The document summarizes protein kinases, specifically focusing on serine/threonine phosphorylation mediated by protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). It describes the structure and activation of PKC, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). PKC contains regulatory domains like C1 and C2 that mediate its translocation to membranes in response to diacylglycerol and calcium. ERK and JNK are activated through phosphorylation by upstream MAPK/ERK kinases in cascades. Scaffold proteins
Carboxypeptidase Y (CPY) catalyzes the following reaction: Peptidyl-L-amino acid + H2O ------> Peptide + L-amino acid.
It is prepared according to the method of Moore & Stein (J. Biol Chem, 211, 907, 1954). It resembles Carboxypeptidase A in its substrate specificity, but it hydrolyzes C-terminal glycine and L-leuicine more rapidly and L-phenylalanine more slowly.
Nucleic Acids, RNA, DNA, Protein Synthesis, DNA Replication, Chromosomes: The images have big font size and reduced background color. Useful for smartphones, classroom and printouts.
Carboxypeptidase Y (CPY) catalyzes the following reaction: Peptidyl-L-amino acid + H2O ------> Peptide + L-amino acid.
It is prepared according to the method of Moore & Stein (J. Biol Chem, 211, 907, 1954). It resembles Carboxypeptidase A in its substrate specificity, but it hydrolyzes C-terminal glycine and L-leuicine more rapidly and L-phenylalanine more slowly.
Nucleic Acids, RNA, DNA, Protein Synthesis, DNA Replication, Chromosomes: The images have big font size and reduced background color. Useful for smartphones, classroom and printouts.
I am a IB HL Biology Student. Our teacher asks students to give lectures, I gave my lecture on Transcription and Translation. Please read through slide notes, should be quite helpful. Also contains some past paper questions.
Molecular and Structural Mechanism for Beta Barrel Proteins Incorporation in ...USTC, Hefei, PRC
Beta Barrel Proteins are important for membrane processes. This presentation is a simplified explanation of research article which elaborate incorporation of beta barrel proteins transport and incorporation and secretion snapshot from outer bacterial cell wall.
I am a IB HL Biology Student. Our teacher asks students to give lectures, I gave my lecture on Transcription and Translation. Please read through slide notes, should be quite helpful. Also contains some past paper questions.
Molecular and Structural Mechanism for Beta Barrel Proteins Incorporation in ...USTC, Hefei, PRC
Beta Barrel Proteins are important for membrane processes. This presentation is a simplified explanation of research article which elaborate incorporation of beta barrel proteins transport and incorporation and secretion snapshot from outer bacterial cell wall.
g protein coupled receptors, ion channels, types of receptors, wnt signalling, cell signalling, tranduction pathway, disorders regarding the signalling
GPCRs are the most dynamic and most abundant all the receptors. The G protein-coupled receptor (GPCR) superfamily comprises the largest and most diverse group of proteins in mammals. GPCRs are responsible for every aspect of human biology from vision, taste, sense of smell, sympathetic and parasympathetic nervous functions, metabolism, and immune regulation to reproduction. GPCRs interact with a number of ligands ranging from photons, ions, amino acids, odorants, pheromones, eicosanoids, neurotransmitters, peptides, proteins, and hormones.
Nevertheless, for the majority of GPCRs, the identity of their natural ligands is still unknown, hence remain orphan receptors.
The simple dogma that underpins much of our current understanding of GPCRs, namely,
one GPCR gene− one GPCR protein− one functional GPCR− one G protein −one response
is showing distinct signs of wear.
Powerpoint Search Engine has collection of slides related to specific topics. Write the required keyword in the search box and it fetches you the related results.
1. SERINE AND THREONINE PHOSPHORYLATION:!
PROTEIN KINASE C, MAP KINASES, AND NF-κB!
!
!
Frank S. Lee!
605 Stellar Chance Labs!
898-4701!
franklee@mail.med.upenn.edu!
2. PROTEIN KINASES:!
The Phosphotransfer Reaction!
PROTEIN! O -! PROTEIN! O!
+!
ATP
ADP
3. PROTEIN KINASE STRUCTURE (PKA)!
2 lobes
N= beta pleated sheets
C=alpha helices
N-terminal
!
cleft = active site
lobe
! Active!
site!
C-
terminal
!
lobe
!
4. PROTEIN KINASES:!
Eleven Subdomains!
• Subdomain I: GXGXXG forms ATP binding
pocket!
• Subdomain II: Lys interacts with α and β
phosphates of ATP! promote nucleophilic attack on gamma phosphate - to inact catalytic act of
kinase, highly conserved site for mutagenesis
• Subdomain VI: Asp deprotonates hydroxyl
of Ser or Thr of protein substrate!
• Subdomain VII: Asp chelates Mg++, which
orients γ phosphate of ATP! -why Mg typically includd in protein kinase rxns
7. PROTEIN KINASE C:!
CATALYTIC (C3/4) AND AUTOINHIBITORY DOMAINS!
CATALYTIC!
AI = portion of PKC that blocks AI!
enz activity of PKC in resting state
Orr and Newton, 1994, JBC 269, 8383!
8. 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...!
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
11. PROTEIN KINASE C:!
C2 DOMAIN!
calcium sensor
also binds to
phosphotidylserine
in addition to Ca
Verdaguer et al., 1999, EMBO J 18, 6329!
12. PROTEIN KINASE C:!
Ca++ ACTIVATION!
MEMBRANE!
PS! Ca
! how it activates PKC in response to these second
messengers
C1! C2!
AI! C3/4!
13. PROTEIN KINASE C:!
C1 DOMAIN!
globular structure
hydrophobic = green
white = hydrophilic
large hydrophobic patch w/
hydrophilic surface
DAG = promotor that's
analog
DAG would bind to same
location (?) - create
GREEN=HYDROPHOBIC!
continuous hydrophobic WHITE=HYDROPHILIC!
surface and translocation of PKC BLUE=ACIDIC!
to membrane RED=BASIC!
Zhang et al., 1995, Cell 81, 917!
14. PROTEIN KINASE C:!
AUGMENTATION OF PKC AFFINITY FOR PS BY DAG!
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
Newton & Keranen, 1994, Biochem 33, 6651!
15. PROTEIN KINASE C:!
DAG ACTIVATION!
MEMBRANE!
DAG! PS! Ca
! DAG binds to C1 domain
C2 - promotoes translocation of PKC to PM, results
in translocationof A1/movement = catalytic activity
C1! C2! of enzyme
AI!
C3/4!
16. MARCKS !
(myristoylated alanine-rich !
protein kinase C substrate)!
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
! S!
S
!
S
K !
K!
K ! K! K!
17. MARCKS REGULATION OFACTIN!
in presence of native
peptide, promote
actin polymerization
MARCKS !
if you take peptide, PEPTIDE!
phosphorylate residues,
this peptide is defective
+!
in promoting actin ACTIN!
polymerization ??
MARCKS !
PHOSPHOPEPTIDE!
+!
ACTIN!
Hartwig et al., 1992, Nature 356, 618!
19. PROTEIN KINASE C
• Autoinhibitory domains as
pseudosubstrates!
• Integration of signals!
• Subcellular localization
imp component PKC from cyt to PM is
translocation of
of enzyme
21. ERK (MAPK): An Insulin-Stimulated Kinase!
Phosphorylated on Thr and Tyr!
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
ERK!
Ray & Sturgill, 1988, PNAS 85, 3753!
22. Activation of the!
Serum Response Element!
Elk is component of ternanry complex
SRF
! Elk-1! DNA element - binds to SRF and binds
to Elk. phosph of ELk (on ser/thr
residue) results in potentiation of
transcriptional activity of protein
ERK!
S 383!
SRF
! Elk-1!
23. ACTIVATION LOOP!
PHOSPHORYLATION IN ERK! in inactive and active state
...DHTGFLTEYVAT...!
UNPHOSPHORYLATED! PHOSPHORYLATED!
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
Canagarajah et al., 1997, Cell 90, 859!
25. ACTIVATION OF ERK BY MEK! examine capacity of MEK
prot kinase assay - s ubstrate in presence
of p32 ATP/ sds page - id of phosph
species
MEK! +! +! +! mek or erk alone - no sig phosph
add both = phosphorylation -
enhancement of cat activity of ERK
ERK! +! +! MEK = dual specificity prot kinase (spec
for Ser and Tyr)
ERK!
MEK ...LIDSMANSFVGT...! 2 residues in SXXS motif -MEk to be
mutate, abolish capcity of
if you
ERK ...DHTGFLTEYVAT...! activated
Crews et al., 1992, Science 258, 479!
26. ?!
MAP2K! MEK1! - has its own phosph loop, implying its target of a
diff prot kinase
phosphorylates
MAPK! ERK!
27. ACTIVATION OF MEK BY Raf!
observations that cells
transformed with Raf have high
ERK act - is Raf activator of
pathway? yes
Raf - transform cells - assay
fractions - MEK = substrate
MEK1!
activity int ransformed cells =
phosph to MEK - cna directly
phosph MEK - specific Serines
Kyriakis et al., 1992, Nature 358, 417!
29. PROTEIN KINASE CASCADES
• Amplification! of small signal
• Multiple layers of regulation!ie. dephosph of ERK
and inact ERK and
• Crosstalk! phosphatases are dual
specific phosphatases
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
31. Raf:RAP COMPLEX! Raf has mult
domains kinase
domain, Nterm
regulatory domain
- interacts w/
small GTPases -
active when
bound to GTP
Rap member of
small GTPase
Nasser et al., 1995, Nature 375, 554!
35. Activation of c-Jun! cJUN has dna binding domain
- leucine zipper and
transactivation domain - large -
Transactivation! DNA binding!
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
1! 331! transcription factors and RNA
PolII
63!73!
CBP!
TF s!
RNA Pol II!
36. C-Jun N-Terminal Kinase (JNK)/!
Stress Activated Protein Kinase (SAPK):!
Homology to ERK!
homology between this and
ERK
degree of conservation
Derijard et al., 1994 Cell 76, 1025!
37. ACTIVATION OF JNK vs. ERK!
are stimuli that active
simialr?
-IP followed by prot
Jnk resp to strssful
kinase assay - Ab
stimuli distinct
against prot kinase of
from growth factors
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
Kyriakis et al., 1994, Nature 369, 156!
38. ACTIVATION OF JNK/SAPK!
BY UV IRRADIATION!
IP jnk, assay it
quantitate
see low basal act and dramatic
induction o fJnk act at around 60
mins
Derijard et al., 1994, Cell 76, 1025!
39. MAPK PHOSPHORYLATION LOOPS!
homology in region of activation loop of jnk - TXY motif - conserved in all isoforms of JNK
helps identify subfamilies of MAPK
abolish activity of JNK/ability to be activated fi you mutate
MKK4! SGQLVDSIAKTMDAGCRPYMAPE
!
41. ACTIVATION OF JNK BY MKK4 (SEK1)!
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
Sanchez et al., 1994, Nature 372, 794!
44. ACTIVATION OF MKK4 (SEK1) BY MEKK1!
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
Yan et al., 1994, !
Nature 372, 798!
