Signal transduction 2nd messengers, PIP and Calcium

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Signal transduction2nd messengers, PIP and
Calcium
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Classical Phosphatidylinositol pathway

• Things to know
• What is phospholipase C b, how is it activated
  and what does it do?
• What does the second messenger DAG do?
• What does the second messenger IP3 do?
• What is protein kinase C and how is it activated?
• What are phorbol esters and what is the
  mechanisms behind their role in carcinogenesis?

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2nd messengers
cAMP IP3 and DAG calcium
2nd messengers
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Phosphatidylinositol
(PI)
Minor phospholipid in cell membranes
Fatty Acids
Glycerol
inositol
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phosphatidyl inositol 4,5-bisphosphate(PIP2)

• Classical pathway depends on the breakdown of
  PIP2
• Polyphosphoinositides are formed by the
  phosphorylation of PI and PI(4)P, respectively.
• Enzymes required
• PI 4 kinase
• PI 4 phosphate 5 kinase
• PI 4,5 bisphosphate (PIP2)
• -mainly in the inner half of the plasma membrane
• -least abundant (less than 10 of total inositol
  lipids)
• -critical breakdown generates two intracellular
  mediators

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PI 4-phosphate
PI 4,5-bisphosphate
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Signal transduction through the
receptor-triggered hydrolysis of phosphatidyl
inositol 4,5-bisphosphate.

• 1. An activated receptor complex leads to the
  activation of one of the
• phospholipase C enzymes.
• 2. Membrane bound phospholipase C hydrolyzes the
  phosphodiester bond linking the phosphorylated
  inositol unit to the acylated glycerol moiety.
• 3. The hydrolysis of PIP2 leads to the production
  of 2 intracellular signaling molecules IP3 and
  DAG.

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G-protein-coupled receptors can activate the
inositol phospholipid signaling pathway through
Gq.

• The activated receptor
• stimulates the GTP for GDP
• exchange on a trimeric G protein
• called Gq.
• activated Gq activates
• phospholipase C-beta.

Gq
Phospholipase C b
GDP
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Phospholipase C (PLC)

• cytosolic enzymes
• act on membrane-inserted phosphoinositide
  substrates.
• Types beta and gamma
• PLC-beta is activated by G-protein-coupled
  receptors, PLC-gamma is activated by receptor
  tyrosine kinases.

Ga
PLC-b1-4
PH domain binds to PIP2
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Phospholipase C cleaves PIP2 to yield two second
messengers (DAG and IP3)
DAG
Phospholipase C-b -cleaves PIP2 to generate
inositol triphosphate (IP3) diacylglycerol (DAG)
IP3
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The signaling pathway splits into two branches
DAG and IP3 each have a job to do.
DAG
PIP2
activated phospholipase C
IP3
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Model of diacylglycerol (DAG) and inositol
1,4,5-triphosphate (IP3)

• DAG
• very nonpolar, in contrast to IP3.
• Remains membrane bound.

IP3
IP3 very high density of negatively charged and
polar groups. IP3 is soluble in the cytosol and
diffuses through it.
DAG
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Inositol trisphosphate (IP3) couples receptor
activation to calcium release from the ER

• IP3
• -rapidly diffuses through the cytoplasm.
• -induces the rapid release of calcium from
  intracellular stores-the endoplasmic reticulum
  and, in smooth muscle cells, the sarcoplasmic
  reticulum.

calcium
IP3
IP-3-sensitive Calcium channel
Endoplasmic Reticulum
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Calcium is released through IP3-gated calcium
release channels

• The IP3 gated calcium release channels are
  regulated by a positive feed back mechanism -
  calcium binds back to the channels to increase
  the calcium release.
• Thus, Ca 2 release is sudden and ALL or NONE

Ca 2
Gated calcium release channels
Ca 2
IP3
Ca 2
Ca 2
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Two mechanisms terminate the initial calcium
response

• Termination of the response
• 1.The lifetime of IP3 in a cell is very short
• IP3 is rapidly dephosphorylated and inactivated.
• 2.The calcium that enters the cytosol is rapidly
  pumped out of the cell.
• 3. Not all IP3 is dephosphorylated some is
  phosphorylated
• to form inositol 1,3,4,5 tetraphosphate (IP4)
• which may promote refilling of the intracellular
• calcium stores.

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What does DAG do?
DAG remains in the membrane -two potential
signaling roles 1. Can be cleaved to release
arachidonic acid -messenger -used to synthesize
eicosanoids Prostaglandins-pain,
inflammation 2. Activates protein kinase C
(major function)
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Protein kinase C (PKC) is activated by DAG and
Ca

• PKC is a serine/threonine kinase.
• Activated by DAG and calcium.

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How is Protein Kinase C activated and what does
it do?
rise in cytosolic calcium -PKC translocates
from the cytosol to the cytoplasmic face of the
plasma membrane. -PKC binds to DAG and is
activated. -activates transcription factors that
regulate genes involved in proliferation.
DAG
DAG
PIP2
PKC
IP3
Ca
ER
Ca
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PHORBOL ESTERS

• The importance of protein kinase C in controlling
  
• cell division and proliferation is revealed by
• the action of phorbol esters.
• phorbol esters
• -polycyclic alcohol derivatives
• - carcinogenic (tumor promoters)
• -activate Protein Kinase C
• mimic diacylglycerol
• not readily degradable and the activation is
• persistent.

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Calcium as a signaling molecule
Things to know

• Calcium DAG activates protein kinase C
• Calcium is used for many signaling purposes in
  the cell-why?
• What kinds of proteins bind calcium?
• What is calmodulin and what does it do?
• Which family of kinases mediate most of the
  actions of Calcium in animal cells?
• Why is CaM-kinase II said to have a memory?
• What is calcineurin and what does it do?
• How do the cAMP and Ca pathways interact?

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Controls on cytosolic Ca

• Calcium is actively pumped out of the cytosol
• Calcium is pumped into the ER, proteins
• bind free calcium

1.
2.
Fig.15-38
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We can monitor calcium levels in a cell

• The concentration of free calcium in a cell can
  be monitored with fluorescent indicators whose
  emissions change markedly when calcium is bound
  (Fura-2 and Fluo-3)

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1. The cytosolic level of Calcium is low in
unexcited cells, but can be rapidly raised in
response to the appropriate signals.
Calcium is used for many signaling purposes in
the cell-why?

• intracellular levels low-
• phosphate esters are abundant and calcium
  phosphate is very insoluble.
• cytosolic level of calcium in unexcited cells is
  typically 100nm, several orders of magnitude less
  than the concentration outside the cell.
• The cytosolic concentration of calcium can be
  rapidly raised by transiently opening calcium
  channels in the plasma membrane or in an
  intracellular membrane.

Ca2 measurements at the single-cell level
3D display
intracellular Ca2 measurement. cells are loaded
with fura-2-AM and excited at 340 and 380 nm
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2. The binding of calcium to a protein can induce
large conformational changes.
Calcium is used for many signaling purposes in
the cell-why?

• Calcium binds tightly to proteins-both negatively
  charged oxygen from glutamate and aspartate and
  uncharged oxygen from glutamine and asparagine
  bind well to calcium.

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Calcium is used for many signaling purposes in
the cell-why?
3. calcium is suited for binding to irregularly
shaped crevices in proteins and can be selected
over Mg 2
Mg 2 is more abundant in cells and is a
potential competitor of Ca 2 - How is Ca 2
chosen over Mg 2 ?

• Mg 2 does not have appreciable affinity for
• uncharged oxygen atoms
• Mg 2 prefers to form symmetric coordination
  shells ,
• whereas Ca 2 can form asymmetric complexes
• having a larger radius

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Proteins with EF hand motifs bind calcium.
What kinds of proteins bind calcium?
EF hand

• The calcium binding domain is formed by two
  helices separated by a loop.
• Calcium binding loop
• is composed of residues containing side chain
  oxygen groups
• The EF hand motif is present in more than 100
  known proteins.
• Example calmodulin

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calmodulin is an EF hand protein that serves as a
calcium sensor.
What is calmodulin and what does it do?

• Calmodulin
• Mutlti-functional calcium receptor
• member of the EF hand family of proteins.
• consists of two globular lobes joined by a long
  alpha helix.
• Each lobe contains two EF hands. There are 4
  calcium binding sites (shown as 1, 2, 3, and 4.)

EF1
1
2
alpha helix
EF2
3
EF3
4
EF4
calmodulin
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A second view of the structure of calmodulin and
the calcium binding loop
calmodulin
Calcium binding loop
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Calmodulin is allosterically activated by Calcium

• Calcium

• Calmodulin
• activated by the binding of three or four calcium
  ions.
• - occurs when the cytosolic calcium level is
  raised above 500 nm.
• Not an enzyme
• Allosteric activator of target proteins.
• Can serve as a permanent component of an enzyme
  complex.

EF1
Calmodulin
alpha helix
EF2
EF3
EF4
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What are calmodulins targets and mechanism of
activation?
jack-knifed calmodulin

• calmodulin targets
• many enzymes
• most important - multifunctional
  calmodulin-dependent protein kinases (CaM Kinase)
  and the plasma membrane Calcium-ATPase pump.

target protein
Fig.15-40
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Most Ca effects are mediated by the CaM-kinases
Which family of kinases mediate most of the
actions of Ca in animal cells?

• The calmodulin-dependent protein kinases
  (CaM-kinases)
• serine/threonine kinases complex of 12 subunits.
• Examples (narrow specificity)
• 1. myosin light-chain kinase activates smooth
  muscle contraction
• 2. Phosphorylase kinase activates glycogen
  breakdown
• Example of multifunctional CaM kinase
• CaM kinase II
• -found in all cells but enriched in nervous
  system
• -Ca influx in neurons activates CaM kinase II
  to phosphorylate tyrosine hydroxylase, the rate
  limiting enzyme in catecholamine synthesis.
• -may play a role in memory and learning mice
  deficient in the a subunit of CaM kinase II
  activity cant remember where they put their car
  keys.

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Calmodulin activates CaM-kinases
Inactive CaM-kinase
Ca/calmodulin
CaM-kinase functions as a molecular memory
device, remembering Ca/calmodulin
activation.
active CaM-kinase
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Why is CaM-kinase II said to have a memory?
Inhibitory domain

• How?
• CaM-kinase becomes activated when calmodulin
  binds to it and remains active after calcium
  withdrawal.

active CaM-kinase
Fully active CaM-kinase
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CaM-kinase has a memory

• Autophosphorylation
• -traps calmodulin so that it does not dissociate
  from the enzyme until cytosolic calcium levels
  remain at base line for 10 seconds.
• -converts the enzyme to a calcium-independent
  form so that the enzyme remains partially active
  even after calmodulin dissociates from it.

Inhibitory domain

• fully active

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A protein phosphatase inactivates CaM-kinase
Inhibitory domain

• CaM-kinase remains partially active until a
  protein phosphatase removes the phosphate
  modification

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How do the cAMP and Ca pathways interact?

• Ways in which the pathways interact
• 1. Some forms of cAMP phosphodiesterase and
  adenylyl cyclase are regulated by
  Ca/calmodulin.
• 2. Share downstream targets.
• Example CaM-kinases can phosphorylate CREB at a
  different site than that phosphorylated by PKA.

PKA
PKA
CaM-kinase
P
P
CREB
P BOX
CREB CRE binding protein transcription factor.
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Calcineurin a calcium regulated protein
phosphatase.
What is calcineurin and what does it do?

• Calcineurin
• Protein phosphatase
• calmodulin binding leads to a conformational
  change in calcineurin that moves the
  autoinhibitory domain of calcineurin away from
  the catalytic site.
• Calcineurin is a major upstream regulator
• of NFAT (nuclear factor of activated T-cells).

Calcineurin is a hetrodimer.It contains one
catalytic subunit, calcineurin A (red) and one
regulatory subunit, calcineurin B.
Ribbon structure
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Role of calcineurin in activation of the NFAT
transcription factor

• NFAT (nuclear factor of activated T-cells)
• -transcription factor
• -plays an important role in inducible gene
  transcription in hematopoietic cells.
• -NHR
• a calcineurin-activated regulatory domain that
  binds calcineurin, is dephosphorylated when
  calcineurin is activated, and controls the
  calcineurin-regulated nuclear translocation of
  NFAT.

DBD DNA binding
DBD
NHR
NHR NFAT homology region
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Role of calcineurin in NFAT activation

• There are three steps involved in the activation
  of NFAT.
• 1. dephosphorylation
• 2. nuclear translocation
• 3. increased affinity for DNA.

Genes regulated by NFAT factors Il-2, IL-4,
IL-5, GM-CSF (growth factors and cytokines
important to hematopoietic cell function)
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Cyclosporin A (CsA) and FK 506

• Immunosuppresive drug used in transplant surgery
  patients.
• Inhibits calcineurin by blocking the active site
  of calcineurin and preventing it from acting on
  any molecules within the cell.
• -major consequence is the inhibition of NFAT
  activity, thus preventing the rejection of
  foreign organs or bone marrow by disrupting the
  signaling pathway that activates T cells.

The photomicrograph illustrates crystallites of
Cyclosporine. This drug is a natural metabolite
of a soil fungus and is isolated from culture
broths. It is currently one of the most useful
drugs of its type.