...DHTGFLTEYVAT...!
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in inactive and active state
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fold phosph on 2 spec residues activation loop - between 7 and 8 - ser and thr key to act of prot kinase overall fold is roughly same but conformation is diff upon phosph of 2 key residues ERk substrates self phosph o nser/thr
Elk-1!
ERK!MAPK!
?!
ACTIVATION OF ERK BY MEK!
Crews et al., 1992, Science 258, 479!
MEK!
ERK!
+! +!+! +!
+!
ERK!
MEK ...LIDSMANSFVGT...!ERK ...DHTGFLTEYVAT...!
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examine capacity of MEK prot kinase assay - s ubstrate in presence of p32 ATP/ sds page - id of phosph species mek or erk alone - no sig phosph add both = phosphorylation - enhancement of cat activity of ERK MEK = dual specificity prot kinase (spec for Ser and Tyr)
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2 residues in SXXS motif - if you mutate, abolish capcity of MEk to be activated
MEK1!
ERK!
MAP2K!
MAPK!
?!
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phosphorylates
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- has its own phosph loop, implying its target of a diff prot kinase
ACTIVATION OF MEK BY Raf!
Kyriakis et al., 1992, Nature 358, 417!
MEK1!
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observations that cells transformed with Raf have high ERK act - is Raf activator of pathway? yes Raf - transform cells - assay fractions - MEK = substrate
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activity int ransformed cells = phosph to MEK - cna directly phosph MEK - specific Serines
Raf!
MEK1!
ERK!
MAP2K!
MAP3K!
MAPK!
PROTEIN KINASE CASCADES
• Amplification!• Multiple layers of regulation!• Crosstalk!
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of small signal
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ie. dephosph of ERK and inact ERK and phosphatases are dual specific phosphatases
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prot that directly interact w/ Raf mult kinases = multiple targets with other pathways PKA can be activated by various hormones and can downregulate ERK pathway thru phosph of Raf
Raf!
MEK1!
ERK!
MAP2K!
MAP3K!
MAPK!
?!
Raf:RAP COMPLEX!
Nasser et al., 1995, Nature 375, 554!
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Raf has mult domains kinase domain, Nterm regulatory domain - interacts w/ small GTPases - active when bound to GTP Rap member of small GTPase
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Raf!
MEK1!
ERK!
MAP2K!
MAP3K!
MAPK!
GTPase! Ras!
MAP KINASE/ERK
• Activation loop phosphorylation!• Protein kinase cascades!
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Growth!Factors! Stress!
ERK! JNK!
Gene!Expression!
Gene!Expression!
Activation of c-Jun!Transactivation! DNA binding!
1! 331!
63!73!
CBP!TF’s!
RNA Pol II!
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cJUN has dna binding domain - leucine zipper and transactivation domain - large - nterminus of protein and imp component is that it has 2 specific ser residues required for potentiation of this activity once cjun is bound to promoters, allows recruitment of CBP and p100 - basal transcription factors and RNA PolII
C-Jun N-Terminal Kinase (JNK)/!Stress Activated Protein Kinase (SAPK):!
Homology to ERK!
Derijard et al., 1994 Cell 76, 1025!
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homology between this and ERK degree of conservation
Kyriakis et al., 1994, Nature 369, 156!
ACTIVATION OF JNK vs. ERK!
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are stimuli that active simialr? -IP followed by prot kinase assay - Ab against prot kinase of interest; IP prot kinase beads and in vitro form prot kinase in presence of p32 atp ie JNK substrate = glutathion Stransferase ie. ERK = mylin based protein incubate, wash identify substrate and by incorporation of p32 can observe quatnification of degree of phosphorylation -induction is what's importnat -FGF - ERK responds to growth factor, Jnk not affected TNF - Jnk responds, ERK only modest
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Jnk resp to strssful stimuli distinct from growth factors
ACTIVATION OF JNK/SAPK!BY UV IRRADIATION!
Derijard et al., 1994, Cell 76, 1025!
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IP jnk, assay it quantitate see low basal act and dramatic induction o fJnk act at around 60 mins
MKK4! SGQLVDSIAKTMDAGCRPYMAPE!
MAPK PHOSPHORYLATION LOOPS!
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homology in region of activation loop of jnk - TXY motif - conserved in all isoforms of JNK
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helps identify subfamilies of MAPK abolish activity of JNK/ability to be activated fi you mutate
Raf!
MEK1!
ERK!
MAP2K!
MAP3K!
MAPK! JNK!
?!
?!
ACTIVATION OF JNK BY MKK4 (SEK1)!
Sanchez et al., 1994, Nature 372, 794!
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prot kinase assays - GST used for JNK; jnk assayed in presence of p32 atp pos control - in presence of anisomycin, induces jnk activity MKK4 has no sig activity against CJUN but if you add to jnk - robust actvity of jnk activation
MKK4! SGQLVDSIAKTMDAGCRPYMAPE!
!!!
MAP2K PHOSPHORYLATION LOOPS!
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SXXXS/T motif
Raf!
MEK1!
ERK!
MAP2K!
MAP3K!
MAPK! JNK!
?!
MKK4!
Yan et al., 1994, !Nature 372, 798!
ACTIVATION OF MKK4 (SEK1) BY MEKK1!
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MEKK1 left = assauys where mKk4 employed as substrate - incubate in presence of p32 - no autophosph. now add mkk1, easily detect phosph on appropriate residues can take protein, isolate, hydrolyze to AA< TLC to sep out ind AA to identify types ofr esidues - S and T are both targts of phosphy -MEKK1 can phosph MK4 - coupledkinase rxn - multiple components of cascade - jnk emplyed as substrate - act site lysine mutated to A. if you take jnk with mkk4, no sig phsph. now add MEKK1, now see phosph of jnk
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Raf!
MEK1!
ERK!
MAP2K!
MAP3K!
MAPK! JNK!
MKK4!
MEKK1!
MAP2K!
MAP3K!
MAPK! JNK!
MKK4, MKK7!
MEKK1, MLK!
MULTIPLE PROTEIN KINASES!OF THE JNK PATHWAY!
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Tanoue et al., 2000, Nat Cell Biol 2, 110!
A CONSERVED DOMAIN IN JNK IS!NECESSARY FOR JNK:MKK4 INTERACTION!
D>N!
JNK:MAP2K INTERACTIONS!
JNK!- -!- -!
MKK4/MKK7!+ + +!
“ED” “CD”!
MAP2K:MAPK INTERACTION!
Chang et al., 2002, Mol Cell 9, 1241!
JNK-INTERACTING PROTEIN-1 (JIP1):!A SCAFFOLD PROTEIN FOR THE JNK PATHWAY!
Whitmarsh et al., 1999, Science 281, 1671!
Whitmarsh et al., 1999, Science 281, 1671!
JIP ORGANIZES A FUNCTIONAL JNK MODULE!
JNK SUBFAMILY OF MAP KINASES
• Multiple MAP kinase cascades!• Docking sites!• Scaffold proteins!
Growth!Factors! Stress!
ERK! JNK!
Gene!Expression!
Gene!Expression!
NF-κB!
TNF-"A
B
NUCLEAR TRANSLOCATION!OF NF-κB!
NF-κB!
NF-κB:IκB!
proteasome!
Activation of NF-κB!
Cytoplasm!
Nucleus!
NF-κB:IκB Complex: Masking of NLS!
Jacobs & Harrison, !1998, Cell 95, 749!
NF-κB! IκB!
DBD!
NLS!
TA!
THE NF-κB/IκB FAMILIES!
p65 (RelA)
RelB
c-Rel
p50/p105
p52/p100
I!B"
I!B#
Bcl-3
Rel Homology Domain
AnkyrinRepeats
IκBε
Brown et al., 1995, Science 267, 1485!
(min)!
Phosphorylation-dependent!Degradation of IκB!
NF-κB!
NF-κB:IκB!
proteasome!
?!
NF-κB:IκB!
32!36!
Brown et al., 1995, Science 267, 1485!
(min)!
Phosphorylation-dependent!Degradation of IκB!
NF-κB!
NF-κB:IκBα!
proteasome!
Feedback Inhibition of NF-κB!
IκBα
?!
IkB Kinases (IKK):!Protein Kinases with !
Protein Interaction Motifs!
ACTIVATION OF IKK!BY CYTOKINES!
DiDonato et al., 1997, Nature 388, 548!
Normal IκB Degradation in IKKα -/- Cells!
Hu et al., 1999, Science 284, 316!
Defective IκB Degradation in IKKβ -/- Cells!
Li et al., 2001, Science 284, 321!
NF-κB!
NF-κB:IκB!
proteasome!
IKKβ
?!
Activation Loops ofIKK-!, IKK-", MEK1, and MKK4
DLGYAKDVDQGSLCTSF-VGTLQYLAPEDLGYAKDLDQGSLCTSF-VGTLQYLAPEDFGVSGQL-IDSMANSF-VGTRSYMSPEDFGISGQL-VDSIAKTRDAGCRPYMAPE
IKK-!IKK-"MEK1MKK4
MAP2K-like Phosphorylation Sites!In the Activation Loops of IKK!
Defective NF-kB Activation IκB Degradation! in TAK1 -/- Cells!
Sato et al., 2005, Nat Immunol 6, 1087!
TAK1!
NF-κB!
NF-κB:IκB!proteasome!
IKKβ!
TNF-α!
THE NF-κB/IκB FAMILIES!
p65 (RelA)
RelB
c-Rel
p50/p105
p52/p100
I!B"
I!B#
Bcl-3
Rel Homology Domain
AnkyrinRepeats
IκBε
Defective p100 Processing in IKKα -/- Cells!
Senftleben et al., 2001, Science 293, 1495
TAK1!
NF-κB!
NF-κB:IκB!proteasome!
IKKβ!
TNF-α!
IKKα!
p100!
NIK TAK1!
NF-κB!
NF-κB:IκB!proteasome!
IKKβ!
LTβ TNF-α!
IKKα!
p100!
MAP3K!
ERK! JNK!
Gene!Expression!
IKK!
NF-κB
• Cytoplasmic-nuclear translocation!• Phosphorylation as a signal for
degradation!
Stimulus!
DAG! Ca++!
PKC!
PLC! MAP3K!
ERK! JNK! IKK!
MEK! MKK4!
