IGP Signaling 2003

1

• IGP Signaling 2003
• Roger Colbran
• Molecular Physiology and Biophysics
• Office724 RRB(MRB1)
• Email roger.colbran_at_vanderbilt.edu
• Friday Jan 31th 900-1000am Protein
  serine/threonine kinases.
• 1000-1100am Kinase cascades and pathways
• Reading
• Active and inactive protein kinases structural
  basis for regulation. Johnson et al., 1996. Cell
  85, 149-158.
• Discussion of glycogen metabolism and its
  regulation in Stryers Biochemistry, Molecular
  Biology of the Cell etc

2
Second messenger signaling by hormones,
neurotransmitters and growth factors
Ion Channels
PLC
A.C.
Modified function
Estimated to be 500 protein kinase ( 330 ser/thr
kinases catalytic subunits), 150 protein
phosphatases (lt40 ser/thr phosphatase catalytic
subunits) in human genome. About 99 of cellular
protein phosphates are on Ser/Thr, most of rest
on Tyr.
3
How does addition of a phosphate modify function?

• 1. Electrostatic involvement in a binding site
• e.g., SH2 Domain binding to P-Tyr domains
• Bacterial Isocitrate Dehydrogenase

P
active
inactive
2. Long-range conformational effects e.g.,
glycogen phosphorylase b -gt a conversion
Phosphorylation at Ser14 prevents N terminal
domain from interacting with surface of each
monomer, allowing a global conformational change
Glycogen phosphorylase b inactive
Glycogen phosphorylase a active
4
Identification of reversible phosphorylation as a
regulatory mechanism I

• 1945 The Coris
• Glycogen phosphorylase catalyzes the
  rate-limiting step in rabbit skeletal muscle
  glycogen breakdown, and adrenaline shown to
  stimulate glycogen breakdown.
• Biochemically purified two forms of glycogen
  phosphorylase.

More from muscle of control rabbits
More from muscle of adrenaline-injected rabbits
As the a form was stored it was converted to the
b form, and showed that this was an enzymatic
process - termed the PR (phosphorylase rupturing
or prosthetic-group removing) enzyme.
1950s Sutherland Identified inorganic phosphate
as another product of the PR enzyme --gt PR
phosphate removing
5
Identification of reversible phosphorylation as a
regulatory mechanism II

• 1955 Sutherland (Liver) and Krebs/Fischer
  (muscle)
• Demonstrated the enzymatic conversion of the b to
  a form of glycogen phosphorylase in
  partially-purified samples - process required ATP
  and Mg2. Termed the enzyme phosphorylase
  kinase
• ATP Mg2 Phos. b ------------------------
  --gt Phos. a ADP Mg2

Phosphorylase kinase
Reversible!!
PR enzyme
1955-68 How does adrenaline increase the Phos
a/Phos b ratio? In principal, adrenaline could
activate phosphorylase kinase or inhibit PR
enzyme. Experiment (Krebs/Fischer, 1959)
Preincubate partially purified muscle extracts
(depleted in glycogen phosphorylase) then assay
phosphorylase kinase activity Phos. Kinase
activity (Units/ml) Control (Mg2) 100
cyclic AMP 200 (Sutherland had just
shown adrenaline increased cAMP)
ATP 1200 ATP, cAMP 3700 Cannot see this
effect with highly purified phosphorylase kinase!
Therefore, another cofactor is removed during
purification - requires cAMP and ATP to activate
phosphorylase kinase.
6
Identification of the missing cofactor - phos.
kinase kinase
Experiment (Krebs/Fischer, 1959-68)
Biochemically fractionate muscle extracts and
recombine fractions with highly purified
phosphorylase kinase GOAL - to recapitulate
cAMP and ATP-dependent regulation of
phosphorylase kinase. (Eventually) Krebs
purified phosphorylase kinase kinase - a
cAMP-dependent protein kinase. Phos. kinase
kinase can phosphorylate Phos. Kinase, casein,
histones and protamine in a cAMP-dependent
manner Ka 70 µM cAMP
100
Now known as cyclic AMP-dependent protein kinase
(cAK), protein kinase A (PKA), or A-kinase.
50
Casein phosphorylation
70 µM
cAMP
Importantly, Sutherland had shown that 70 µM cAMP
existed in cells
7
Activation of muscle glycogen phosphorylase by
adrenaline - the first protein kinase cascade
8
Protein phosphorylation is a ubiquitous
regulatory mechanism

• First thought that protein phosphorylation was a
  unique way of regulating glycogen metabolism.
  THEN
• 1. PKA shown to phosphorylate many proteins
  physiologically
• Other metabolic enzymes such as glycogen
  synthase (glycogen synthesis), pyruvate kinase
  (glycolysis), hormone sensitive lipase
  (lipolysis)
• Transcription factors such as CREB (gene
  expression)
• Receptors such as b-adrenergic receptor
  (receptor desensitization)
• Ion channels such as voltage gated calcium
  channels (increased cardiac function)
• Cytoskeletal/contractile proteins
• etc,
• Now know there are about 500 protein kinase genes
  in the human genome - almost all are
  evolutionarily related.

9
Almost all protein kinases share sequence homology
Eleven clusters (subdomains) of highly conserved
residues in almost all protein kinases (some
identical in gt90). Minimal protein kinase
catalytic domain is approximately 260 amino acids.
IGXGXXgXV
tXDFG
Many kinases phosphorylated in catalytic domain
(see later)
P
C
N
AXKXI
tXXyI(X)6tXDIXXXNI
ATP-Binding lysine (K) in subdomain II is
essential and invariant (frequently mutated to
give a kinase dead protein)
The XXX sequence in subdomain VI usually defines
the selectivity for tyrosine or serine/threonine
phosphorylation. This is typically a KPE
sequence in serine/threonine kinases, but a AAR
sequence in tyrosine kinases. There are also
dual specificity kinases that phosphorylate
both Ser/Thr and tyrosine residues, typically in
other protein kinases.
10
generic protein kinase structure
Phosphate anchor (domain I) interacts with
phosphate in ATP
Inserts into catalytic domains of various kinases
occur on loops between major secondary structural
features
Catalytic cleft
Activation loop - phosphorylated in many kinases
Catalytic loop (domain VII)
11
If all 330 Ser/Thr kinases function with similar
enzymatic mechanism, how is specificity achieved?

• Appropriate regulation
• Substrate recognition.
• Colocalization with regulators and with substrates

12
Mechanisms contributing to specificity of protein
phosphorylation

• Substrate recognition
• Consensus phosphorylation sequences
• Identify phosphorylated residues in many
  proteins, and obtain sequence of surrounding
  residues.
• In many cases (especially many Ser/Thr kinases)
  these sequences fit a pattern.
• Chemically synthesized small peptides (10-20
  amino acids) with these sequences are often
  phosphorylated as effectively as full-length
  protein.
• PKA ARG- Arg/Lys- Xaa- Ser/Thr- Hyd
• PKC (Arg/Lys)1-3 (Xaa)1-2 Ser/Thr (Xaa)0-2
  (Arg/Lys)1-3
• CaMKII ARG- Gln Xaa Ser/Thr
• Clearly some overlap in specificity, but this can
  account for physiological specificity in some
  cases
• e.g., Phospholamban - inhibitor of the
  Ca2-ATPase in the cardiac sarcoplasmic reticulum
  - phosphorylation by PKA or CaMKII blocks
  inhibitory function and thereby increases Ca2
  uptake into SR. Strictly phosphorylated by PKA at
  Ser16 and CaMKII at Thr17 in the sequence
• Arg Arg Ala Ser Thr
• Other kinases prefer acidic residues (e.g.,
  casein kinase II) or prolines (e.g., MAP-kinases)
  close to their target Ser/Thr

13
2. Protein kinases have divergent regulatory
sequences
homologous catalytic domain
C-subunit
AID
PKA
cAMP
cAMP
R-subunit
Ligand-binding domain
PKG
cGMP
cGMP
PKC
Ca2, diacylglycerol, phospholipids
MLCK
CaM
CaMKII
CaM
Casein kinase II
TM
EGFR
c-src
14
Regulation of protein kinases

• Want to keep kinases inactive and only activate
  them when necessary

e.g., PKA is an inactive tetramer in the absence
of cAMP
Regulatory subunits dimerize via an N-terminal
dimerization (DIM) domain
Catalytic cleft of C-subunit is occluded by
inhibitory domain of R-subunit so substrates
cannot bind.
DIM
catalytic subunit
INH
site 1
site 2
Core regulatory (R) subunit dimer - two genes RI
and RII. a/b splice variants of each
Two cAMP-binding sites per R subunit.
15
Mechanism of inhibition of PKA C subunit by R
subunit

• Inhibitory domains of R subunits, and of a
  naturally occurring PKA inhibitor protein (PKI),
  share sequence similarity with the PKA consensus
  phosphorylation sequence
• PKA consensus ARG Arg/Lys Xaa Ser/Thr Hyd
• RI subunit ARG ARG GLY ALA ILE (a
  pseudosubstrate)
• RII subunit ARG ARG VAL SER(P) VAL
• PKI ARG ARG ASN ALA ILE (a pseudosubstrate)
• Proteolytic studies, as well as site-directed and
  deletion mutagenesis experiments, indicated this
  region is essential for potent inhibition by RI,
  RII and PKI.
• However, small synthetic peptides containing only
  this region are much weaker inhibitors than the
  intact RI or RII subunit or longer PKI peptides.

(function of PKI still debated may suppress
basal PKA activity, and may play a role in export
of catalytic subunit from nucleus)
16
Co-crystalization of PKI-tide with the PKA C
subunit

• PKI a natural and specific PKA inhibitor

F10 is critical for potent inhibition by PKI but
is not conserved in R subunits
C-subunit - red Conserved residues - white ATP
and PKI - yellow Thr197 in C-subunit is
phosphorylated
17
Activation of PKA by cAMP
DIM
catalytic subunit
INH
4cAMP

site 1
site 2
2
cAMP binds first (i.e., at lower cAMP) to site
1 due to a higher affinity, inducing a
conformational change which increases the
affinity of site 2 for cAMP. Full activation
requires occupation of all four cAMP binding
sites and dissociation of the C subunit. Thus,
activation displays positive cooperativity
100
PKA activity
50
cAMP
70 µM
18
Activation of kinases by catalytic domain
phosphorylation

• PKA C subunit is constitutively phosphorylated at
  thr197 - the phosphate makes essential
  interactions that stabilize the bilobal
  structure/orientation (see crystal structure 2
  slides previous).
• Many other kinases have conserved Thr and/or Tyr
  residues in the activation loop (i.e., in the
  same location as Thr197) - phosphorylation of
  these residues is dynamically modulated to
  regulate kinase activity.
• PKA KGRT------WTLCGT autophosphorylation
  (and/or PDK)
• ERK2 DPDHDHTGFLTEYVAT MEK (MAP kinase
  kinase)-dual specificity kinase
• CaMKI GSVL------STACGT CaM-kinase kinase (CaMKK)

P
P
P
P
CaMKI has an autoinhibitory domain AND requires
both binding of calcium/calmodulin and
phosphorylation in the activation loop for full
activation
19
Structural comparison of inhibited CaMKI and PKA
4Ca2 plus calmodulin
Ca2/ CaM
PKA PKItide
CaMKI
20
(No Transcript)
21
Integrated regulation of muscle glycogen
metabolism
ADRENALINE (epinephrine) Fight or flight
response --gt activation of phosphorylase and
inactivation of synthase --gt glycogen broken
down to provide energy for contraction. INSULIN
After feeding --gt inactivation of phosphorylase
and activation of synthase --gt more glycogen
stored
22
Phosphorylase kinase integrates cAMP and Ca2
signals
Calmodulin is a ubiquitous Ca2-binding protein
that binds many proteins, including several
kinases
23
Mechanisms of (Glycogen) Phosphorylase Kinase
regulation
Complex oligomeric protein native molecular
weight 1.34 X 106. Holoenzyme contains 4 copies
each of 4 distinct subunits, ?, ?, ?, ?.

• ?, ? Regulatory subunits --gt both phosphorylated
  by PKA
• ? catalytic subunit also binds exogenous
  calmodulin in presence of Ca2
• ? calmodulin associated with holoenzyme even in
  absence of Ca2

Ca2 activates phosphorylase kinase by 1.
Binding to ? subunit (integral CaM) 2.
Allowing binding of additional CaM (or troponin C)
100
PKA Phosphorylation Increases Vmax 20X Decreases
Ka(Ca2) 20X
Phosphorylated by PKA
Activity of purified Phos. Kinase
50
Nonphosphorylated enzyme further stimulated by
extra CaM or troponin C
100
50
Nonphosphorylated
Ca2
20 µM
1 µM
24
Mechanism of phos. kinase regulation by PKA
PKA phosphorylates the a and b subunits, but with
a different time course a simple case of
multi-site phosphorylation.
? subunit
response
activity
a subunit
Lag in a subunit phosphorylation phosphorylation
of b subunit required first?
time
suggests ? subunit phosphorylation primarily
responsible for activation
Possible roles of a subunit phosphorylation?
Silent? Modulates dephosphorylation? PP1
selectively dephosphorylates the ??subunit ---gt
inactivation. However, this reaction is slower
when the a??subunit is also phosphorylated. PP1
and Phos. Kinase are both associated with the
glycogen particle - PP1 activity is regulated
(see Wadzinski) Type 2 phosphatases selectively
dephosphorylate a??subunit.
25
Advantages of multi-site phosphorylation

• Many proteins are phosphorylated at multiple
  serine, threonine and/or tyrosine residues. This
  can have a number of advantages
• Allows multiple inputs into a single process
  (signal integration)
• Allows more graded responses
• Allows opposing regulation of a process by
  distinct pathways
• Phosphorylation of one residue can create a new
  consensus site for another kinase. e.g., GSK3
  recognizes the sequence Ser-Xaa-Xaa-Xaa-SerP
• Phosphorylation by one kinase can block
  phosphorylation by a second kinase at a distinct
  site.
• Phosphorylation of additional sites may modulate
  dephosphorylation.
• Thus, knowledge of the specific sites
  phosphorylated is critical to understanding
  regulation.

26
Regulation of skeletal muscle glycogen synthase.

• Glycogen synthase was the second substrate for
  PKA that was identified (1963)

PKA
Active I-form
Inactive D-form Can be stimulated by G-6-P
Immediate hypothesis Elevation of cAMP in
response to adrenaline activates PKA and provides
coordinated regulation of glycogen metabolism by
activating glycogen breakdown (via Phos. Kinase)
and inactivating glycogen synthase.
27
In vitro studies of glycogen synthase
phosphorylation
Glycogen synthase (GS) is phosphorylated at more
than nine serine residues by many protein kinases
that are regulated by diverse signal transduction
pathways (took gt 20 years to work out).
Chemically digest phosphorylated protein with
cyanogen bromide - specifically cuts at Met
residues. Two fragments (CB-N and CB-C) contain
all phosphorylation sites. Sites can be further
separated by reverse phase HPLC and identified
after proteolytic digestion with trypsin cuts at
basic (R/K) residues
N
C

• Also measure effects of different kinases on G.S.
  activity surprisingly PKA has relatively little
  effect blowing the simple early hypothesis out of
  the water. More detailed analyses showed that
• Phosphorylation of Ser46, Ser87 and Ser100 has
  little direct effect on activity.
• Phosphorylation of Ser7 is moderately
  inactivating.
• Phosphorylation of Ser10 and Ser30/34/38/42 is
  potently inactivating.
• However, CK1 (casein kinase 1) and GSK3 (glycogen
  synthase kinase 3) were not know to be regulated
  at that time.

28
Which sites in GS are phosphorylated in vivo?

• Use rabbit skeletal muscle as a model system.
  Experiment
• Anesthetize the animal.
• Inject hormones, agonists or antagonists for
  brief time.
• Dissect skeletal muscle and freeze - must be
  done rapidly at 4C.
• Purify glycogen synthase while blocking
  phosphorylation and dephosphorylation in extract.
• Digest protein with cyanogen bromide and then
  digest CBB and CBC with trypsin.
• Analyze peptide mixture on reversed phase HPLC.
• Measure extent of phosphorylation in each peptide
  by chemical phosphate estimation, mass
  spectrometry or back phosphorylation (3
  different labs in early 1980s).

Starved/propanalol (b blocker) Starved/adrenaline
Phosphorylation Stoichiometry (mol/mol)
Activation of PKA in response to adrenaline may
account for changes at Ser7, Ser87 and Ser100.
What about changes at Ser10 (casein kinase 1) and
Ser30-38 (GSK3), which likely account for
inactivation in vivo?
29
Phosphorylation of Ser10 is potentiated by Ser7
phosphorylation
See the same thing with a synthetic peptide
containing Serines 7 and 10. CK1 recognizes a
phosphorylated Ser in the consensus
phosphorylation sequence SerP-Xaa-Xaa-Ser. Terme
d hierarchal phosphorylation
N-terminal sequence of glycogen
synthase N-Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ser-S
er-Leu-Pro-..
P
P
PKA
CK1
This mechanism could explain increased
phosphorylation at Ser10 in glycogen synthase
following administration of adrenalin. Adrenalin
--gt elevated cAMP --gt PKA activation --gt
increased Ser7 phosphorylation --gt increase in
availability of Ser10 as a CK1 substrate.
30
How does adrenaline increase phosphorylation at
Ser30-38?
These sites are exclusively phosphorylated by
GSK3, which also recognizes a phospho-Ser in its
consensus phosphorylation sequence Ser -- Xaa
-- Xaa -- Xaa -- SerP. GSK3 phosphorylation of
glycogen synthase is potentiated by prior casein
kinase 2 (CK2) phosphorylation - also hierarchal
BUT Phosphorylation of Ser46 does not change
following adrenaline injection in vivo. How
does cAMP elevation result in increased Ser30-38
phosphorylation?
31
The phosphorylation-dephosphorylation cycle
Protein phosphatase phosphoprotein phosphatase
(more properly) PP PrP
The overall level (or STOICHIOMETRY) of
phosphorylation of any cellular protein is
determined by the BALANCE of the activities of
specific protein kinase and protein phosphatases
involved. This is generally a dynamic process,
even under basal conditions. Thus, there is
so-called futile cycling of cellular ATP - not
really futile since it allows for fine and rapid
regulation by subtle changes in the activities of
EITHER the kinase OR the phosphatases an
advantage in many cases. Although protein
phosphatases were actually discovered prior to
protein kinases (see PR enzyme in first hour),
our knowledge of phosphatases has lagged behind
that of the kinases.
32
Adrenaline (via PKA) inhibits protein phosphatase
1G -the glycogen synthase phosphatase
cAMP
Adrenaline
Adrenaline induces increased phosphorylation at
Ser10 and Ser30-38 inhibiting glycogen synthase
activity, even though these sites are not
directly phosphorylated by PKA. Thus, more
glucose remains available for energy production
by glycolysis
PKA
glycogen
glycogen
Soluble INACTIVE
G.S.
G.S.
Glycogen- associated ACTIVE
Glycogen- associated
Constant GSK3 and CK1 activity